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Expanding Indications for the New Macrolides, Azalides, and Streptogramins edited by Stephen H. Zinner Brown Univenity Rhode Island Hospital Roger Williams Medical Center Providence, Rhode Island
Lowell S. Young Kuzell Institutefor Arthritis and Infctious Diseases California Pacific Medical Center San Francisco, California
Jacques F. Acar Fondation HSpital Saint-Joseph Paris, France
Harold C. Neu College Physicians &' Strtgeons Columbia Univenity N m York, Neprlr York
MARCELDEKKER, INC. D E K K E R
NEWYORK BASEL HONGKONG
Library of Congress Cataloging-in-Publication Data Expanding indications for the new macrolides, azalides, and streptogramins / edited by Stephen H. Zinner... [et al.]. p. cm. - (Infectious disease and therapy ;v. 21) (Infectious disease and therapy ;v. 21) “The Third International Conference on Macrolides, Azalides, and Streptogramins (ICMAS111) was held January 24-26,1996, in Lisbon, Portugal”-Pref. Includes index. ISBN 0-8247-0056-2 (hardcover :alk.paper), ISBN 0-8247-0141-0 (soficover .:alk. paper) 1. Macrolide antibiotics-Congresses.2. Antibiotics-Congresses. I. Zinner, StephenH. 11. International Conference on the Macrolides,halides, and Streptogramins (3rd: 1996: Lisbon, Portugal) 111. Series. IV. Series: Infectious disease and therapy; v. 21. [DNLM: 1. Antibiotics, Macrolide-congresses. 3. Virginiamycincongresses. W1IN406HMNv.2119971 RM666.M25E97 1997 615’.3294c21 DNLMDLC for Libraryof Congress 97-12233 CIP The publisher offers discountson this book when ordered in bulk quantities. For more information, write to Special SalesProfessional Marketing at the address below. This book is printed on acid-free paper. Copyright
1997 by Marcel Dekker, Inc. All Rights Reserved.
Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Marcel Dekker, Inc. 270 Madison Avenue, New York, New York 10016 Current printing (last digit): 10987654321 PRINTED IN THE UNITED STATES OF AMERICA
Series Introduction
Marcel Dekker, Inc., has for many years specialized in the publication of high-quality monographs in tightly focused areas in a variety of medical disciplines. These have been great valueto both the practicing physician and the research scientist as sources of detailed andup-to-date information presented in an attractive format. During the last decade,there has been a veritable explosion in knowledge inthe various fields relatedto infectious diseases and clinical microbiology. Antimicrobial resistance, antibacterial andantiviralagents,AIDS, Lyme disease,infectionsinimmunocompromised patients, and parasitic diseases are but a fewof the areas in which an enormous amount significant work has been published. The Infectious Disease and Therapy series covers carefully chosen topics that should be of interest and valueto the practicing physician,the clinical microbiologist, and the research scientist. Brian E. Scully, M . B., B.Ch. Harold C. Neu, M.D.
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Preface
The Third International Conference on Macrolides, Azalides, and Streptogramins (ICMAS 111) was held January 24-26, 1996, in Lisbon, Portugal. This meetingattracted over 1300 scientists, physicians, and clinical investigators from all over the world. The program included state of the art lectures, roundtable discussions, eight workshop symposia, research updates fromindustry,andover 300 poster presentations. Since the first ICMAS meeting in January, 1992, enormous progress has been made the in research and development ofnew compoundsandtheirapplication to clinical medicine. This volume includes plenary papers, summariesof the workship symposia, and selectedextended.abstractsfrom the meeting. The macrolide antibiotics were amongthe earliest antibioticalternatives to penicillin. Erythromycin has certainly maintained an important place in the worldwide therapeutic armamentarium. Certain therapeutic disadvantages of erythromycin were overcome by the introduction of the new macrolides and azalides, clarithromycin, roxithromycin, and azithromycin.Theseagentshaveprovidedexcellentintracellularantibacterial activity-with a broader bacterial spectrum than erythromycin-and considerably fewer unwanted side effects. The streptogramins, currently used extensively in some countries, haveattracted a tremendous levelof interest. Quinupristiddalfopristin,the newest streptogramin, has important activity against staphylococci resistant to methicillin andEnferococcwsfueciurnresistant to vancomycin. The latest clinical and microbiological data on this and related agentsare summarized in this volume. As resistant bacteria continue to challenge clinicians worldwide, additional antibioticswill surely be needed. The ketolide antibiotics, introduced in this volume, provide activity against penicillin-resistant pneumococci as V
vi
Preface
well asother important gram-positive and gram-negative pathogens. Several other uses for macrolide and azalide type antibiotics have been discovered in recent years. The nonbacterial effectsof macrolideshzalides on immunity and on theproduction of mucoid bacterial products were discussed at this conference andare included in this book. This conference provided an opportunityfor the exchange of scientific informationabout laboratory and clinical research among the pharmaceutical industry and clinical and laboratory investigators. Many new aspects of the use of macrolides, azalides, and streptogramins were presented and are discussed. Through the active discussion at this meeting we recognize the major accomplishments with these compounds and also the new problems on the horizon to challenge basic and clinical investigators in industry and academe. Interest in the macrolide, azalide, streptogramin, and new ketolide compounds undoubtedlywill increase as we struggle to maintain our advantage over bacterial pathogens. Many of these challenges are set forth within these pages and we look forward to pursuing their solutions in future, similar meetings.The organizers are grateful formajor contributions fromAbbott Laboratories, Hoechst Marion Roussel, Pfizer, Inc., and Rh6ne-Poulenc Rorer, which made possible the organization of ICMAS 111. Other support from Pliva Pharmaceuticals and Eli Lilly is greatly appreciated. The editors are grateful to our General Secretariat, Ann Wallace, for her style, substance, and tireless efforts on behalf ICMAS. Stephen H. Zinner Lowell S. Young. Jacques F. Acar Harold C. Neu
Contents
Series Introduction Preface Contributors to Plenary Sessions and Workshops
iii
PLENARY SESSIONS 1. Overview of the Clinical Use of Macrolides and Streptogramins Roger G. Finch 2. Postantibiotic Effects and the Dosing of Macrolides, Azalides, and Streptogramins William A . Craig Ketolides:NewSemisynthetic14-MemberedRing Macrolides Andrt! Bryskier, Constantin Agouridas, and Jean-FranCois Chantot 4. Streptogramins: From Parenteral to Oral Daniel H . Bouanchaud
51
Azalides: Basic and Clinical Research Michael W; Dunne
vii
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Contents
7. Nonantibiotic Effects of Macrolide Antibiotics: Suppression of the Bacterial Glycocalyx of Pseudomonas aeruginosa Isolates from Patientswith Cystic Fibrosis Charles W. Stratton
91
8. Effects of Macrolides on Leukocytes and Inflammation Marie-Thdr2se Labro
101
9. Animal Uses of Macrolides and Related Antibiotics Jean-Pierre Lafont, Elisabeth Chaslus-Dancla, and Jean-Louh Martel
117
10. The New Macrolides for Treatment of Pediatric Infections: Roundtable Discussion George H . Mecracken, Jr., Urs B. Schaad, and M. J. Tarlow The Molecular Mechanism of Action of Streptogramins and Related Antibiotics C. Cocito, M. Di Giambattista, E. Nyssen, and l? Vannuflel
12. Early Clinical Results withQuinupristinlDalfopristin for the Therapy of Bacteremia Due to Resistant Gram-positive Bacteria Robert C. Moellering, Jr., and Sharon L. Cenvinka 13. Drug Interactions of Macrolides and halides Ethan Rubinstein and Shlomo Segev 14. Macrolides, halides, and Streptogramins in Treatment of Opportunistic Infections in Immunocompromised Patients Jack S. Remington
131
145
173 177
189
WORKSHOPS
15. Legionella and Spirochetes I. M. Hoepelman and Daniel M. Musher
207
16. Mycoplasma and Chlamydia J. Thomas Grayston and Pentti Huovinen
219
17. Staphylococci, Streptococci, and Pneumococci Jacques E Acar and Josd Melo-Cristino
225
18. Pharmacokinetics and Pharmacodynamics Charles H . Nightingale and Fritz Sorgel
233
ix
Contents Campylobacter and Helicobacter pylori Jean-Paul Butzler and Francis Mkgraud
Haemophilus influenzae, Enterococci, and Anaerobes Vincent T. Andriole and Ian Phillips The Newer Macrolides, Azalides, and Streptogramins for the Management of HIV-Associated Opportunistic Infections Kenneth H. Mayer and J. Allen McCutchan 22. Special Pathogens John E. McGowan, Jr., and Joel Ruskin EXTENDED ABSTRACTS
I. NEW AGENTS Ketolides: A New Class of Macrolide Antibacterials-Structural Characteristics and Biological Properties of RU 004 Constantin Agouridas, X Benedetti, A. Bonnefoy, P. Collette, A. Denis, P. Mauvais, G. Labbe, and Jean-Francois Chantot Isolation of an Antifungal Macrolide from Soil Sample Nocardioides Strain: Production and Structure Elucidation K Loppinet, L. Hilali, N. Youssef, R. Bonaly, and C. Finance
JI. LEGIONELLA, SPIROCHETES Azithromycin in the Treatment of Community-Acquired Legionnaires’ Disease I. Kuzman, S. Schonwald, and . l &.dig Temperature Dependence of MIC and MBC of Roxithromycin Against Borrelia burgdo$eri In Vitro I. Wendelin, R. Gasser, and E. C. Reisinger Tolerability of Treatment with g of Azithromycin Given in 5 Days Franc Strle, Vera Maraspin, Stanka Lotrit-Furlan, and JoZe Cimperman Azithromycin in the Treatment of Erythema Migrans J. GoriSek and J. Rogl
Contents Follow-up Study of Patients with Syphilis Treated with Azithromycin A. L. Mashkilleyson and M. A. Gomberg
m. MYCOPLASMA, CHLAMYDIA A Comparison of the In Vitro Sensitivity of Chlamydia pneumoniae to Macrolides and a New Benzoxazinorifamycin, KRM-1648 C.-C. Kuo, J Thomas Grayston, T. Hidaka, and L. M. Rose Susceptibilities to Azithromycin of Isolates of Chlamydia pneumoniae from Patients with Community-Acquired Pneumonia l? M. Roblin, N. Sokolovskaya, and M. R. Hammerschlag Azithromycin in Control of Trachoma Julius Schachter Microbiologic Efficacy of Azithromycin forthe Treatment of Community-Acquired Pneumonia Due to Chlamydia pneumoniae in Children M. R. Hammerschlag, P. M. Roblin, Michael Campbell, Antonia Kolokathis, M. Powell, and the Pediatric Pneumonia Study Group Community-Acquired Pneumonia in Children: Underestimation of Mycoplasma Infection Efficacy and of Macrolides Dominique Gendrel, Josette Raymond, Florence Moulin, JeanLuc Iniguez, Sophie Ravilly, Pierre Lebon, and Gabriel Kalija
Ureaplasma urealyticumIsolations from Young Children with Respiratory Sharon A. Poulin, Ruth B. Kuna'sin, and Rita D. DeLollis
Asthma? Clarithromycin PreventCould Rita D. DeLollis, Ruth B. Kuna'sin, and Sharon A. Poulin Azithromycin in the Treatment of Chlamydia trachomatis Infection Dajek Single-Dose Azithromycin inthe Treatment of Genital Chlamydial V: Ferianec, K. Holoman, J. Chmurny, V: Grba, and F. Stano
xi
Contents Single-Dose Azithromycin Treatment: A Solution for Uncomplicated Lower Genital Tract Chlamydial Infection B. Kobal Report of a Multicenter Clinical Evaluationof Azithromycin in the Treatment of Nongonococcal Urethritis Men in M. Urban, J. Flek, V. Herman, M. Chaloupka, B. Krhny, J. Klamo, D. Pactk, and P. Tomdtik Efficacy of Roxithromycin in Urogenital Infection I. I. Derevianko
IV. STAPHYLOCOCCI,
STREPTOCOCCI, PNEUMOCOCCI
Dissociated Macrolide and Lincosamide Resistance in Streptococcus pneumoniae and Variation of Susceptibility Testing Detecting Clindamycin Resistance Methods in E. L. Fasola, S. Bajaksouzian, P. C. Appelbaum, and M. R. Jacobs Prevalence of Antibiotic Resistance inStreptococcus pyogenes: Results of a National MulticenterSurvey Conducted in Italy During E. A. Debbia, P. Cipriani, G. l? Gesu, G. Ortisi, M. G. Menozzi, E. Nani, V . Nicolosi, R. Rigoli, R. Serra, M. Toni, K E. Vigand, and G. C. Schito In Vitro Activityof Some Macrolides Against S. pyogenes G. Tempera, P. M . Fumeri, V. Nicolosi, and G. Nicoletti Antipneumococcal Activity of RP (an Injectable Streptogramin), Erythromycin, and Sparlloxacinby MIC and Rapid G. A. Pankuch, M. R. Jacobs, and l? C. Appelbaum Effect of Macrolide Resistance on the Activity of the Oral Streptogramin RPR and Its Components Against Streptococcus M. R. Jacobs, S. K . Spangler, and P. C. Appelbaum
In Vitro Antibacterial Activity of RU 004, a New Ketolide piratory ns Against Active Constantin Agouridas, A. Bonnefoy, and Jean-Fraqois Chantot
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Contents
Antistaphylococcal Activity of QuinupristidDalfopristin, a WellDefined Mixture of Chemically Modified Streptogramins Christof von Eiff and Georg Peters E-Test for Susceptibility Testingof Streptococcus pyogenes to Azithromycin, Clarithromycin, Erythromycin, and Roxithromycin Gerard J. van Asselt and Jacobus H. Sloos Efficacy of Clarithromycin Against Experimental Pulmonary Infection Caused by Streptococcus pneumoniae Strains with a Novel Macrolide Resistance Mechanism J. A. Meulbroek, M. J. Mitten, A. Oleksijew, K D. Shortridge, S. K. Tanaka, and J. D. Alder Macro- and Microautoradiographic Studies on Penetrationof Azithromycin in Bacterially Infected Mice Issei Nakayama, Emiko Yamaji, Kaoru Shimada, Shuichi Yokoyama, Kazumi Miura, Hideya Muto, Kazunori Enogaki, Masatoshi Ogawa, and Kino Shimooka In Vivo Antibacterial Activity of RU 0 0 4 , a New Ketolide Active Against Respiratory Pathogens Constantin Agouridas, A. Bonnefoy, and Jean-Franco& Chantot Open Noncomparative Study of the Efficacy and Safety of Azithromycin in the Treatment of Adult Tonsilitis (Epidemiological Study of the Responsible Bacteria) Desaulty Open Study of Clarithromycin in the Treatment of Pneumonia Due to Streptococcus pneumoniae Murat Hayran, Mustafa Erman, Deniz Giir, Murat Akova, and Serhat Una1 Clinical Study of Rokitamycin on Pneumococcal Upper Respiratory Tract Infectionsin Pediatrics Yoshikiyo Toyonaga
402
407
411
415
421
425
429
432
V. PHARMACOKINETICS, PHARMACODYNAMICS, DRUG INTERACTIONS
Comparison of the Bronchopulmonary Pharmacokinetics of Clarithromycin and Azithromycin Kalpana B. Patel, Dawei Xuan, Charles H. Nightingale, Pamela R. Tessier, John H . Russomanno, and Richard Quintiliani
439
Contents
xiii
Kinetics of Dirithromycin: Concentrations in Tonsils, Bronchial Secretions. Multiple-Dose Mucosa, and Studies C. Muller-Serieys, E. Bergogne-Berezin, E Lemaitre, M. Derriennic, A. Ohman, P. Gehanno, C. Le Royer, and J. Clavier
447
A Human Model of Local Abscess Using Skin Chambers: The Penetration of Azithromycin and the Chemiluminescence Response of Neutrophils (PMN) K. Takahashi, V: Duchateau, M. Husson, A. M. Bourguignon, and F. Crokaert
455
Separation of Presystemic and Post-Absorptive Influenceson the Bioavailability of Azithromycin Cynomolgus in Monkeys G. Foulds, A. G. Connolly, J. H. Former, and A. M. Fletcher
460
Clinical Pharmacologyof Azithromycin Given at Various Sites Along the Gastrointestinal Healthy Tract in Subjects David R. Luke, G. Foulds, Joseph Scavone, Hylar L. Friedman, and William J. Curatolo Effect of Food and Formulationon Bioavailability of Azithromycin G. Foul&, David R. Luke, S. A. Willavize, William J. Curatolo, Hylar L. Friedman, M. J. Gardner, R. A. Hansen, R. Teng, and .l Vincent
ubjects Healthy Azithromycin inRectal David R. Luke, G. Foulds, G. Melnik, Paden C. Going, and Valerie Lawrence Comparative Pharmacodynamics of Clarithromycin and Azithromycin Against Respiratory Tract Pathogens Bauernfeind, R. Jungwirth, and E. Eberlein
464
469
474
478
VI. HELJCOBACTER PYLORI, CAMPYLOBACTER Comparison of Clarithromycin Efficacy in Ferrets and Humans for Treatment of Helicobacter-Induced Gastritis P. Ewing, J. D. Alder, M. J. Mitten, A. Conway, A. Oleksijew, K. Jarvis, L. Paige, and S. K. Tanaka Azithromyciflanitidine Combined Treatmentof Helicobacter pylori in Patients with Duodenal Ulcer and Chronic Gastritis: A Pilot B. Desnica, V: Burek, and N. Makek
487
493
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Contents
Azithromycin Promising as a Part of Helicocidal Regimens 0.Shonovd, P. Petr, and 0. Hausner
498
ANAEROBES, ENTEROCOCCI, HAEMOPHILUS INFLUENZAE
Comparative In Vitro Activity of Five Macrolides Against Gram-positive Cocci, Campylobacter Species, and Anaerobes M. Marina, K. Ivanova, N. Hadjieva, and A. Urumov
505
Prophylactic Effect of Azithromycin on Experimentally Induced Infection Intraabdominal in Rats N. Panovski, P. MiloSevski, and N. Labatevski
511
Are Macrolides Active Against Haemophilus infiuenzae? Are In Vitro E Crokaert, M. Aoun, K Duchateau, H. Goossens, P. Grenier, A. Vandennies, and J. Klastersky
516
Macrolide Susceptibility of Isolates of Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis from Patients with Community-Acquired Lower Respiratory Tract Infection-Results of an International Multicenter Study(the 523 Project), 1992-1994 Alexander D. Felmingham, J. Linares, and the Alexander Project Group Resistance of Common Respiratory Pathogens to Erythromycin and Azithromycin Milan &.&an, Ana Zlata Drag&, Katja Seme, Andreja Orafem, and Metka Paragi Efficacy of Clarithromycin and Azithromycinat Human Pharmacological Dosage Against ExperimentalHaemophilus Infection infruenzae Pulmonary J. D. Alder, M. J. Mitten, A. Conway, A. Oleksijew, K . Jarvis, L. Paige, and S. K. Tanaka
528
533
VIII. MYCOBACTERIA AND OTHER INFECTIONS IN HIV-INFECTED PERSONS
Clarithromycin 500 mg BID as Prophylaxis for MAC DiseaseA Follow-up John T. Sinnott, Douglas A. Holt, Sally H. Houston, Gary Bergen, Pamela Sakalosky, Julie A. Larkin, and Richard Oehler
539
Contents
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Clarithromycin Prophylaxis for Disseminated Mycobacterium avium Infection i? M. File, Jr., D. C. Claypoole, S. . I Longstreth, D. J. Signs, W. H. Ruby, and A. S. Indorf Azithromycin + Pentamidine + Pyrimethamine in the Prophylaxis of Opportunistic Infections in HIV-Positive Patients with CD4 Count Less Than G. Barbarini, G. Garavelli, G. Barbaro, B. Grisorio, A. Lucchini, S. Lopez, G. Del Buono, S. Edo, and R Alcini
544
549
M. SPECIAL PATHOGENS Human and Animal Bite Wounds: Bacteriology, Macrolide Susceptibility, and Therapeutic Potential Ellie J. C. Goldstein and Diane M. Citron
555
Azithromycinin the Treatment of PertussisinChildren: A Pilot Study A. Bade, N. Kuzmanovid, and i? Zmid
559
The Use of New Macrolides in Experimental Brucella melitensis Infection R. Lang, D. Torten, B. Shasha, and Ethan Rubinstein
563
X. NONANTIBACTERIAL EFFECTS In Vivo Effect of Azithromycin Subinhibitory Concentrations on the Mortality of Experimental Pseudomonas aeruginosa Sepsis M. N. Marangos, M. E. Klepser, D. l? Nicolau, Charles H . Nightingale, and Richard Quintiliani
571
Clarithromycin Reduces Cl-Dependent Transepithelial Potential Difference in Tracheal Mucosa of Anesthetized Rabbits J. Tamaoki, H . Takemura, E. Tagaya, Y. Takeda,and K . Konno
576
Inhibition of Adherence of Klebsiella pneumoniae Strains to Intestine-407 Cell Linesby Roxithromycin S. Favre-Bonte, C. Forestier, A. Darfeuille-Michaud, C. Rich, J. Sirot, and B. Joly Clinical and Immunological Study of Roxithromycin on Infectious Diseasesin Obstetrics and Gynecology K . Izumi, H . Mikamo, K. Kawazoe, and T Tamaya
581
586
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Contents
XI. CLINICAL STUDIES OTITIS MEDIA Five-Day Treatment of Acute Otitis Media in Children with Clarithromycin Dimitris A. Kafetzis, Theodore Bairamis, Dimitra Dinopoulou, Stamatina Vlachou, and Nicholas Apostolopoulos Azithromycin Versus Amoxicillin for Acute Otitis Media Prophylaxis Nicola Principi, Paola Marchisio, Emanuela Sala, Luisa Lanzoni, and Stefania Sorella
593
597
XII. CLINICAL STUDIES: BRONCHITIS Azithromycin for the Treatment of Community-Acquired Bronchitis: A Large Multicenter Efficacy-Tolerance Trial E Rafi Safety and Efficacy of Clarithromycin Compared with Cefpodoxime Proxetil in the Treatment of Acute Exacerbation ofObstructive Chronic Pulmonary Disease R Ltophonte and J. P. Chauvin
605
609
m.CLINICAL STUDIES: PNEUMONIA Randomized Comparative Trial of the Safety, Efficacy,and Cost of Intravenous Cefuroxime Plus Intravenous Erythromycin Versus Intravenous Cefuroxime PlusOral Clarithromycin in the Therapy of Community-Acquired Pneumonia D. Skupien, A. Margulis, N. Kaczander, S. Jaworski, A. Ekleberry, J. Pypkowski, and M. J. Zervos
Azithromycin in the Treatment of Community-Acquired Pneumonia K. Golec, M. Rzeszutko-Grabowska, and D. BukowskaNierojewska
A
617
626
Azithromycin: 3-Day Versus 5-Day Dosage Regimen for Community-Acquired Children Pneumonia in B. Ficnar, N. Huzjak, I. Klinar, and M. Matrapazovski
629
Single-Dose Azithromycin inthe Treatment of Atypical Pneumonia: S. Schonwald, I. Kuzman, V Car, J. tulig, and K. Oreskovid
634
Azithromycin in the Treatment of Patients with Upper Respiratory Tract Infections, Comparisonof 3-Day and 5-Day Regimens P. Dole2a1, M. Kro#l&k,and J. KlaZanskj Clinical and Bacteriological Evaluation of Clarithromycin (50-mg Tablets) in Children with Lower Respiratory Tract Infection Yoshikiyo Toyonaga Experience with Improved Complianceof Clarithromycin Granules in Children Kei3uke Sunakawa, Hironobu Akita, Satoshi Zwata, Yoshitake Satoh, Tatsuo Aoyama, and Ryochi Fujii Efficacy and Tolerability of Azithromycin Versus Josamycin in the Treatment of Children with Lower Respiratory Tract Infections Stt?p&nKutflek, Jozef Hoza, Daniela Markov&,and Milan Bayer Azithromycin in the Treatment of Respiratory Tract Infections in Children K. Galova, I. Marinova, J. Hractova, Kukova, S. Krifan, S. SuJliarska, H. Cintalanova, and A. Nogeova Efficacy of Roxithromycin in Elderly and Middle-Aged Patients with Respiratory Tract Infections S. Yakovlev
641
645
649
656
660
664
OTHER CLINICAL STUDIES Experience with Azithromycin as Prophylaxis in Transrectal Biopsy of the Prostate M . Kvarantan and C. Dohoczky
671
Author Index Subject Index
675 681
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Contributors to Plenary Sessions and Workshops
JacquesF.Acar
Fondation Hdpital Saint-Joseph, Pans, France
ConstantinAgouridas Research Center, Roussel-Uclaf, Romainville, France Vincent T. Andriole Yale University School of Connecticut
Medicine, New Haven,
Daniel H.Bouanchaud* Rhdne-Poulenc Rorer, Vitry-sur-Seine, France Andr6Bryskier
Research Center, Roussel-Uclaf, Romainville, France
Jean-Paul Butzler University Hospitals, St. Pierre, Brugmann, and Queen Fabiola, Brussels, Belgium Sharon L. Cerwinka Rhdne-Poulenc Rorer, Collegeville, Pennsylvania Jean-FranqoisChantot Research Center, Roussel-Uclaf, Romainville, France Elisabeth Chaslus-Dancla Institut National de la Recherche Agronomique, Nouzilly, France C. Cocito University of Louvain Medical School, Brussels, Belgium
J Carl Craft Abbott Laboratories, Abbott Park, Illinois WilliamA.Craig
University of Wisconsin, Madison, Wisconsin
*Currentaffiliation:MEDICOM, Paris, France
Contributors
Michael W. Dunne Pfizer Central Research, Groton, Connecticut Roger G. Finch The City Hospital and University of .Nottingham, Nottingham, United Kingdom M. Di Giambattista University of Louvain Medical School, Brussels, Belgium J. Thomas Grayston University of Washington, Seattle, Washington I. M.Hoepelman
University Hospital, Utrecht, The Netherlands
Pentti Huovinen National Public Health Institute, lkrku, Finland INSERM U294, CHU X. Bichat, Paris, France
Marie-Th6rrbeLabro
Jean-Pierre Lafont Institut National de la Recherche Agronomique, Nouzilly, France Jean-Louis Martel Centre National d’Etudes Veterinaires et Alimentaires-LYON, Lyon, France KennethH.Mayer Rhode Island
Memorial Hospital of Rhode Island, Pawtucket,
George H. McCracken, Jr. University of Texas Southwestern Medical Center at Dallas, Dallas, Texas J. Allen McCutchan University of California School of Medicine, San Diego, California John E. McGowan,Jr.
Emory University, Atlanta, Georgia
FrancisMt5graud Hopital Pellegrin et Universite de Bordeaux 2, Bordeaux, France Jod Melo-Cristino University of Lisbon, Lisbon, Portugal
Robert C. Moellering, Jr. Boston, Massachusetts
Beth Israel Deaconess Medical Center,
Daniel M. Musher Baylor College of Medicine and Veterans’ Affairs Medical Center, Houston, Texas CharlesH.Nightingale
Hartford Hospital, Hartford, Connecticut
E. Nyssen University of Louvain Medical School, Brussels, Belgium Ian Phillips United Medical and Dental Schools of Guy’s and St. Thomas’s Hospitals, London, United Kingdom Jack Remington Stanford University School of Medicine, Stanford, and Palo Alto Medical Foundation, Palo Alto, California
Contributors
mi
Ethan Rubinstein Sheba Medical Center, Tel-Aviv University, School of Medicine, Tel-Hashomer, Israel JoelRuskin Kaiser Permanente Medical Center and Medicine, Los Angeles, California
UCLA School of
Urs B. Schaad University of Basel, Basel, Switzerland .
ShlomoSegev ShebaMedical Center, Tel-AvivUniversity,Schoolof Medicine, Tel-Hashomer, Israel Fritz Sorgel Institute for Biomedical and Pharmaceutical Research, Nurnberg-Heroldsberg, Germany
Charles W. Stratton Vanderbilt University School of Medicine, Nashville, Tennessee
M. J. Tarlow Birmingham Heartlands Hospital, Birmingham, United Kingdom P. Vannuffel University of Louvain Medical School, Brussels, Belgium
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PLENARY SESSIONS
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Overview of the Clinical Use of Macrolides and Streptogramins Roger G. Finch The City Hospital and University Nottingham Nottingham, United Kingdom
INTRODUCTION Among the macrolides, erythromycin has been extensively used for approximately 45 years. the prototype drug, it has defined the place of this class of antibiotic in therapeutics. It provides an effective alternative to thepenicillins in the treatment of many common community infections of the skin and respiratory tract, and to a lesser extent in the treatment of various sexually transmitted infections. It has been the mainstay for treating atypical infections such as those causedby Mycoplasma pneumoniae, the ureaplasma, Legionella spp., Coxiella burnetii, and, more recently, Chlamydia pneumoniae. It is used in surgical prophylaxis in some countries in relation to colonsurgeryandhasoccasionalusesin the control of pertussisanddiphtheria.However,itslimitationshaveincluded low biovailability, poor gastrointestinal intolerance, and the emergence of resistance among common target pathogens such as staphylococciandstreptococci,as wellasexhibitingunreliableactivityagainst Haemophilus influenzae.
3
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DEVELOPMENT OF STREPTOGRAMINS AND MACROLIDES The streptogramins belong to the peptide group of antibiotics and include the mikamycins,ostreomycins,virginiamycins,andpristinamycins. The latter naturally occurring antibiotics derived from Streptomyces pristinaespiru. Pristinamycin and virginiamycin have been used in continental Europe as oral agents for the treatment of staphylococcal and streptococcal infections. The pastfewyearshaveseenmajordevelopmentsinboth the macrolide and peptide classesof compounds,' most notably withthe availability of azithromycin,clarithromycin,androxithromycinamong the macrolides, and inthe case of the streptogramins, the development RP 59500 (quinupristiddalfopristin) from the original pristinamycins. The importance of thesedevelopmentscan be viewed fromseveral perspectives.First,theyprovideanexpansion of the macrolidelincosamide-streptogramin (MLS) group antibiotics at a time whenthe development of new anti-infective agents, and in particular new classes of drug, has shown a considerable slowing in comparison with former years. Second, we are currently faced with a number of new infectious challenges such as the emergence of multidrug-resistant enterococci and mycobacteria, and serious staphylococcal infections, particularly those caused by multidrug-resistant Staphylococcus aweus and coagulase-negative staphylococci. Furthermore, new diseases have been identified such as Lyme disease and HIV disease,withits attendant microbialcomplications,and Helicobacter pylori-associated gastroduodenal disease. Likewise, the reemergence of epidemic diphtheria in the countries of the former Soviet Union emphasizes the continuing need for notonlyeffectiveimmunoprophylaxis but also judicious chemoprophylaxis.
Structure Activity Issues The macrolides, lincosamides, and streptogramins are structurally diverse but are conventionally grouped together because of their common ability to bind to bacterial ribosome to inhibit protein synthesis and thereby bacterial multiplication. The macrolides form a large groupof related antibiotics, largelyderived from Streptomycetes. Structurally, they consist of a macrocyclic lactam ring to which various aminosugars are attached. The macrolides are classified into 14 and 16 carbon-containing rings. Among the C-l4 compounds are erythromycin, oleandomycin, and, more recently, clarithromycin,roxithromycin,troleandomycin,flurithromycin,anddirithromycin,
MacrolideslStreptogramim-Overview
5
which is a pro-drug of erythromycylamine. Among the C-l6 compounds are josamycin, midecamycin, rosaramycin, and spiramycin, as well as the semisynthetic miocamycin, characterizedby a single aminosugar. Azithromycin is a 15-membered compound with a methyl-substituted nitrogen at position which is therefore strictly an azalide, although for all practical purposes it can be considered as a macrolide. The lincosamides,lincomycinandclindamycin, are derivativesof Streptomyces lincolnis. Although there is some overlap in the areas of clinical use with those ofthe macrolides and streptogramins, theyare sufficiently distinct and will not be discussed further in this chapter. The streptogramingroup of antibioticsincludes the mikamycins, pristinamycins, oestreomycins, and virginiamycins which have been produced as secondary metabolites from a variety of natural Streptomycetes. They have a complex and often unstablestructure but lend themselves to modification without major loss of antimicrobial activity. Recent interest has focused on pristinamycin, which has two macrolactone componentspristinamycin I, and pristinamycin 11,. RP (quinupristin) and RP (dalfopristin) are the respective chemically derived water soluble components which when combined are bactericidal and synergistic against avariety RP [quinupristiddalfopristin target,pathogens. (Synercid it is undergoing clinical trials. The mode action of RP is to bind to the bacterial ribosome to form a stable dalfopristinribosome-quinupristin complex which irreversibly inhibits protein synthesis resulting in bacterial cell death. It would appear that this mechanism of action is unique and, therefore, distinguishes RP from other antibiotic classes. The focus of this chapter will be largely on the recently available macrolides, namely azithromycin, clarithromycin, and roxithromycin, and also quinupristiddalfopristinwith emphasis on their clinical use.The constraints of space do not permit a review of the safety aspects of these drugs. In order to appreciate their therapeutic potential, an understanding Of their in vitro performance and pharmacokinetic behavior is a necessary prerequisite.
In Vitro Activity The spectrum of activity of the recently available macrolides andstreptogramins is sufficiently distinctive among the major classesof anti-infectives, perhaps withthe exception tetracyclines, inthat it encompasses not only many common bacterial pathogens but also includes atypical pathogens, Mycobacteria spp., spirochaetes, and selective protozoa. Furthermore, the observed high tissue and intracellular concentrations broaden their clinical
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utility. The growing importance of drug resistance to other classes of antibiotic further emphasizes their growing importance. These resistance problems include penicillin-resistant pneumococci, multiresistant enterococci, and, in particular, vanocmycin-resistant strains, methicillin-resistant staphylococci, and multiresistant coagulase-negative staphylococci, as well as drug-resistant Mycobacteria spp. Macrolides
The in vitro activity of erythromycin encompasses most streptococci, including Streptococcus pneumoniae, Streptococcus pyogenes , and the viridans streptococci, but excludes the enterococci. Staphylococcus aureus is also susceptible with the exception of methicillin-resistant strains (MRSA) which are often multiresistant. Other sensitive gram-positive pathogens include corynebacterium and Listeria monocytogenes. Susceptible gram-negative pathogens include Neisseria gonorrhoeae, Moraxella catarrhalis, Legionella pneumophila, and Bordetella pertussis. Activity against Haemophilus influenzae is equivocal. Gram-positive anaerobic bacteria are often sensitive. The Enterobacteriaceae are not considered susceptible. Other susceptible pathogens include Mycoplasma pneumoniae, Chlamydia trachomatis, Chlamydia pneumoniae, Treponema pallidum, Borrelia burgdorferi, and Mycobacterium spp. The most striking in vitro differences for the most recently available macrolides is their enhanced activity against many common pathogens (Table 1). This is particularly so for clarithromycin, which is approximately twice as active as erythromycin against staphylococci and streptococci, and fourfold to eightfold more active than azithromycin. It should be emphasized that methicillin-resistant isolates of S . aureus are uniformly resistant to these new macrolides, as are the enterococci. The activity of roxithromycin most closely resembles that of the parent compound, erythromycin. Whereas the activity of erythromycin against H . influenzae has been unpredictable, azithromycin is twofold to eightfold more active and roxithromycin remains of comparable activity. The 14-hydroxy metabolite of clarithromycin has biological activity, which is twice as active as clarithromycin against H . influenzae. Bordetella pertussis also remains sensitive to these new macrolides. Some of the most striking differences in activity are seen against atypical respiratory pathogens; clarithromycin is the most active agent against C. pneumoniae, L. pneumophila, and M . pneumoniae. Erythromycin is an alternative agent for the treatment of gonococcal infections and also provides useful activity against nongonococcal genital tract pathogens such as Chlamydia. trachomatis and Ureaplasma urealyticum. Clarithromycin again demonstrates the greatest instrinsic activity and
MacrolideslStreptogramins- 0vervie w
7
Tuble I Macrolide Activity (MIC,,, mg/L) Against Common Bacterial Pathogens
Organism
Erythromycin
S . areus
Coagulase-negative staphylococcus S . pneumoniae S . pyogenes S . agalactiae E . faecalis E . faecium N . gonorrhoeae L . monocytogenes H . injluenzae B . pertussis H . pylori M . catarrhalis C . jejuni
Anaerobic streptococci B . fragi1i.s C. perfringens C . trachomatis U.urealyticum H . ducreyi
Azithromycin
Clarithromycin
0.5 0.5
0.12 0.25
0.25 0.25
0.02-0.06 0.03 0.03 >64 >64 2.0 0.5 4.0-8.0 0.03 0.25 0.25 2.0 4.0 2.0 1 .o 0.125 2.0 0.06
0.12 0.12 0.06 >64 >64 0.25 2 1 .o 0.06 0.25 0.06 0.5 2.0 2.0 0.25 0.25 2.0 0.003
0.02 0.06 0.06 >64 >64 0.5 0.25 8.0 0.03 0.03 0.25 2.0 4.0-8.0 2.0 0.5 0.016 0.2 0.015
Roxithromycin 1.o 0.25 0.03 0.06 0.25 >64 >64 1.0 1.0 8.0 0.03 0.5 1.o 4.0 32 32 2.0 0.125 2.0 0.06
~
Source: Data based on Refs. 3, 29, 51, and 62.
includes Haemophilus ducreyi. Azithromycin is the most active agent against N. gonorrhoeae whereas roxithromycin offers no microbiological advantage over erythromycin against this group of pathogens. Although the macrolides are inactive against common gram-negative pathogens, Cumpylobucter jejuni is susceptible. Furthermore, Helicobacter pylori is also susceptible, with clarithromycin showing the greatest activity (1). Of considerable interest is the activity of the new macrolides against Mycobucteria spp. M. tuberculosis is relatively insensitive to clarithromycin and azithromycin, for which the MIC,s are >10 mg/L and 32 mgIL, respectively. Roxithromycin inhibits most strains of M . tuberculosis at 4 mg/L, which with the addition of rifampicin or isoniazid is further reduced to 0.25 mg/L (2). Of greater interest is the activity of these compounds against atypical mycobacteria, in particular the Mycobucterium avium complex (MAC).
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Azithromycin and roxithromycinare relatively inactivein vitro withM I G s of 32-62m&. However,clarithromycindemonstrates greater activity with M I G s of 2-8 m& (3). The activity of clarithromycin against MAC is pH and media dependent. The MI& lies between2 and 8 m&, whereas the minimum bactericidal concentrationvanes between S6 and m@ (4) which emphasizes the importance of intracellular concentrations of drug in relation to any clinical utility (5). This activity has been supported by in vivo studies in beige mice, where bacterial counts the spleen in are reduced by clarithromycin and potentiated by clofazimine and rifabutin (6). The performance of azithromycin againstMAC, although less striking in vitro, has also reduced bacterial counts in beige mice (7), whereas macrophages infected with MAC of azithromycin inthe presence of interferonhave shown increased uptake gamma or tumor necrosis factor-alpha (8). Against other Mycobacterium spp., roxithromycin hasshown'potentially useful activity with MIC values against M. malmoenese, M. scrofulaceum, M. szulgai, M. xenopi, and M.kansasii but excluding M.marinum (9). Clarithromycin has demonstrated potential clinically useful activity against M. marinum resistant to other antituberculous agents-". kansasii (MIC90 0.25m&) (lo), M.chelonei spp., and M. forfuitum spp. (11). Other Organisms. Azithromycin, in keeping with other macrolides, has activity against amoebas and other protozoans. For example, the growth of Entamoeba histolytica trophozoites is inhibitedby concentrations of 40 m&, which is comparable to erythromycin (12). Giardia spp. appear to be insensitive. However,of greater interestis the susceptibility ofToxoplasma gondii to the macrolides. In the case of azithromycin, 50% inhibition of nucleotide synthesis has been demonstrated in macrophage grown cells at concentrations of 140 m& compared with a range of 54-246 mgL for clarithromycin, roxithromycin, and spiramycin. (13). Streptogramins
As-noted earlier, pristinamycin is composed of two components, PIA and PII,. Recently,thesetwofermentationproductshavebeenchemically modified to produce the water-soluble injectable derivatives quinupristin (RP 57669), derived from PI,, and dalfopristin (RP54476), derived from PIIA. When formulated a 30 : 70 ratio, they are synergistic and have been developed as RP 59500 (SynercidB). Quinupristiddalfopristin individually exhibit weak bacteriostatic activity against gram-positive bacteria. In combination, they are rapidly bactericidal against S. pneumoniae,Enterococcusfuecium, methicillin-sensitive(MSSA) and resistant (MRSA) S. aureus, and coagulase-negative staphylococci.
MacrolideslStreptogramins-Overview
9
The MIC,s are usually 1 m& and include strains resistant to erythromycin, ciprofloxacin, gentamicin, and rifampicin. The activity against staphylococci is comparable to vancomycin, erythromycin,and ciprofloxacin but less than ampicillin and erythromycin against streptococci. The in vitro activityof RP 59500 is summarized in Table 2. Considerable interest has focused on the activity of RP 59500 against enterococci.Althoughmany are susceptible,MICconcentrationshave ranged from 0.25-32 m@. E . faecium is usually more susceptiblethan E . faecalis. Likewise,vancomycin-resistant E. faecium are often sensitive. Those sensitive to vancomycin are often twofold or more susceptibleto RP 59500. Multiresistant strainsof E. faecium remain susceptibleto RP59500 with an MIC90 of1.0 m@. Gram-negative and intracellular pathogens such as-N. meningitidis, N. gonorrhoeae, Legionella spp., M . pneumoniae, and Chlamydia spp. are also susceptible with MIC, 1.0 m& (Table 2). H.influenzae and M . catarrhalis are less susceptiblethan other pathogens withMIC, of 4 m&. Table 2 Susceptibility of Pathogens toQuinupristinDalfopristin(m59500)
Dositive Gram ~~
MIC,lto4to95% and 88%, respectively (47,48), have been published, including comparability with erythromycin and penicillin V. Acute mmillary sinusitis has also responded with satisfactory clinical responses of up to 92%, matched by similar bacteriological eradication rates (49); comparators have included amoxicillin-clavulanic acid. Acute tonsillitis and otitis media have shown similar cure rates of up to 87% and99%, respectively, in comparison to treatment with josamycin (50). The target pathogens have been representative of these infections, most notably S. pneumoniae, S. pyogenes, and S. aureus. Lower Respiratory Tract Infections. A variety of comparative and noncomparative studies have evaluated clarithromycin in such clinical syndromes as acute bronchitis, infective exacerbationsof chronic bronchitis, and community-acquired pneumonia. Clarithromycin250 or 500 mg twice daily for 1-2 weeks has proved effective in all these conditions. For example, clinical cure rates of up to 83% and bacteriological eradication rates of up to 100% have been recorded in community-acquired pneumonia (51), whereas in patients with acute exacerbations of chronic bronchitis, these figures have been of the order of96% and 100%, respectively. In the
Macrolides/Streptogramins-Overview
17
treatment of acute bronchitisor acute exacerbationsof chronic bronchitis, comparators have included josamycin, roxithromycin, erythromycin, ampicillin, cefuroxime axetil, and cefixime. With regard to atypical pneumonia, the results have been encouraging, with satisfactory responsesto infections causedby Chlamydia pneumoniae, Mycoplasma pneumoniae, and Legionella pneumophila. In the latter, a nonblinded multicenter study demonstrated clinicalcure in patients in doses of mg twice dailyfor periods of days With regard to H.infruenzae infection in patients withacute exacerbations of chronic bronchitis, clarithromycin was equivalent to cefaclor, cefuroxime axetil, or cefixime this emphasizesthe importance of the hydroxy metabolite against this pathogen.
Skinand Soft TissueInfections. A number of comparative multicenter studies have demonstrated the comparability of clarithromycin to agents such as erythromycin and cefadroxil with clinical successrates of or greater, and bacteriological curerates of Target pathogens have largely includedStaphylococcus aureus. Urogenital Infections. A number of studieshaveassessed the performance of clarithromycin in patients with nongonococcal and gonococcal urethritis/cervicitis. Response rates of up to have been recorded in patients with chlamydial urethritis and in persons with predominantly ureaplasma infections In the case of mixed gonococcaland chlamydial infections, response rates have been lower.
MycobacterialInfections. Atypical mycobacterial infections and, in particular, those caused by MAC complicating HIV disease have been a particular focusof assessment. Pilot studies have included randomized, some placebo controlled, double-blind crossover studies with clarithromycin dosages of g twice daily, either alone or in combination with clofazamine mgdaily, ethambutol 20 mglkgdaily,isoniazid mglkgdaily,and rifampicin mglkg daily, administered for up to 6 weeks in comparison with placebo These studies have monitored blood for colony-forming units of MAC. Bacteremia was eliminated in all recipients of clarithromycin and the four other antimycobacterial agents. In an additional small study of patients withAIDS, clarithromycin and clofazamine, and ethambutol and isoniazidfor 6 weeks, followed bymaintenance with clarithromycin and rifabutinfor weeks, followed by clarithromycin alone indicated that those who received clarithromycin responded clinically and developed negative blood cultures(56). Tho important studies have beenreported from Canada and France. The former prospective, randomized study compared ciprofloxacin mg
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18
bid, ethambutol mgkg qid, rifampin 600 mg qid, and clofazimine 100 mg qid with clarithromycin g bid, rifabutin 600 mg qid, and ethambutol mgkg qid in evaluable HIV-infected patients with MAC bacteremia followed to death. The clarithromycin-containingregimen was significantly better with regard to clearance of bacteremia, 67/97 (69%), and median survival months) In the French study, clarithromycin2 g/day g/day after months) and clofazimine mg/day mg/day after months) was compared with a similar clarithromycin regimen, combined with rifabutin mg qid and ethambutol mg qid.End points included decrease in fever and negative blood cultures and survival. No statistical significant differences were observed at 2 and 6 months terms in of the end points, although clarithromycin resistancewas more frequent (p < in the clofazime/clarithromycinregimen Clarithromycin has also shown some promise with early responses in immunocompromized patients infected withM.chelonae Helicobacter pylori. Clarithromycinhasshownconsiderablepromisein the treatment of H . pylori gastroduodenal disease.It is the most activeof the new macrolides against this pathogen (MIC, 0.03 m&). The objectives of therapy are not only to heal the associated H . pylori gastritis or duodenalulcerbutalso to prevent recurrence and alter the long-term natural history of the disease. monotherapy, eradication rates of have been observed 4-6 weeks posttreatment (60).Various strategies involving clarithromycin are under investigation or comparative clinical trials; the combinations are with acid-controlling agents suchas omeprazole, ranitidine-bismuth citrate, or bismuth.
Toxoplasmosis. A small number of patients with AIDS and complicating toxoplasma encephalitis have been treated with pyrimethamine and clarithromycin g twice daily for up to 6 weeks. Clinical and radiological response rates have been of the order of (61). Roxithromycin
Roxithromycin has antibacterial activitythat closely resembles erythromycin but with improved plasma, tissue, and body fluid concentrations and a much longer half-life.It has been evaluated for a variety of clinical indications and, in particular, infections of the upper and lower respiratorytracts skin and soft tissues, and the urogenital tract. selection of the published studies are reviewed.
Upper Respiratory Tract Infections. Pharyngitis, tonsillitis, sinusitis,and otitis media have been studied using 300 mg once or mg twice daily regimens with clinical response rates that have exceeded 92% for these
MacrolideslStreptogramins-Overview
19
conditions. Likewise, bacteriological response rates have been high, rangingfrom 9 0% to Incomparativestudies,comparability to clarithromycin-and amoxicillin-clavulanic acid has been demonstrated.
Lower Respiratory Tract Infections. Several trials have investigated the performance of roxithromycin in the treatment of acute bronchitis, acute exacerbations of chronic bronchitis, and pneumonia. These have been comparative against agents such as azithromycin, doxycycline, and amoxicillin. In the case of acute bronchitis, clinical efficacy rates of and have been reported for a regimen of mgbid. In treating acute exacerbations of chronic bronchitis, roxithromycin has been equivalent to amoxicillin; doxycycline, and azithromycin. Several small studies treating community-acquired pneumonia have also demonstrated efficacy rates of approximately withcomparability to erythromycin,azithromycin, clarithromycin, and amoxicillin. difference has been demonstrated between mg once daily and mg twice daily when treating atypical pneumonias; these have included infections causedby chlamydiae, mycoplasma, Legionella spp., and Coxiella burnetii. Responseshavebeen prompt, with efficacy rates comparable to erythromycin Despite modest in vitro activity against H . infiuenzae, analysis of the performance against this pathogen in various studies has demonstrated an response rate in treated patients. Intention to treat analysis also remains satisfactory at by meta-analysis There have been relatively few studies in children, although conditions such as tonsillitis, pharyngitis, impetigo, pneumococcal pneumonia, and pyoderma have responded satisfactorily, with response rates exceeding and bacteriological eradication rates ranging from to
Helicobacterpylon' Infections. Helicobacter pylori is susceptibleto roxithromycin with an MIC, of mg/L at pH H . pylori-associated duodenal ulcers have been treated with roxithromycin in combination with omeprazole and bismuth subnitrate. Eradication rates of approximately have been reported in small numbers of patients (66). Additional studies have confirmed healing month posttreatment using a regimenof roxithromycin forthe first weeks combined withthe addition of metronidazole forthe first days and lansoprazole for total a of weeks.
Skinand Soft Tissue Infections. Althoughprospective,comparative, double-blind studies have been few, roxithromycin has demonstrated clinicalefficacyratesvaryingbetween and in noncomparative studies In the treatment of erysipelas, roxithromycin mg bid has been as effective as penicillin with overall efficacy rates of and respectively.
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UrogenitalInfections. Roxithromycinhasbeenassessedinavarietyof gynecological and venereal infections. These have included nongonococcal urethritiskervicitis,genitalchlamydialinfections,andgonococcalcervicitis. Roxithromycin300 mg per day as a single dose or in divided dosesfor days has been shownto be effective inthe treatment of all these infections (67). Response rates have varied between 74% and 100%. Comparators have included doxycycline, minocycline, and erythromycin. OtherInfections. Roxithromycin150 mgsbd hasbeencomparedwith penicillin V in patients with uncomplicated erythema chronicum migrans; one study was stopped prematurely becauseof a less favorable response in the roxithromycin recipients (68). Further studies have indicated a more favorable response (69). The use of roxithromycin asthe primary preventionfor Pneumocystis curinii pneumonia (in combination with dapsone) and cerebral toxoplasmosis has been evaluated in a small number of patients with HIV infection (CD4 countsof pg/ml) was slightly higher(RP MIC, = ml) than that of the strains with intermediate erythromycin resistance(RP MIC, = pg/ml). For all strains tested, the MIC range was &m1 also reported by Boswellet al. for S. aureus the susceptibility to RP was found in this study to be greater for E . faecium growing exponentially thanfor bacteria in stationary phase It was also reported that activity against E. faecium was increased if RP were combined with vancomycin, gentamicin, or teicoplanin, even if the strains being tested were resistant to one or more of these antibiotics For example, Hill et al. reported that the combina-
58
Bouanchaud
tion of RP plus 8 pglml of teicoplanin reduced MICs to 0.06 pglml for 31 of vancomycin-resistant and gentamicin-resistant strains In a study intracellular activity, Herrera-Insuaet al. foundthat RP was more effective against polymorphoneclear-ingested vancomycin-sensitive E. faecium than against the ingested vancomycin-resistant strains.At four times the MIC, RP killed the former strains but only inhibited the latter. It was suggested that intracellular concentration and intracellular activity may not be directly related for this antibiotic (20).
Resistance to QuinupristidDalfopristin:Biochemical Mechanisms and Detection The rapidity with which resistance genes are disseminated through a bacterial population, especially in the hospital setting, is unpredictable. Both the frequency at which genes encoding proteins involved in resistance appear and the hardiness and transfer capacity of the genetic element carrying such genesare involved. The gene erm encodes a methylasethat modifies a base in 23s ribosomal RNA, reducing the affinity of the ribosome for macrolides,lincosamides,andgroup B streptogramins, such that these antibiotics cannot bind to their target (the MLS, resistance phenotype) (21). Other genes, frequently carriedby plasmids,. have been identified in staphylococci; these encode enzymes that can inactivate streptogramin components (22), aswell as an efflux mechanism. An inactivating enzyme has also been found inEnterococcus (23). For the streptogramins, it might be supposed that a prerequisite of resistance would be the appearance, in the same bacterial population, of genes that encode resistance to both components and, consequently, that resistance to RP would occur less frequently than it does for single agents. Resistanceto RP is apparently rare, but the low frequency of resistance observed far does notsimply reflectthe putative requirement for two coexisting mechanisms. It has been found that bacterial strains harboring genes that encode resistance to the group B streptogramins are susceptible to RP whereas those harboring genes encoding resistance to the group A streptogramins are resistant (24). In one in vitro study, itwas reported that, upon repeated exposure to concentrations of two or four times the MIC of RP E. faecium strains having MICs increased 16-foldor more could be derived from susceptible strains. Exposure to an initial drug concentration of at least 16 times the expected MIC prevented the emergence such resistant strains The implications of this study, however, are not entirely clear with respect to the clinical setting, wherethe genesis of resistance by mutation (presumably point mutation) may play a role of lesser importancethan the
Parenteral Streptogramins: From
to Oral
59
dispersal of genetic elements, such as transposons or plasmids, that encode complex resistance mechanisms. Susceptibility breakpoints forRP 59500 are now being determined.It has been suggested,on the basis of a study in which disks were loaded with and a disk zone diameter of 15 pg of RP 59500, that an MIC 2 1 18 mm could serve as provisional susceptibility breakpoints for S. pneumoniae, S. aureus, and Enterococcus spp. (26).
IN VITRO ACTIVITY OF RPR 106972, A NEW ORAL STREPTOGRAMIN, DERIVED (AS QUINUPRISTIN/ DALFOPRISTINE) FROM NATURAL PRISTINAMYCIN RPR 106972 is a cocrystalline associationof two natural molecules (in a4/2 molar ratio, or 45/55 in weight): type I pristinamycin (RPR 112808) containing PI, (RP 13919, > 95%)as the major component, and type I1 pristinamycin (RPR 106950) containing PIIB (RP 13920, > 95%) as the major component.
In Vitro Bacteriostatic Activity* Staphylococci
Minimal inhibitory concentration determination demonstrated that RPR 112808 inhibited the growth of sensitive and inducibly resistant S. aureus strains, with MICs ranging from 4 to 32m& but was inactive againstMI&constitutivelyresistant S. aureus strains. It inhibited S. epidermidis strains, with MICs ranging fromto264 m&, except the MLS, constitutively resistant strains (MIC> 128 m&). RPR 106950 inhibited all the S. aureus strains tested, with MICs ranging from 4 to 16 m&,and the S. epidermidis strains, withMICs ranging from 1 to 16 m a , except strains Mau., N 52, N 100, and N 122 (MIC > 128 m&). RPR 106972 (RPR 112808RPR 106950) was uniformly active against MLS,-sensitive and M L S , inducibly resistantS. aureus strains (MIC range: 0.12-1 m&), MLS, constitutively resistantS. aureus (MIC range:1-1 mg/ L),and S. epidermidis strains (MIC range:0.12-0.50 m&).
M L S ,
Streptococci The MIC determination demonstratedthat, against streptococci (groups B, C , G ) ,RPR 112808 was active, with MICs ranging from 0.50 to 8 m&, *Data on File, RhBne-Poulenc Rorer.
Bouanchaud
60
except against the strains resistant to erythromycin (MIC 128 m&). RPR 106950inhibited the tested strains, with MICs ranging from 0.50 to m&. RPR 106972 demonstrated a potent activity against all the strains tested (MIC range: 0.03-0.12 m&), comparable to that of erythromycin (MIC range: 0.03-0.50 m&). Against Str. pneumoniae, RPR 112808 wasactive, with MICs ranging from 1to m& against the strains sensitiveto erythromycin. Against the strains resistant to erythromycin, it was less active (MIC range: > 128 m&). RPR 106950 did not demonstrate significant activity against anyof the Str. pneumoniae strains tested (MIC range: m&), but the RPR 112808/RPR 106950 association was strongly synergistic:RPR 106972 was uniformly and potently active against all the strains studied whatevertheir resistant profile (MIC range:0.06-0.50 m&). Against strains sensitive to the reference compounds, its activity was comparable to that of erythromycin (MIC range: 0.12-0.25 m a ) , about to four times inferiorto that of oxacillin (MIC range: 0.015-0.25 m&), and two to eight timessuperior to that of ciprofloxacin (MIC range: 0.50-1 m&).
Enterococci RPR 112808 inhibited the strains tested, with MICs ranging from 1 to 16 m&. RPR 106950 was slightly active against E . faecium, E. durans, and E . hirae (MIC range: 2- > 128 m&) but was inactive against E . faecalis (MIC range: 128- > 128 m&). RPR 106972 demonstrated potent activity against E . faecium, E durans, and E . hirae (MIC range: 0.06-0.50 m&). It was less active against E . faecalis than against the other enterococci (MIC range:0.50-2 m&).
Neisseria
Pasteurella species
Against Neisseria species, RPR 112808 was weakly active (MIC range: > 128 m&), whereas RPR 106950 demonstrated potent activity (MIC range: < 0.12-1 m&). RPR 106972 demonstrated strong activity (MIC range: 0.015-0.12 m&). Against P . multocida, RPR 112808 was inactive (MIC < 128 m&). RPR 106950 andRPR 106972 were poorly active (MIC ranges: 16-128 and 4-16 mg/L, respectively).
Moraxella catarrhali RPR 112808 did not demonstrate bacteriostatic activity against this species (MIC range: 32-128 m&). RPR 106950 inhibitedthe strains tested, with MICs ranging from 0.50to 2 m&. RPR 106972 demonstrated an activity (MICrange:0.12-0.25m&) tofourtimessuperior to that of erythromycin (MIC range: 0.12-1 m a ) .
Streptogramins: From Parenteral to Oral
61
Haemophilus influenzae RPR 112808 did not demonstrate activity ( M C range: > 128 m&). RPR 106950 was moderately active (MIC range: 4-16 m&). RPR 106972 (MIC range: 0.50-2 m&) was 2 to 16 times more active than erythromycin (MIC range: 4-16 m&). Enterobacteria and Pseudomonas aeruginosa Neither RPR 112808 (MIC > 128 m&), RPR 106950 (MIC > 128 m&), nor RPR 106972 (MIC range: 128- > 128 m&) demonstrated any activity against enterobacteriaor Pseudomonas aeruginosa. Anaerobes RPR 112808 and RPR 106950 were active against gram-positive anaerobes with MICs ranging from 0.50 to 64 m& and 1to 128 m&, respectively. They were generally inactive against gram-negative anaerobes (MIC ranges: 32- > 128 m& and > 128 m&, respectively.
Mycoplasma
Ureaplasma species
Against the M. pneumoniae strains tested, RPR 106972showed potent bacteriostatic activity (MIC = 0.12 m&). Against the M. hominis strain tested, RPR 106972 had an MIC of 1 m&. Against the U. urealyticum strain tested, RPR 106972 (MIC = 2 m&) demonstrated activity.
Legionella species RPR 112808and RPR 106950 demonstratedpooractivityagainst the strains tested (MIC ranges: 4-128 m& and 8-32 m&, respectively), and RPR 106972 (MIC range: 0.12-1 m&) was two to four times less active than erythromycin (MIC range: 0.06-0.25 m&).
Mycobacteria species PR 106972 was inactive (MIC range: 16> 128 m&) against the 15 strains tested [M.intracellulare (1 strain), M. avium (6 strains), M.fortuitum (1 strain), M. scrofulaceum (1 strain), M. simiae (1 strain), M. kansasii (2 strains), M.malmoense (1 strain), M.szulgai (1 strain), and M.xenopi (1 strain) (expt XWOl 079).
Synergism of the RPR 112808
RPR 106950 Combination
The RPR 112808 and RPR 106950 combination showed synergism for all bacterial strains tested.The FIC index obtained with the lowest concentration of eachcompoundrangedfrom0.034 to 0.13 for Staphylococcus aureus (seven m,-sensitive and MLS,-resistant strains), from 0.008 to
Bouanchaud
62
0.12 for Streptococcuspneumoniae (four strains including one eythromycinresistant strain and one penicillin-intermediate strain), and from 0.017 to for Enterococcus spp.(sevenstrainsincluding one multiresistant strain of Enterococcusfaecium).
Influence of the Ratio of RPR 112808 to RPR 106950 The RPR112808 andRPR 106950 combination was found to be synergistic over a wide range of ratios against all the strains tested (9040 to 80/20).
In Vitro Bactericidal Activity Staphylococcus aureus Against MU,-sensitive andMLS,-inducibly resistant strains (two strains of each), RPR 106972 was bactericidal at concentrations of 0.25-2 m& [two to four times the MIC accordingto the strain tested]in a 6-24-h period. A dose-dependent effect was found at concentrations corresponding to two and four times the MIC, but no difference in the effectwasobserved between the concentrations of four and eight times the MIC. AgainstM L S , constitutively resistant strains,RPR 106972 at the concentration of 8 m@ was bactericidal within 24 h (starting from of 6 contact) h againstone strain (8 m& = four times the MIC) and decreased viable counts by 2 log,, CFU/ m1 for the other (8 m& = eight timesthe MIC).
Streptococcus pneumoniae Against erythromycin-resistant strains (two strains), RPR 106972 wasbactericidal at the concentrations of 0.50 m& (two to four times the MIC according to the strain tested) within the first3h of contact. Against penicillin-intermediate strains (two strains), RPR 106972 was bactericidal at concentrations of 0.25-0.50 m& (two to four timesthe MIC) in a 6-8-h period. Against the two strains with a low levelof resistance to ciprofloxacin, RPR 106972 was bactericidal at the concentration of 0.25 m& (one to two times the MIC)ina3-6-h period.Againststrainsresistant to erythromycin, ciprofloxacin, and/or penicillin (three strains), RPR 106972 decreased viable countsby 2 log,, CFU/ml at the concentration of 2 m& (four to eight timesthe MIC) in a 6-8-h period.
Enterococcus species Against E . faecalis (two strains tested), RPR 106972 did not demonstrate any bactericidal activity. However, atthe concentration of eight hours the MIC (4-16 m&) according to the strain tested), it decreased viable counts by 2 log,, CFU/ml.
Streptogramins: From Parenteral Oral to
63
Against E . faecium (four strainstested), RPR was bactericidal period (one strain, at theconcentration of eight timesthe MIC in 24-48-h a MIC = m&) and decreased viable countsby to log,, CFU/ml (three strains, MICs = m&). Haemophilus influenzae .
Against the strains tested (including strains producing p-lactamases), RPR was bactericidal at the concentrations of or 4 m& or 4 the MIC accordingto the strain tested) in a to 6-h period strains) or in a to 24-h period strain). Conclusion
RPR has potent in vitro bacteriostatic activity against aerobic grampositive cocci, including staphylococci resistant to oxacillin and macrolideslincosamides-streptogramin, (MU,), gram-positive anaerobes, certain gram-negative bacteria responsible for respiratory tract infections (Neisseria spp., Moraxella catarrhalis, Haemophilus injluenzae), and fastidious due to the bacteria asLegionella spp. andMycoplasma spp. This activity is synergism of its components, RPR (containing PI, asthe major component) and RPR (containing PII, as the major component). This synergistic activity occurred over a wide range of RPR to RPR ratios and was affected only slightly by variations in culture conditions. RPR demonstrated in vitro bactericidal activity at concentrations ranging fromto to four times the MIC againstStaphylococcus aureus sensitive or resistant to oxacillin andM L S , (five ofsix strains tested), Streptococcus pneumoniae (six of nine strains tested, including erythromycinresistant penicillin-intermediate, ciprofloxacin-resistant,and multiresistant strains) and Haemophilusinfluenzae (six strains tested including strains producingp-lactamases).Againstenterococci (six strains tested), RPR was notbactericidalexceptagainstone strain of Enterococcus faecium; nevertheless it decreased viable counts by log,, CFU/mL at concentrations of eight timesthe MIC. RPR demonstrates promising in vitro activity against most of the bacteria responsible for respiratory tract, cutaneous and genital community-acquired infections. It may also be considered for oral follow on therapy for quinupristiddalfopristin.
ACKNOWLEDGMENTS I thank K. Pepper for helpful comments, and J.F. Desnottes and N. Berthaud for providing newdata from their laboratory.
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64
REFERENCES 1. Bamere JC, Bouanchaud DH, Desnottes JF, Paris JM. Streptogramin analogues. Exp Opin Invest Drugs1994; 3(2):115-131. 2. Aumercier M, BouhallabS, Capmau ML, Le Goffic F. RP 59500: a proposed mechanism for its bactericidal activity. J Antimicrob Chemother 1992;30 (Suppl A):9-14. 3. Cocito C, Di Giambattista M,Vannufel P. The mechanism of action of streptogramins. 3rd International Conference on the Macrolides, halides and Streptogramins, Lisbon, 1996; abstr 12. 4. Barriere JC, Bouanchaud DH, Paris JM, Rolin 0, Hams Smith C. Antimicrobial activity againstStaphylococcus aureus of semisynthetic injectable streptogramins: RP 59500 and related compounds. J Antimicrob Chemother 1992; 30 (suppl A):1-8. 5. Shonekan D, Handwerger S, Mildvan D. Comparative in vitro activities of RP 59500 (quinupristiddalfopristin), CL 329,998, CL 331,002, CP-99,219, clinafloxacin,teicoplaninand vancomycinagainstgram-positive bacteria. 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, 1995; abstr E-124. 6. von Eiff C, Peters G. The in vitro activity of RP 59500, a new semisynthetic injectable pristinamycin, against staphylococci. 3rd International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 3.25. 7. Berthaud N, Charles Y,Gouin AM, Houdebine C, Rousseau J, Desnottes JF. In vitro bactericidal activity of RP 59500 (quinupristiddalfopristin) against adherent Staphylococcus aureus. 35th Interscience Conferenceon Antimicrobial Agents and Chemotherapy, San Francisco,1995; abstr E-122. 8. Hamilton-Miller JMT, ShahS. Killing ofStaphylococcus epidermidisin biofilm by RP 59500 (quinupristiddalfopristin)and other antibiotics. 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, 1995; abstr A-104. 9. Boswell,FJ, Sunderland J, Andrews JM, Wise R. Time kill kinetics of RP 59500 (quinupristiddalfopristin)on Staphylococcus aureuswith and without a raised MBC evaluated using two methods. 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco,1995; abstr E-123. 10. Reinert R, Kresken M, Lemperle M, Lutticken R.In vitro antibacterial activity of RP 59500 (quinupristiddalfopristin),a semisyntheticstreptogramincombination, against Streptococcus pneumoniae. 3rd International Conference on the Macrolides, halides and Streptogramins, Lisbon,1996; abstr 3.15. 11. Pankuch GA, Jacobs MR, Appelbaum PC. Antipneumococcal activityof RP 59500 (an injectable streptogramin), erythromycin and sparfloxacin by MIC and rapid time-kill. 35th Interscience Conference on Antimicriobial Agents and Chemotherapy, San Francisco,1995; E-117. resistant clones of 12. Tarasi A, Tomasz A. Activity of RP59500 against multidrug Streptococcus pneumoniae. 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco,1995; abstr E-116.
Streptogramins: From Parenteral to Oral
65
13. Dever L, Tarasi A, Tomasz A. Bactericidal activityof RP 59500 (RP)against Streptococcuspneumoniaein the rabbit modelof experimental meningitis. 3rd International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 3.31. 14. Grimm H. In vitro activity of quinupristiddalfopristin(RP 59500) and 8other antibiotics againstEnterococcus faecium. 3rd International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 6.18. 15. Hill RLR, Smith C, Casewell MW. Bactericidal and inhibitory activityof RP 59500 (quinupristiddalfopristin)and its components against vancomycin- and gentamicin-resistant Enterococcus faecium. 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, 1995; abstr E-118. 16. Williams JD, Maskell JP, Whiley AC, Sefton A M . Comparative in vitro activity of RP 59500 (quinupristiddalfopristin)against Enterococcus spp. 3rdInternational Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 6.19. 17. Caron F, Gold HS, WennerstenCB, Moellering RC, Jr., Eliopoulos GM.Role of growth phase and of susceptibility to erythromycin on the bactericidal effect of RP 59500, a combinationof quinupristin and dalfopristin, against vancomycin-resistant Enterococcusfaecium strains. 3rdInternational Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996;abstr 6.17. 18. Kang SL, Rybak MJ. In vitro bactericidal activity of RP 59500 (quinupristid dalfopristin) alone and in various combinations against resistant strains of Enterococcus species and Staphylococcus aureus. 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, 1995; abstr E-121. 19. Lorian V, Fernandes F. Synergistic activity of injectable streptogramin RP 59500 (quinupristidda1fopristin)-vancomycin combination. 35th Interscience Conference on AntimicrobialAgentsandChemotherapy,SanFrancisco, 1995; abstr E-126. 20. Herrera-InsuaI, Jacques-Palm K, Murray BE, Rakita RM. Intracellular activityof RP 59500 (quinupristiddalfopristin), aninjectablestreptogramin, against Enterococcus faecium. 35th Interscience Conferenceon Antimicrobial Agents and Chemotherapy, San Francisco, 1995; abstr E-119. 21. Weisblum B. Inducible resistance to macrolides, lincosamides and streptogramin type B antibiotics: the resistance phenotype, its biological diversity and structural elements that regulate expression-areview. J Antimicrob Chemother 1985; 16 (supplA):63-90. 22. Allignet J, Loncle V, El Solh N: Sequence of a staphylococcal gene, vat, encoding an acetyltransferase inactivating streptogramin A and related antibiotics (RP 54476). 33rd Interscience Conferenceon Antimicrobial Agents and Chemotherapy, New Orleans, 1993; abstr 218. 23. Leclercq R, Nantas L, Soussy CJ, Duval J. Activity of RP 59500, a new parenteral semisynthetic streptogramin, against staphylococci with various mechanisms of resistance to macrolide-lincosamide-streptogramin antibiotics. J Antimicrob Chemother 1992; 30 (suppl A):67-75.
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24. El Sohn N, Loncle V, Aubert A, Casetta A, Allignet J. Analysis of staphylococcal elements conferring resistance to streptogramin A (RP 54476). 33rd Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, 1993; abstr 217. 25. Millichap J, Ristow Noskin T, G, Peterson L.Selectionof Enterococcus fuecium strains with stable and unstable resistance to RP 59500 (quinupristiddalfopristin) using stepwise exposure in vitro. 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco,1995; abstr E-120. 26. Tenover FC, Baker CN. Development of provisional disk diffusion breakpoints for testing RP 59500 (quinupristiddalfopristin). 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, 1995; abstr D-30. 27. Neu HC, Chin N, Gu J. The in vitro activity of new streptogramins, RP 59500, RP 57669 and RP 54476, alone and in combination. J AntimicrobChemother 1992; 30 (Suppl A):83-94. 28. Dubois J, Joly JR. In vitro activity of RP 59500, a new synergic antibacterial agent, against Legionellu spp. J Antimicrob Chemother 1992; 30 (suppl A): 77-81. 29. Renaudin H, Boussens B, Bebear C. In vitro activity of RP 59500 against Mycoplasma. 31st Interscience Conferenceon Antimicrobial Agentsand Chemotherapy, Chicago,1991; abstr 897. 30. Nougayrede A, Berthaud N, Bouanchaud DH. Post-antibiotic effects of RP 59500 with Staphylococcus uureus. J Antimicrob Chemother 1992; 30 (suppl A):101-106. 31. Craig W, Ebert S. Pharmacodynamic activities of RP 59500 in an animal infection model. 33rd Interscience Conferenceon Antimicrobial Agents and Chemotherapy, New Orleans, 1995; abstr 470.
Azalides: Basicand Clinical Research Michael W. Dunne Pfirer Central Research Groton, Connecticut
The field of research inthe area of macrolides has beenquite productive in the last 10 years, as evidencedby the clinical developmentof a number of novel compounds. Among these agentsare included the azalides. The azalides as a class are defined by the inclusion of a nitrogen into the macrolidenucleus.Thisaminosubstitutionaddsanadditional positive charge to the compound and can be added to a 14membered, 15-membered, or, theoretically, a 16-membered ring, with the most productive substitutions occurring the at 8 and 9 position. An interest in altering the macrolide nucleus of erythromycin arose because of the acid instabilityof this compound. In fact, 10% of erythromycin is degraded in an acidic environment in approximately S, whereas substitution at the 9 position into the 15-membered ring, the structure of azithromycin, increases the time to acid degradation to about 20 min (1).
MICROBIOLOGIC ACTIVITY The azalides as a classhave the potential for improved gram-negative activity (Table 1). The minimal inhibitory concentrations (MICs) for the Enterobacteriaceae dropped into a range now potentially achievable with in vivo concentrations of the drug. Forthe compounds displayedin Table 1, 67
68
Dunne
Table l
MIc (cLg/ml)B halide Microorganism Enterococcus faecalis 5407 Staphylococcus aures MB 2865 S. haemolyticus MB 5412 Streptococcus pyogenes MB 2874 S. pyogenes MB 5403 (r. wn)b S. pyogenes MB 5406 (r. indy Enterobacter cloacaeCL 4298 Escherichia coli MB 2884 Klebsiella pneumoniae MB 4005 0.25-1 Haemophilus influenzae MB 5363 Pseudomonas stutzeri MB 1231
Erythromycin
Azithromycin analog
1-2
2-4
4
0.25
0.5-1
2-4
0.125 0.015
0.25 0.03
0.5 0.125
>l28 32 32-64 32-64 32-64
>l28 16 0.5-1 1-2 2
2-4 0.25-1.0
>l28 16 4-8 4-8 8
2-8 0.03-0.06
0.25
*Determinedby a standard broth microdilution assay. con: constitutive macrolide resistance. ind: inducible macrolide resistance. Source: From Ref. 2.
this enhancementof gram-negative activity did not depend on thering size, as both 14- and 15-member azalide rings were active (2). Although these azalides maintain activity against gram-positive organisms as well, there is a concomitant 1-2 tube dilution increase in MIC's over erythromycin.For example, the MIC of erythromycin to S. pneumoniae is pg/ml, whereasthat for azithromycin is 0.12 pg/ml. This typeof trade-off between gram-negative and gram-positive activity has been seen with other macrolide and azalide analogs. The mechanism for enhancement of gram-negative activity is not known; however, there is speculationthat the negative charge inthe outer surface of gram-negative bacilli attractsthe positively charged azalide nucleus. It does not appear, however, that the azalides are actively pumped into the gram-negative organism Although there is a slight enhancement of binding to gram-negative ribosomes,there is not enough improvement to explain all but a small degree of potency. It is important to note
Azalides: Basic and Clinical Research
69
that nitrogensubstitutionhasnotovercome the M L S , phenotype of macrolide resistancedue to methylation of ribosomal RNA.
PHARMACOKINETICS The nitrogen substitution within the azalide nucleus has also had a significant impact on thepharmacokinetic propertiesof the class. While in extracellular environments, the azalides have a relatively neutral pH, allowing for passive diffusion through the lipophilic cell membrane. Once inside an acidified vacuole,however,both of the aminoconstituents of the ringbecome charged, preventing diffusion back through the membranes, a phenomenon referred to as the ion-trapping mechanism(4). The azalides are sequestered intracellularly and slowly released back into the circulation (5). Consequently, a large reservoir of drug, free from degradation, is available to provide prolonged tissue exposure while extending the effective dosing interval. A second consequence of this sequestrationof drug involves its concentrationwithin the neutrophil. These cells concentrate the drug and subsequently accumulateat the site of infection, providing a targeted delivery to infected tissues (6). Third, the accumulation of antibiotic within the cells extends the microbiologic spectrumof activity to cover a variety ofintracellular pathogens includingMycobacterium avium, legionella, chlamydia, and rickettsia. Activity against these organisms is achieved because of a combination of the high intravacuolar levels as well as the prolonged retention in that space, maximizing exposureof the bacterium to the drug. This prolonged exposure distinguishes the azalides from other macrolides and quinolones which reach elevated peaks but diffuse more rapidlythe from cell Table 2 displays the murine pharmacokinetic properties of three azalide analogs and the preferential accumulation of drug within tissues(7). Table 2 Murine Pharmacokinetic Propertiesof Three halide Analogs and Accumulation of Drug in Tissues
AUcu-24 (PLg Wml)
Antibiotic
L-701,677 L735,659 L-708,365 azithromycin) G689,108 Source: From Ref. 7.
138 320
6 5
7
1038 2423 2768
271
70
Dunne
METABOLISM The metabolism of azalides is quite different from other macrolides. In vitro studies have demonstrated a lack of degradation of azithromycin within the cytochrome P450 system(8). In addition, numerous drug interaction studies have shown no impact of azithromycinon the metabolism of theophylline, terfenadine, estrogen, carbamazepine, rifabutin, and zidovudine (9-11). With regardto azithromycin, mostof the drug is not metabolized at all, with two-thirds being excreted unchanged the in bile. Metabolismof the remaining drug occurs via demethylation of the nitrogen groups.
CLINICAL STUDIES
The azalides have distinguished themselves with improved acid stability, enhanced gram-negative activity, and a pharmacokinetic profile allowing for prolonged exposure with less frequent dosing. The prototype compound in this class is azithromycin and its early clinical development was focused on the treatment of respiratory tract disease, buildingon the enhancedactivityagainst Haemophilusinfluenzae,Moraxellacatarrhalis, Chlamydia pneumoniae, andlegionella,whileshortening the course of therapy from 10-14 days, typical with erythromycin,to 5 and even 3 days. Its activity against sexually transmitted pathogens, both intracellular, such as Chlamydia trachomatis, and gram-negative, such as Neisseria gonorrhoeae and Hemophilus ducreyi, was developed with single-dose strategies, important in this area where lack of compliance remains a significant public health issue. Further development has expanded on the pharmacokinetic advantages of this drug by exploring single-dose therapiesfor other indications, by taking advantageof the potential for intermittent dosingwell as as by intervening to prevent new infections, all while broadening the scope of potential pathogens to include parasites and enteric gram-negative organisms. Single-dose therapies for treatment of chlamydia, gonorrhea, and chancroid have been studied and published, as thehas activity of azithromycin in the eradication of trachoma (12-15). Trialsare underway looking at singledose treatments of otitis media and shigellosis. Based on pharmacokinetic modeling, there is reason to believe that in addition to higher peak levels, the delivery of drug in shorter courses may provide a greater area under the plasma concentration versus time curve as well, possibly providing enhan activity comparedto similar total doses given over longer periods of time. These shorter courses of therapy must be studied carefully, however,the as inflammatory componentof manyinfectious diseases and the symptoms that come with them may continue beyond the first 24 h of therapy.
71
Azalides: Research Clinical Basic and Table
Pathogens in Intestinal Tract and Their MICs ~~
Azithromycin
Pathogen
Erythromycin
4.0 4.0
Salmonella typhi Salmonella enteritidh Shigella Shigella jejuni Campylobacter Vibrio cholerae ticus Vibrio Yersinia enterocolitica Escherichia coli
1.o 0.5
8.0
4.0
Enterotoxigenic Enteroinvasive
4.0
Clostridium difficile Aeromonas spp. Source: Data from Refs.
and 27.
Intermittent therapies may be of some advantage where prolonged therapy is required or direct observed therapy is considered necessary. This possibility is being explored in the treatment of M . avium disease with three times per week dosing (16). Azithromycinhasdemonstratedactivityin the prevention ofdisease caused by a variety of pathogens. A 1200-mg once weekly regimen has been shown to prevent the development of disseminated Mycobacterium avium infectioninpersonsinfectedwith AIDS. Prevention of bothsinusitisandpneumoniawasalsofoundinthesetrials(17,18). Efficacyin the prevention of malaria due to P. falciparum hasbeen demonstrated both in a controlled Phase I1 study in the United States (19)andafieldtrialinWesternKenya(20).Duringthis latter study, efficacy over placebo or doxycycline was also demonstrated in the prevention of shigellosis (21). Another area of early clinical research involves the treatment of bacterial infections of the gastrointestinal tract. Table 3 lists a variety these pathogens and the MI& are all generally < 4 Most of these are actually intracellular pathogens and consequently are located where azithromycin concentrationsare the highest. Activity has been seen the in treatment of Campylobacter enteritis (22), typhoid fever (23), and shigellosis (25). There are no data yet available concerningE . coli enteritis; however, levels of azithromycin in ileostomy fluidare in the range of 550-1000
72
Dunne
ml, as a consequence of both unabsorbed drug anda transintestinal route of elimination (24). In conclusion, the azalides have provided the clinician with an improvement over therapy with erythromycin. Azithromycin has shown activity in the treatmentof a wide variety of diseases while offering advantages in dosing that improve theease of administration.
REFERENCES 1. Fiese EF, Steffen SH. Comparison of the acid stability of azithromycin and erythromycin, A J Antimicrob Chemother1990; 25 (suppl A):39-47. 2. Jones AB, Herbert CM. J. Antibiot 1992;45:1785-1791. 3. Capobianco JO, Goldman RC. Erythromycin and azithromycintransport into Haemophilus injluenzae ATCC 19418 under conditions of depressed proton motive force. Antimicrob. Agents Chemother 1990;34:1787-1791. 4. Tulkens PM. Intracellular distribution and activity of antibiotics. Eur J Clin Microbiol Infect Dis 1991;lO:lOO-106. 5 . Gladue RP, Snider ME. Intracellular accumulation of azithromycin by cultured human fibroblasts. Antimicrob. Agents Chemother 1990;34:1056-1060. 6. Gladue RP, BrightGM,Isaacson RI, NewborgMF. In vitroandinvivo uptake of azithromycin (CP-62,993) by phagocytic cells: possible mechanisms of delivery and release at sites of infection. Antimicrob Agents Chemother 1989;33:277-282. 7. Pelak BA, Cerckens LS, Kropp H. Tissue distribution and pharmacokinetics of novel h a l i d e 9-Deoxo-8a-Aza-8a-Homoerythromycin derivatives. 33rdInNew Orterscience Conference on Antimicrobial Agents and Chemotherapy, leans, 1993. Amacher DE, Schomaker SJ, Retsema JA, etal. Comparison of the effects of the new azalide antibiotic, azithromycin, and erythromycin estolate on rat liver cytochrome P-450. Antimicrob Agents Chemother 1991;35:1188-1190. 9. Gardner M, Coates P, Hilligoss D, Henry E.Lack of effect of azithromycin on the pharmacokinetics of theophylline in man. 9th Mediterranean Congress on Chemotherapy, Athens, 1992. 10. Honig P, Wortham D, Zamani K, Conner D, Cantilena L. Effect of erythromycin, clarithromycin, and azithromycinon the pharmacokinetics of terfenadines. Clin PharmacolTher 1993;53:161. 11. Chave JP, Munafo A, Chatton J Y , Dayer P, Glauser M, Biollaz J. Once-aon weekazithromycinin AIDS patients:tolerability,kineticsandeffects zidovudine disposition. Antimicrob Agents Chemother 1992;36:1013-1018. 12. Martin DH, Mroczkowski TF, Dalu ZA, etal. A controlled trial of single dose azithromycin for the treatment of chlamydial urethritis and cervicitis. N. Engl J Med 1992;327:921-925. 13. Martin DH, et al. Comparison of azithromycin and ceftriaxonefor the treatment of chancroid. Clin Infect Dis 21:409-414.
esearch Clinical Azalides: and Basic
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14. Handsfield HH, Dalu ZA, Martin DH, Douglas JM, McCarty JM, Jr, Schlossberg D, Azithromycin Gonorrhea Study Group. Multicenter trial of single-dose azithromycin vs. ceftriaxone in the treatment of uncomplicated gonorrhea. Sex. Transm Dis 1994;21(2):107-111. 15. Bailey RJ, Arullendran P, Whittle HC, Mabey DCW. Randomized controlled trial of single-dose azithromycin in treatment of trachoma. Lancet 1993;342: 453-456. 16. Wallace RJ, Jr, Griffith DE, Brown BA, et al. Initial results of three times weekly azithromycin(AZM) in treatment regimens for Mycobacterium aviumintracellulare (MAI) lung diseasein non-AIDS. 3rd International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996. 17. Havlir DV, Dube MP, Sattler FR, Forthal DN, KemperCA, Dunne MW, et al. Mycobacterium qavium complex with weekly Prophylaxis against disseminated agithromycin, daily rifabutin, or both. N. Engl. J. Med. 1996;335:392-398. 18. Oldfield EC, Dickinson G, Chung R, etal. Once weekly azithromycinfor the prevention of Mycobacterium avium complex (MAC) Infection in AIDS patients, 3rd Conferenceof Retroviruses and Opportunistic Infections, Washington, DC, 1996. 19. Andersen SL, Berman J,Kuschner R, et al.Prophylaxis of Plasmodium falciparum malaria with azithromycin administeredto volunteers. Ann Intern Med 1995;123:771-773. 20. Andersen SL, 0100 AJ, Gordon DM, et al. A double blinded, placebo controlled trial of azithromycin comparedto doxycycline for malaria prophylaxis in Western Kenya (submitted). 21. Shanks GD, Ragama OB, Aleman GM, Andersen SL, Gordon DM. Azithromycin prophylaxis prevents epidemic dysentery, Trans Roy SOC Trop Med Hygiene: 1996;90:316. 22. Kuschner RA, Trofa AF, et al. Use of azithromycin for the treatment of Campylobacter enteritis in travelers to Thailand, an area where ciprofloxacin resistance is prevalent. Clin Infect Dis 1995;21:536-541. 23. Tribble D, Girgis N, Habib N, Butler T. Efficacy of azithromycin for typhoid fever. Clin Infect Dis 1995;21:1045-1046. 24. Luke DR, Foulds G, Going PC, Connolly A. Oral absorption profile and disposition of azithromycin (AZM) in ileostomy subjects. 3rd International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996. (AZR) is 25. Khan WA, Seas C, Dhar U, Salam MA, Bennish ML, Azithromycin equivalent to ciprofloxacin (CIP) in the treatment of shigellosis: Results of a randomized, blinded, clinical trial. Abstracts of the 36th InterscienceConference on Antimicrobial Agents and Chemotherapy.1996. 26. Kitzis MD, Goldstein F W , MiCgi M, and AcarJF. In-vitro activity of azithromycin against various Gram-negative bacilli and anaerobic bacteria. J Antimicrob chemother 1990; 25, supplA: 15-18. 27. Jones K, Felmingham D, and Ridgway G. In vitro activity of azithromycin (CP-62, 993), a novel macrolide againstenteric pathogens. Drugs Exptl Clin Res 1988;14:613-615.
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Basic and Clinical Research on Macrolides J Carl Craft Abbott Laboratories
Abbott Park, Illinois
INTRODUCTION In 1952, a soil sample from the Philippineswas foundto contain astrain of Succhuropolysporu erythrueu(formally Streptomyces erythreus) which produced an antibacterialeffect. The discoveryof erythromycin, the first macrolide, set a high standard from which all subsequent macrolides have been judged. Erythromycin is very safe and useful in treating both outpatient and inpatient infections. Although it was initially developed for the treatment of staphylococcal infections in patients allergicto penicillin, it has been found to have much broader unanticipated uses and has in vitro activity against many organisms (Table 1).
ERYTHROMYCIN the full potential of erythromycin was defined by use, were several major disadvantages. These disadvantages include mainly variable pharmacokinetics, prokinetic activity, and unpredictable activity against HuemophiZ u s influenme. Pharmacokinetic studies have shown erythromycin to have variable absorption fromthe gastrointestinal tract dependent on formulation. Bioavailability ranges from 18% to 45%, with peak serum concentrations of 0.5-1.9 pg/ml after the administration 250-500-mg doses every 75
76 Table I
Craft Microbiology of Erythromycin
Streptococcus pyogenes (group A beta-hemolytic streptococcus) Alpha-hemolytic streptococcus (viridans group) Staphylococcusaureus Streptococcus pneumoniae Mycoplasma pneumoniae Chlamydia trachomatis Chlamydia pneumoniae Corynebacteriumdiphtheriae Corynebacteriumminutissimum Campylobacterjejuni Entamoeba histolytica Plasmodium species Listeria monocytogenes Bordetella pertussis Legionella pneumophilia Neisseria gonorrhoeae Treponema pallidum
6 h(1,2). The most frequent side effectsof oral erythromycinpreparations are gastrointestinal and are dose related. They include nausea, vomiting, abdominal pain, diarrhea, and anorexia. These are related to erythromycin’s activity as a motilin receptor agonist. The overall resultof this motilinlike activity isthat erythromycin may not be toleratedby up to one-third of patients Erythromycin has activity against Huernophifus influenzae with a minimal inhibitory concentration (MI%) of pg/ml (5). Due to the relatively high MICs of some isolates and variable pharmacokinetics, erythromycin does not always successfully treat H.influenzae infections. Macrolide research has focused for many years on developing macrolides which would overcome these problems while maintaining the many advantages of erythromycin.
CLARITHROMYCIN Clarithromycin, the 6-0-methyl semisynthetic derivative erythromycin, was discovered by Taisho Pharmaceutical Co., Ltd and developed worldwide byAbbott Laboratories. This relatively minor modification of the molecule provides substantial improvements over erythromycin. Clarithromycin has improved pharmacokinetics when comparedto erythromycin while maintaining the advantages. Orally administered clarithromycin is rapidly absorbed from the gastrointestinal tract and is not affected by food (6-8).
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77
Absorption is characterized by a brief lag time with maximum plasma concentrations usually occurring 2 h after dosing (6-8). Substantial amountsof a pharmacologically active metabolite, 14(R)-hydroxyclarithromycin, are formed followingthe oral administrationof clarithromycin(6-8). The absolute bioavailability of clarithromycin is 55% for the parent compound (g), but this does not take into account the first-pass metabolismof clarithromycin to 14(R)-hydroxyclarithromycin.Because boththe parent and metabolite are active, the true bioavailability is closerto 80-90% (5). Mean peak plasma concentrationsof clarithromycin are 1.0-2.8 pg/ml for the 250-mg bid dose and the 500-mgbid dose,respectively(5-8). The mean peak concentrations of 14(R)-hydroxyclarithromycinare 0.6-0.9 pg/ml for the 250-mg biddose and500-mg biddose, respectively (5-8). The pharmacokinetics of clarithromycinare nonlinear andare characterized by increases in half-life and greater than dose-proportional increases in area under the plasma concentration versus time curve (AUC) with increasing dose size (6-8). The nonlinearity of clarithromycin pharmacokineticsatisleast partially due to the capacity-limited formationof 14(R)-hydroxyclarithromycin (6-8). and AUC values forthe metabolite show lessthan proportional increases with increasing dose size, whereas the apparent elimination halflife increases with increasing dose. Despite the nonlinearity of the pharmacokinetics of clarithromycin,the steady state appears to beachieved by the fourth day of either once or twice daily dosing (6-8). Clarithromycin has a higher affinitythan erythromycin for penetration into cells and tissue. The excellent penetration into cells combined with 80-90% bioavailability and high serum concentrations results in even greater intracellular and tissue concentrations(10) (Table 2)Thus, clarithromycin has balanced pharmacokinetics achieving high intracellular concentrations needed for intracellular infections such as Chlamydia pneumoniae and disseminated Mycobacterium avium, withoutsacrificingserumand interstitial concentrations neededfor Streptococcus pneumoniae and Haemophilus infruenzae infections. Macrolides such as erythromycin and oleandomycin have been known to induce strong muscular contraction inthe gastrointestinal tract of dogs and humans. Intravenous erythromycin given at a O.OZmg/kg dose to dogs induced a pattern of migrating contractions in the gastrointestinal tract similar to the effect of motilin, a 22aminoacid peptide hormone, on gastrointestinal contractile activity (11). When given intravenously at a dose of 7 mg/kg, an immediate increase in contractile activity in the whole length of the intestinal tract was observed (12). The prokinetic activityof 14-membered macrolides and 15-membered azalides can also be related to their ability to bind to motilin receptors inthe gastrointestinal tract. These findings in dogs relate well to the frequent side effects of erythromycin
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Tub& 2 Mean Bronchopulmonary Pharmacokinetics (pg/ml) for Clarithromycin, 14(R)-Hydroxyclarithromycin,and Azithromycin
Plasma Clarithromycin 14-Hydroxyclarithromycin Azithromycin Epithelial lining fluid Clarithromycin Azithromycin Alveolar cells Clarithromycin 14-Hydroxyclarithromycin Azithromycin ~~
4h
8h
a
a
h
a
24h
a
~~
.Below limit of assay (0.01 Source: From Ref.
which include nausea, vomiting, abdominal pain, diarrhea, and anorexia. Clarithromycin activity in the dog model is only 60% of that for erythromycin. Clarithromycin has a decreased affinity for the motilin receptors when compared to erythromycin and azithromycin (13) (Table 3). This decreased affinitywas shown to have clinical significance in two clinical studies comparing clarithromycin to erythromycin forthe treatment of pneumonia. In pneumonia studies conducted in adults comparing clarithromycin to erythromycin base or erythromycin stearate, there were fewer adverse events involving the digestive system in clarithromycin-treated patients compared to erythromycin-treated patients (13% versus 32%;~ < .01) (3,4). llventy percent the erythromycin-treated patients discontinued therapydue to adverseeventscompared to 4%of clarithromycin-treatedpatients. In other studies, clarithromycin was found to have gastrointestinal tolerance comparable to oral cephalosporins such as cefaclor (14,15). Clarithromycin is approximately equal to or one tube dilution less active than erythromycin againstH . influenzae and H . paruinfluenzae (1619). However, the 14-hydroxy metabolite has activity equal to orone tube dilutionmoreactivityagainst H . injruenzue aserythromycin, with an MIC, of 2-4 pg/ml (20). The combination of clarithromycin and 14(R)hydroxyclarithromycin produces additive and sometimes synergistic effects against H . influenzae both in vitro (21,22) and in vivo in a gerbil model of otitis media and a mouse model of pneumonia (23). To explain the ergistic effect between clarithromycin and 14(R)-hydroxyclarithromycin, their interactionwith gram-negative and gram-positive bacterial ribosomes
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on Macrolides
Tab& 3 Motilin Activity of Antibiotic Macrolides ~~
~
Clarithromycin Erythromycin Azithromycin
Smooth muscle (PED,,)
Dog (force/freq) stomach
5.85
1.00
Motilin binding
jejunum
PIC50
1.09
*p < .05 relative to azithromycin. Source: From Ref. 13.
was studied. It wasfound that for H. influenzae (1879) strain), 14(R)hydroxyclarithromycin bindsto two different sites. Similar to erythromycin and clarithromycin,it binds to the subunit of the ribosome. The 14(R)hydroxyclarithromycin molecule also bindsto the 70s ribosomal initiation complex which may provide the potential for a synergistic effect in some organisms (24). This increased activity against a full range of respiratory pathogens has allowed clarithromycin to obtain more extensive indications than erythromycin as shown below.
Clarithromycin Indications and Usage (3) Clarithromycin tablets and oral suspension are indicated for the treatment ofmild to moderate infections caused by susceptible strains of microorganisms in the conditions listed below: Adults: PharyngitislTonsillitis due to Streptococcus pyogenes (The usual drug of choice in the treatment and prevention of streptococcal infection and prophylaxisof rheumatic feveris penicillin administered by either the intramuscular or the oral route. Clarithromycin is generally effective in the eradication of S. pyogenes from the nasopharynx; however, data establishing the efficacy of clarithromycinin the subsequent prevention of rheumatic fever are not available at present.) Acute maxillarysinusitis due to Haemophilus influenzae, Moraxella catarrhalis, or Streptococcuspneumoniae. Acute bacterial exacerbationof chronic bronchitis due to Haemophilus influenzae, Moraxella catarrhalis,or Streptococcuspneumoniae. Pneumonia due to Mycoplasma pneumoniae or Streptococcus pneumoniae.
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Uncomplicated skin and skin structure infectionsdue to Staphylococcus aureusor Streptococcus pyogenes. Disseminated mycobacterial infections due to Mycobacterium avium or Mycobacterium intracellulare. Children:
PharyngitislTonsillitis due to Streptococcus pyogenes. Acute maxillarysinusitis due to Haemophilus infiuenzae, Moraxella catarrhalis,or Streptococcus pneumoniae. Acute otitis media due to Haemophilus infiuenzae, Moraxella catarrhalis, or Streptococcuspneumoniae. Uncomplicated skin and skin structure infectionsdue to Staphylococcus aureus or Streptococcus pyogenes. Disseminated mycobacterial infections due toMycobacterium avium, or Mycobacterium intracellulare. Prophylaxis: Clarithromycin tablets and oral suspension are indicated for the prevention of disseminated Mycobacterium aviumcomplex (MAC) disease in patients with advanced HIV infection.
Helicobacterpylori Clarithromycin in combination with ompeprazole are indicated for the treatment of patients with an active duodenal ulcer associated with H. pylori infection. The eradication of H. pylori has been demonstrated to reduce the risk of duodenal ulcer recurrence. Clarithromycin also has activity in vitro and in vivo activity against several other new pathogens of clinical importance. These pathogens include many of the environmental mycobacteria, which have become a major problem because of the AIDS epidemic and Helicobacterpylori which is associated with peptic ulcer disease. Unlike erythromycin, clarithromycin has activity against mycobacteria. The MICsof clarithromycin for M. chelonae, M. kansasii, and M. xenopi are generally 1 pg/ml(25-27). The MI&, for M. avium complex using pH-adjusted media in Bactec is 0.5 pg/ml (28). Clarithromycin has been shownto have activity againstM. leprae in the mouse foot pad model (29). This activity has been very useful in the treatment and prophylaxis against these previouslyuntreatable infections inAIDS patients. Clarithromycin, however, does not have sufficient activity against M. tuberculosis to be of clinical benefit.
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Macrolides for Peptic Ulcer Disease Peptic ulcer disease results in a high degree of morbidity and attendant economic cost. Early in this century,the pathogenesis of this disorder was believed to be related to diet complicated by stress.Palliative therapy focused on bed rest and a diet of bland foods. Subsequently, it was suspected that the gastric secretion of hydrochloric acid producedthe tissue injury associated with peptic ulcer disease, and antacids became'a standard therapy. In the early H,-receptor antagonists replaced antacids as the mainstay of therapy. More recently, inhibitorsof the proton pump gastric parietal cells, for example, omeprazole and lansoprazole, have also been shown to be effective in accelerating ulcer healingand, when continued, reduced the rateof recurrence. Even after complete ulcer healing has been obtained using thesepotent therapeutic agents, peptic ulcer disease recurs. The natural history of ulcer had been described as "once an ulcer, always an ulcer." Since the early research into gastritis andpeptic ulcer disease has been dominated by Helicobacter pylori. H . pylori is a small, gramnegative, spiral-shaped, urease-producing bacterium that inhabits the mucus layer coveringthe gastric mucosa.The association between this organism and human diseasewas first reported by Marshall and Warren in Initially, identification of the bacterium included it with the genus Campylobacter, but further research demonstrated a difference and the name Helicobacter pylori was adopted. Infection ofthe human stomachby H . pylori occurs worldwideand is virtually ubiquitous in many developing countries. Infection seems to be acquired early in life and is generally inversely proportional to social economic status. Means of transmission is unknown but is believed to be by way of the fecal-oral route, and infection, although acquired in childhood, generally persists unlesstreated.
Clarithromycin Treatment of Peptic Ulcer Disease Clarithromycin is very active against H . pylori, with aMIC, of pg/ml Clarithromycin and 14(R)-hydroxyclarithromycinare bound tightly to H . pylori ribosomes, having aKd value of 2 M which isthe tightest binding interaction observed for a macrolide-ribosome complex A comparison of kill kinetics and MICs demonstratedthat clarithromycin (with early bactericidal activityof at least a log reduction by h) was more active than azithromycin and erythromycin against H . pylori When tested againstH . pylori at pHvalues from5.5 to 8.0, clarithromycin was found to be the most activeof the macrolides tested (Table
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82 Table 4 Comparative Activityof Macrolides Against H . pylon
M C (pdml) Antimicrobial pH
pH 7.3 pH 5.5 6.5 pH
Clarithromycin 0.03 14-OH-clarithromycin Azithromycin 0.12 Roxithromycin 0.12 Erythromycin 0.25 0.25 Josamycin 1.00 Dirithromycin
8.0
< 0.015 0.06 0.06
0.25 1.oo
0.12
1.00 1.00 2.00 4.00 8.00
0.03
0.12 0.12 0.25
< 0.015 0.03
0.03 0.03 0.03
0.12 0.03
Source: From Ref.
Clarithromycin has been studied extensivelythefor treatment of peptic ulcer disease associated with H.pylori infection. Early pilot studies demonstrated clarithromycin to be the most active agent when used as monotherapy for the eradication of H . pylori. Clarithromycin monotherapy demonstrated eradication rates from15% to 54%, dependent on both dosage and frequency (34,35). Morefrequent dosing showing the highest eradication rates (Table 5). eradication rates of even 50% did not appear to be adequate for the treatment of peptic ulcer diseases associated with H.pylori, other agents were addedto clarithromycin. The addition of omeprazole to clarithromycin has improvedthe efficacy of clarithromycin (36,37) (Table6 ) . This is probably related to theincrease inthe mean pH of the stomach to 5.7, an effect of omeprazole which improvesthe activity of clarithromycinand by increasingthe concentration of clarithromycin at the site of the infection (38). Omeprazole also has limited activity againstH . Table
Results of Clarithromycin Monotherapy Studies Eradication
Study number MW-484” M91-602‘
rate dosage Clarithromycin (2 weeks)
Eradl evala
42mg QID 250 15 mg BID 500 1000mg BID 500 mg QID
5/12 U13 4111 7/13
.Eradicated/evaluable at 4 weeks after last dose. bData from Ref. CDatafrom Ref. 35.
36 54
Basic and Clinical Research on Macrolides
83
Table 6 Treatment of H.pylon Associated Duodenal Ulcer: Clarithromycin 500 mg tid and Omeprazole40 mg qid for2 Weeks
Studies United States8 Europeb
94% 99%
(60/64) (1481149)
72% 78%
(41157) (114/146)
29% 8%
(16155) (10/127)
'Data from Ref. bData from Ref.
pylori with MIC valuesof approximately 25 pg/ml. Clarithromycin in combination with ranitidine bismuthcitrate has also been shown to improve the activity of clarithromycin (Table 7). Smaller increasesin pH caused by ranitidine are augmented by the anti-H. pylori activity of bismuth citrate and probably account for the synergy seen with ranitidine bismuth citrate. Clarithromycin has become the cornerstone H . pylori eradication therapy. The best regimen using clarithromycin is yet to be determined, but many studies are under way.
PROKINETIC DRUGS Motility of the gastrointestinal tract is modulatedby the gastric pacemaker and spread distally by the myentericplexus. After feeding and during digestion, the plexus initiates the grinding of solids into chyme. Emptying of chyme from the stomach and progression through the gastrointestinal tract is proceeded by waves of electrical activity grouped in Phases I to IV, and known as the migrating myoelectric complex (MMC). Controlof gastrointestinal motilityis associated with cholinergic, adrenergic, and motilin receptors (Fig. 1). Stimulation of cholinergic nervesby acetylcholine leads to increase in motor activity, contractions fromthe esophagus to the small
Tab& 7 Treatment of H.pylon Associated Duodenal Ulcer Clarithromycin500 mg tid or250 mg qid and RBC400 mg bid for2 weeks
United Statesa Clarithromycin 500 mg tid Europeb Clarithromycin 250 mg qid 'Data from Ref. bData from Ref. 40.
Healing
Eradication
72%
82%
89%
94%
84
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Muscle
Figure l
Mode of action of prokinetic agents.
intestine, and maintenance of general muscle tone. This activity is countered by stimulation of dopamine receptors,which leads to gastrointestinal atony, lossof antroduodenal coordination, and reduced peristalsis (Fig. 1). Hence, exogenous cholinergic receptor agonists or dopamine antagonists lead to increased gastrointestinal motor activity and the induction of Phase I11 of the MMC (41), stimulation of coordinated antroduodenal contractions, and gastric emptying. Motilin is 100-fold more active than acetylcholine in stimulationof the gastrointestinal tract (42). Motilin is believedto be the major physiological factor inthe initiation and coordinationof the interdigestive muscle contractions which propel gastric contents through the gastrointestinal tract (Fig. 1). Exogenous administration of motilin results in an increase in lower esophageal sphincter pressure(44,45) and increased motility of the lower esophagus (46). In addition, induction of coordinated contractions resembling PhaseI11 the MMC leads tohccelerated gastric emptying(47). Erythromycin mimics the activity exogenous motilin when administered intravenouslyto rabbit, dog, and man (11). In humans, the intravenousadministration of erythromycininducesPhase I11 activity of the MMC (48) and coordinates contractile activities inthe esophagus, gastric antrum, and the intestine. Erythromycin specifically inhibits the binding of {lZI}-motilin to rabbit duodenal muscle strips (49) and displaces bound motilinfromisolatedrabbitcolonmonocytes (50). Erythromycinalso demonstrated clinical effectiveness in diabetic gastroparesis 32). (51 The prokinetic activityof the macrolide molecule is chemically unrelated to the antibacterial activityof the class of compounds. Thus, modifica-
85
Basic and Clinical Research on Macrolides Table 8 Ribosomal Binding Studies
Relative binding Compound Erythromycin ABT-229
1.5
10-7
1.5
10 M
M
lo00 lo00
Source: From Ref. 42.
tion of erythromycin led to compounds with improved prokinetic activity and reduced antibacterial activity, as demonstrated by reduced ribosomal binding (5334) (Table 8). ABT-229 is an excellent candidatefor use in humans withabnormal gastrointestinalmotility. In vitro data indicate that ABT-229displaces bound {'~I}-motilinfrom receptors in isolated rabbit antral smooth-muscle tissues; this displacement was logs greater than for erythromycin. Binding of ABT-229 is within an order of magnitude of that of motilin, indicating that ABT-229isa potent synthetic motilin agonist. In addition, tissue assays using rabbit duodenal smooth muscle indicate that ABT-229 is several orders of magnitude more potent than erythromycin as a prokinetic agent. ABT-229 has minimal affinity for bacterial ribosomes, which are the binding sitesof the antimicrobial activityof macrolides, withan affinity 1000-4000-fold lower than erythromycin. (Table 8). In addition, in vitro data against standard laboratory strainsof bacteria indicate that the mean MIC is 50 to > lOOpg/ml for ABT-229, comparedto erythromycin, which had a mean MICof 0.04-0.5 &m1 for susceptible organisms.Thus, there is minimal riskof altering the normal gut floraby long-term administration of ABT-229. Potency in animals has been demonstrated in the opossum by placement of strain gauge transducers inthe lower esophageal sphincter. ABT229 and erythromycin were each administered as1.0-mgkg a dose intravenously. Both drugs induce motility in the lower esophageal sphincter,but ABT-229 produces higher amplitude contractions and a more prolonged effect than erythromycin. In addition, ABT-229 also increasedthe toneof the esophageal smooth muscle. Thus, ABT-229 would be expected to be more effectivethan erythromycin for improving esophageal peristalsis and sphincter tone in man. The anesthetized dog model has been usedto evaluate the effect of ABT-229 and erythromycin on gastrointestinal motility. A single dose of eachcompound (0.4 mgkg) wasgiven intravenously.Bothdrugs had qualitatively similar activity, but ABT-229 induced more potent and sus-
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86
tained smooth-muscle contractions in the stomach, duodenum, and jejunumthanerythromycin. The response to ABT-229wasalso compared with that of cisapride in the same model; ABT-229 produced a higher motility index in the stomach, duodenum, and jejunum of the anesthetized dog. The motilin activitiesof erythromycin was always considered to be an unwanted side effect, but more recently erythromycin has been used successfully to treat diabetic gastroparesis. Thus, prokinetic macrolides have a potential to be usedto treat disorders of gastrointestinal motility in humans such as diabetic gastroparesis and gastroesophageal reflux.
THE FUTURE OF MACROLIDES The macrolides, after more than 40 years of use as antibiotics, are now on the verge a new age. New macrolides with improved activity against the resistant Streptococcus pneurnoniae will continue the antibiotic uses this class of drugs. Macrolides have become widely used as immunomodulators in the treatment of rejection during organ transplantation. Their usefulness in the treatment of inflammation associated with diseases such as asthma and Crohn's diseaseare just being defined andthe possibilities of treating some forms of cancer with combination therapy including macrolides has been reported. The prokinetic macrolidesare extending the nonantibiotics uses of the macrolides; this suggests that the potential may be unlimitedfor these extremely versatile molecules.
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35. Peterson WL, Graham DY, Blaser MJ, Genta R M , Klein PD, Stratton CW, Drnec J, Prokocimer P, Siepman N. Clarithromycin as monotherapy for eradication of Helicobacterpylon: a randomized, double-blind trial. Am GastroJ enteroll993; 88(11):1860-1864. 36. Hunt R, Schwartz H, Fitch D, Fedorak R, A1 KawasF, Vakil N. Dual therapy of clarithromycin and omeprazole for treatment of patients with duodenal H.pylori infection. 3rdInternational Conference on the ulcers associated with Macrolides, halides, and Streptogramins, Lisbon,1996; abstr 5.18. 37. O'Morain C, Logan RPH, the Clarithromycin European H . pylori Study Group. Clarithromycin in combination with omeprazole for healing of duodenal ulcers, prevention of duodenal ulcer recurrence, and eradication of H . pylori in two European studies. 3rd International Conference on the Macrolides, Azalides, and Streptogramins, Lisbon,1996; abstr 5.25. 38. Gustavson LE, Kaiser JF, Edmonds AL, Locke CS, DeBartolo ML, Schneck DW. Effect of omeprazoleon concentrations of clarithromycin in plasma and gastric tissue at steady state. Antimicrob Agents Chemother 1995;39(9): 2078-2083. 39. Peterson WL, Sontag SJ, Ciociola M, Sykes DJ, McSorleyDD, Webb DD, H. pylonUlcer Group. Ranitidine bismuth citrate plus clarithromycin is effective inthe eradication of Helicobacter pylori and prevention of duodenal ulcer relapse.3rd International Conference on the Macrolides, halides, and Streptogramins, Lisbon, 1996; abstr 5.32. 40. Bardhan K D , Dallaire C, Eisold H, Duggan AE. The treatment of duodenal ulcer with GR122311X (ranitidine bismuth citrate) and clarithromycin. 3rd International Conference on the Macrolides, halides, and Streptogramins, Lisbon, 1996; abstr 5.33. In: Physiology, Diagno41. Champion, M. Treatment of gastric motility disorders. sis andTherapy in G.I. Motility Disorders. Toronto:MES Medical Education Services, 1987:13-14. 42. Fox JET, Daniel EE, Jury J, Fox AE, Collin SM. Site and mechanisms of action of neuropeptides on canine gastric motility differin vivo and in vitro. Life Sci 1993; 33:819-825. 43. Lee K, Chey WY, Tail HH, Yajima H. Radioimmunoassay of motilin. Validation and studies on relationship between plasma motilin and interdigestive myoelectric activityof the duodenum of dog. Am J Dig Dis 1978; 23:789-795. 44. Lux G, Rosch W, Domsche S, Domsche W, Wunsch E. Intravenous 13-Nlemotilin increases the human lower esophageal sphincter pressure. Scand J Gastroenterol 1976; 11 (suppl39):75-79. 45. Jennewein HM, Bauer R, Hummelt H, Lepsin G, Siewert R. Motilin effects on gastrointestinal motility and lower esophageal sphincter (LES) pressure in dogs. Scand JGastroenteroll976; 11(suppl39):63-65. 46. Jennewein HM, Hummelt H, Siewert R, Waldeck F. The uniform effect of natural motilin inthe lower esophageal sphincter, fundus, antrum, and duodenum in dogs. Digestion 1975;13:246-250. In: Itoh Z, ed. 47. Yamagishi T,Debas HT. Biological activity in gastric emptying. Motilin. San Diego: Academic Press, (1990):122-132.
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48. Tomomasa T, Kuroume T, Arai H, Wakabayashi K, Itoh Z, Erythromycin induces migrating complexin human gastrointestinaltract. Dig Dis Sci 1986; 31:157-161. 49. Kondo Y, Toni K, Amura S , Itoh Z, Erythromycin and its derivatives with motilin-like biological activities inhibit the specific binding of 1-125 motilin to duodenal muscle. Biochem Biophys Res Commun 1988; 150:877-882. 50. Hasler W, Heldsinger A, Chung OY. Erythromycin contracts rabbit colon monocytes via occupationof motilin receptors. Am Physioll992; J 262:G50G55.
51. Urbain JL, Vantrappen G , Janssens J, VanCutsem E, Peeters TL, De Roo M. Intravenous erythromycin dramatically accelerated gastric emptying in gastroparesis diabeticorum and normals and abolishesthe emptying discrimination between solids and liquids. Nucl J Med 1990; 31:1490-1493. 52. Janssens J, Peeters TL, Vantrappen G, Tack J, Urbain JL, De Roo M, Mulus E, Bouillon R. Improvement of gastric emptying indiabetic gastroparesis by erythromycin. Preliminary studies.N Engl J Med 1990; 322:1028-1031. 53. Omura S, Tsuzuki K, SunazukaT, ToyodaH, Takahahsi I, Itoh Z. Gastrointestinal motor-stimulating activity of macrolide antibiotics and the structureactivity relationship. JAntibiot (Tokyo) 1985; 38:1631-1632. 54. Omura S, Tzuzuki K, Sunazuki T, Marui S, Toyoda H, Inatomi N, Itoh Macrolides with gastrointestinal motor stimulating activity. J Med Chem 1987; 30:1941-1943.
7 Nonantibiotic Effects of Macrolide Antibiotics: Suppression of the Bacterial Glycocalyx of Pseudomonas aeruginosa Isolates from Patients with Cystic Fibrosis Charles W. Stratton Vanderbilt University Schoolof Medicine Nashville, Tennessee
INTRODUCTION Macrolide antibiotics such as erythromycin, clarithromycin, and azithromycin, as well as clindamycin have been notedto have the ability to decrease sputum production in patients with chronic respiratory infections (1-3). Macrolide antibiotics also have beenreported to decrease the in vitro expression of mucoid exopolysaccharide (MEP), also called alginate, slime, and glycocalyx, produced by Pseudomonas aeriginosa (4,s). Although the decrease of sputum production in patients with chronic respiratory infections has beenattributed to a direct effect on the production of sputum it may, instead, be due to inhibition of MEP production by respiratory pathogens. Pseudomonas aeruginosa is a common cause of chronic pulmonary tract infections in patients with cystic fibrosis (CF) (6).Indeed, the predominant in vivo phenotype of P . aeruginosa that invariably emerges in chronic pulmonary infectionsof CF patients is this mucoid strain which is character91
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bed by the copious production of MEP (7,8). Emergence of this strain is associated with progressive respiratory distress and a poor prognosis (9). Suppression of MEP, therefore, may prove importantfor the therapy of CF patients with chronic pulmonary infections caused by this pathogen. This study usedthe E-strip agar diffusion method (10) to evaluate the suppressiveeffects of erythromycin,clarithromycin,azithromycin,and clindamycin on the in vitro productionof pseudomonal MEP using mucoid. strains of P . aeruginosa that werefreshlyisolatedfrom CF patients. Macrolide-containing E-strips were placed on the surface of inoculated agar plates and periodically assessed during 24 h of incubation. Other antimicrobial agents that inhibit protein synthesis (tetracycline and chloramphenicol)werealsoassessed.Inaddition,selectedcombinations of macrolides and antipseudomonal agents were evaluated using a doubledisk/E-strip agar diffusion technique.
MATERIALS AND METHODS Isolates
lbenty-five fresh mucoid strainsof P.aeruginosa were obtained and evaluated after initial isolation from cystic fibrosis patients. Because mucoid strains of P . aeruginosa often become nonmucoid after being subcultured in the clinical microbiology laboratory (11,12), only freshly isolated mucoid strains that had not been subcultured were used. The ATCC strain of P . aeruginosa (ATCC 27853) and other well-characterized strains [PsSOSAI', PsSOSAI-, and PsSOSAICONisogenic strains(13),kindlysupplied by Doctor David M. Livermore] were included as nonmucoid controls.
Antimicrobial Agents
Antimicrobial agents used in this study included erythromycin, clarithrom cin, azithromycin, and clindamycin, as well as tetracycline, chloramphenicol, andthe commonly used antipseudomonal agents gentamicin, ciprofloxacin, and piperacillin. Each agent was obtained as a graded carrier strip containing a predefined antibiotic gradient (AB Biodisk, Solna, Sweden) or on a susceptibility disk containing a fixed concentrationthe antibiotic (Difco) .
Media The agar media selected for this assessment included media limited in nutrients which promotes the growthofmucoidsessileformsof P. aeruginosa (1 1,12); these were cation-supplemented Mueller-Hinton agar
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(CSMHA, BBL) and Batco minimal agar Davis (BMAD, Difco). In addition, Bactec nutrient agar (BNA, Difco) was used; this medium nutrientis rich and promotesthe growth of nonmucoid planktonic forms(llJ2).
Assessment of Suppression The methodology selectedfor measuring the suppressive effectsof macrolides on MEP production of P . aemginosa was agar-based because agar promotes the microbial growthof mucoid sessile forms by providing attachment sitesfor these sessile forms as opposed to growth inbroth, which does not provide such attachment sites, thus promoting the growth of nonmucoid planktonic forms(14). The E Test (AB Biodisk) agar diffusion method (10) was used,as it providedlowandhighconcentrations of the selected macrolide and allowedthe determination of MIC (minimal inhibitory concentration) values. After inoculation with mucoid strains and placement of macrolide-containing E-strips, agar plates were incubated in ambient atair 35°C and evaluated after6, 8, 10, 12,24, and 48 h of incubation. Selected combinations of macrolides and antipseudomonal agents were evaluated cm) using a double-disk/E-strip agar diffusion. This technique used(10small plates of CA-MHB. These plates were inoculated with a mucoid stain by triple cross-streaking with a cotton swab asper the NCCLS recommendations forthe disk diffusion test(15). An E-strip containing the macrolide to be tested is placed on the agar surface and a disk containingthe antipseudomonal agent is placed at each end of the E-strip on opposite sides. The zone sizesof these two disksare assessed at 6,8,10,12,24, and 48 h of incubation. This allowed assessment ofthe effect of high and lowconcentrations of each macrolide on the inhibitory effect of the antipseudomonal agent.
RESULTS The results of this studyare summarized as follows:
1. Initial clinical isolates ofmucoid strains of P . aeruginosa from CF patients exhibited a characteristic mucoid appearance (wet, smooth, confluent colony growth) on CSMHA and BMAD; in contrast, these initial isolates exhibited a nonmucoid appearance (dry, rough, nonconfluent colony growth) when grownon BNA. 2. Each macrolide studied initially was noted to suppress the production of MEP at 6-12 hours of growth on CSMHA as evidenced by an eliptical zone aroundthe E-strip in which discrete nonmucoid colonies were seen. Nonmucoid colonies withinthe inhibitory zonesof the macrolides when suspended in an India inkhaline solution and viewed by
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Tabk Z Evaluation of the Potential for Tetracyclineand Chloramphenicol to Suppress Alginate Production in25 Mucoid Isolates of Pseudomonas aeruginosa. Results of Potential Glycocalyx Suppressionon the Antimicrobial Activity for Selected Antipseudomonal Agents
tested
Zone size (mm): top; bottom; -t standard
Antipseudomonal Suppressive Difference (mean agent Gentamicin Piperacillin Imipenem Pipltazo Ciprofloxacin Gentamicin Piperacillin Imipenem Pipltazo Ciprofloxacin
4.
5. 6.
7.
Tetracycline Tetracycline Tetracycline Tetracycline Tetracycline Chloramphenicol Chloramphenicol Chloramphenicol Chloramphenicol Chloramphenicol
'
19 (2 1.2); 18 (2 1.1); l ( * 0.8) 19 (2 1.3); 20 (2 1.2); l ( + 1.0) 26 (+ 1.5); 25 (2 1.6); none 27 (2 1.4); 28 (2 1.7); l ( + 1.5) 20 (+ 1.2); 19 (+ 1.4); 1 (2) 0.9) 19 (2 1.2); 20 ( 2 1.3); 1(+ 1.1) 20 (+ 1.4); 18 ( 2 1.7); 2 (+ 1.6) 25 (2 1.5); 25 (+ 1.7); none 27 (2 1.6); 27 (+ 1.5); none 20 (2 1.4); 19 (2 1.5); 1(-t 1.3)
phase-contrast microscopy were found to exhibit a marked reduction in capsular size in comparison with mucoidsurroundcolonies ing the inhibitory zones. These suppressive effectsof the macrolides studied wereranked as clarithromycin = azithromycin > erythromycin > clindamycin. At 24 h, this suppressive effectwas almost nonexistant withthe exception of azithromycin. Tetracycline and chloramphenicol did not suppress the production of mucoid exopolysaccharide. Instead, tetracyclinewas noted to inhibit the growth of these mucoid strains of P. aeruginosa at concentrations of 16-32 @m1 for up to 24 h after incubation on all three types of agar. Chloramphenicol was inhibitory at 24 h with pg/ml on BNA only. The inhibitory effectsof tetracycline werealsonotedfornonmucoidstrains,including the ATCC strain. In contrast, the inhibitory effectsof chloramphenicol were not seenfor any nonmucoid strains. (See Table 1.) Slimy strains of Pseudomonas often were found to besusceptible to gentamicin, ciprofloxacin, and piperacillidtazobactam at 24 h when tested on CS-MHA and BMAD but were resistant when tested on BNA. Nonmucoid strains were invariably resistant to gentamicinwhentested on BNA,yetweresusceptiblewhen tested on CS-MHA and BMAD. (See Table 2.)
etested
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Table 2 Evaluation of the Suppressive Effectof Macrolides on MEP Production in 25 Mucoid Isolatesof Pseudomonas aeruginosaand Their Effecton the Inhibitory Activity of Selected Antipseudomonal Agents
Zone size (mm); top; bottom; 2 standard
Antipseudomonal Suppressive difference (mean agent Gentamicin Piperacillin Imipenem Pipttazo Ciprofloxacin
Erythromycin Erythromycin Erythromycin Erythromycin Erythromycin
19 (2 1.2); 19 ( a 1.3); l ( + 1.1) 18 1.1); 17 (2 1.2); 1 (+ 0.8) 26 ( a 1.4); 27 (2 1.5); 1 (+ 0.9) 28 (2 1.5); 28 ( 2 1.6); none 23 ( a 1.4); 19 ( a 1.2); 4 (2 1.0)
Gentamicin Piperacillin Imipenem Pipltazo Ciprofloxacin
Clarithromycin Clarithromycin Clarithromycin Clarithromycin Clarithromycin
19 (2 1.3); 21 (+ 1.6); 2 (2 1.5) 20 (+ 1.4); 20 (2 1.5); none 27 (+ 1.3); 25 (2 1.9); 2 (2 1.7) 28 (2 1.1); 27 ( a 1.4); 1 (+ 1.2) 26 (+ 1.5); 19 (2 1.3); 7 (+ 1.4)
Gentamicin Piperacillin Imipenem Pipltazo Ciprofloxacin
Azithromycin Azithromycin Azithromycin Azithromycin Azithromycin
18 (+ 1.4); 19 (+ 1.5); 1(2 0.8) 20 (2 1.5); 19 (2 1.3); 1 (2 1.4 26 (a 1.4); 26 (+ 1.8); none 29 (+ 1.2); 28 (2 1.2); 1 (+ 1.1) 26 (2 1.6); 19 (2 1.3); 7 (2 1.4)
Gentamicin Piperacillin Imipenem Pipltazo Ciprofloxacin
Clindamycin Clindamycin Clindamycin Clindamycin Clindamycin
20 (2 1.7); 19 (+ 1.8); 1(2 1.7) 19 (a 1.6); 19 (2 1.4); none 26 (+ 1.4); 27 (2 1.8); l ( + 1.6) 28 (2 1.6); 28 (2 1.4); none 20 (a 1.3); 19 (+ 1.2); 1 (2 0.9)
8. Double-diskE-strip tests revealed that macrolides enhanced the inhibitory effects of ciprofloxacin on CS-MHA and, to a lesser
degree, on BMAD. In contrast, these macrolides were noted to be antagonistic for gentamicinon CS-MHA and BMAD. appreciable effects were seen for the p-lactam agents tested. 9. Hypersusceptible strains recovered from a number of CF patients and were characterized as follows: Mucoid appearance Lack of pigments Increasedsusceptibility to p-lactamagents(meanMICs to carbenicillin = 4 pg/ml) with the exception of imipenem to which susceptibilities remainedthe same.
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Stratton Increased susceptibility to tetracycline (MIC = 6 pg/ml) and chloramphenicol (mean MICs= 8 pg/ml) Moderatelyresistant to aminoglycosides(meanMICs = 4 Pg/ml)
DISCUSSION Macrolide exopolysaccharides produced by P. aeruginosa is essentially an anionicpolymericdiffusionbarrierandcanbethoughtofasanionexchangeresin of almostinfinitesurfacearea(16).In addition, MEP protects Pseudomonas from heavy-metal toxicity (17) from most bacteriophages, from phagocytic white blood cells (18,19), from antibodies and or complement (20), and from an inhospitable milieu such as osmotic, pH, or enzymatic dangers (17). P . aeruginosa is able to excrete into its MEP and the surrounding medium several different classes of molecules, includingexopolysaccharides (the buildingblocks of MEP),pigmentswhich function as siderophores, protein enzymes, and toxins (21). Pseudomonal MEP also appears to serve as a repository for defensive substances such as p-lactamase (22). Finally, electron microscopy has revealed that there is an interaction of pseudomonal MEP with the mucus in the airways of the human lung (23). This allows mucoid phenotypes of P. aeruginosa to more readily and avidly attach to lower airways than nonmucoid strains. The synthesis of MEP by P. aeruginosa thus appears to be an important virulance factor (7-9). Exposure of planktonic cellsof Pseudomonas aeruginosa to a biofilm surface produced by cells of the same species triggersthe expression of at least genes, aZgC and aZgG (24,25), which inducethe synthesis of MEP. Nutrient limitation in isolates of P . aeruginosa also has been shown to result in increased synthesisof MEP, whereas availability of nutrients results in decreased MEP (11,12). Of interest is that several porin proteins (D-l and D-2 OMPs) of P . aeruginosa are induced in minimal media and repressed in nutrient media. The mucoid phenotypeof P. aeruginosa has been shownto be due to the production of copious amounts of MEP which is composed of one to four linked moietiesof D-mannuronate and L-glucuronate (26,27). MEP is well known for its abilityto form highly viscous aqueous solutions. Indeed, physicalpropertiescharacterizingpseudomonal MEP include(1)water retention, (2) ion binding, and (3) ability to form gels. The water retention and gel volume are at a minimum when the proportion of glucuronic acid residues is approximately 30%. MEP from mucoid P. aeruginosa always lacks poly-G blocks which have buckled polysaccharide chains and form so-called “egg-carton structures.” Instead, pseudomonal MEP has poly-M
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blocks which have extended “ribbon-like” polysaccharide. Poly” blocks are elastic and have high water-retention capability which is thought to be related to an increased amountof side-chain sugars The presence of 0 side-chain sugarson P . aeruginosa lipopolysaccharide (LPS) imparts hydrophilic properties on this LPS which, in turn, results inthe LPS being a virulence factor which can impart resistance to human serum Among other changes that occurunderstarvationconditions are those associated withthe stringent response The stringent responseis an adaptation to conditions amino acid starvation This response includes induction of specific enzymes such as guanosine tetraphosphate (Gp4) in the initial stagewhich then increases the transcription of both inducible and repressible enzyme operons involved the in stringent response Examples of the stringent response include phenotypic changes seen in marine bacterial cells These changesare characterized by rapid multiple divisions of starved cells leading to the formation of “ultramicropm in diameter) which are also called “dwarf” forms. bacteria” (< Dwarf forms ofP . aeruginosa have been described in microcolonies within the lung tissueof patients with CF Rapid formationof multiple copies is presumed to improve the chances of individual genomes surviving. These cell are dormant forms and are quite resistant to many antimicrobial agents and to osmotic stress. Of interest is that whereas long-term-starved cells are resistant to cellwall active agents as well as agents that inhibit DNA synthesis, they remain somewhat susceptibleto agents that inhibit protein synthesis. This may be due to the fact that maintenance of MEP involves synthesisof the precursors within the cytoplasm, translocation of these precursors to the outer portions of the cell walVmembrane, and final assembly of the biofilm matrix. Studies have shown that MEP maintenanceis dependent on a carbohydrate source, an energy source, certain enzymes, and a functioning efflux pump. There is an increasing body of evidence that macrolides interfere with the synthesis and repair of pseudomonal MEP (43) as well as the elaboration of exotoxins due to their codon-anticodon interactions in which translation of mRNA for inducible enzymes is inhibited. The results of this study confirm earlier reports and, more importantly, demonstratethat the suppressive effectof macrolides on pseudomonal MEP productionof is seen in the phenotypic forms typicallyfound in chronicpulmonaryinfections.Thisstudyalsoillustrates the mediumdependent variability of susceptibility test resultsthat has long been recognized asone of the major problems inherent with in vitro testing. However, if the medium can be selected to promote the growth of those microbial phenotypes actually found in infected patients, the results will be clinically relevant. The suppression of pseudomonal MEP production bymucoid
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strains bymacrolidesoffersafeasibleapproachfor CF patients with chronic pulmonary infections now that newer macrolides with better absorption andfew side effectsare available. Moreover, nonantibiotic effects such as suppressionof the MEP clearly will be clinically importantfor the therapy of other chronicinfectionsinvolvingglycocalyx-encasedsessile forms.
REFERENCES 1. Sawaki M, Mikami R, Mikasa K, Kunimatsu M, Ito S, Narita N. The longterm chemotherapy with erythromycin in chronic lower respiratory tract infections-second report: including cases with Pseudomonas infections. J Japan. Assoc Infect Dis1986; 60:45-50. 2. Goswami SK, Kivity S, Marom Erythromycin inhibits respiratory glycoconjugate secretion from human airways in vitro. Am rev Respir Dis 1990; 141:72-78. 3. Tamaoki J, Takeyama K, Tagaya E, Konno K. Effect of clarithromycin on sputum production and its rheologicalproperties in chronic respiratory tract infections. Antimicrob Agents Chemother1995; 39:1688-1690. 4. Yasuda H, Ajiki Y, Koga T, Kawada H, Yokata T. Interaction between biofilms formed by Pseudomonas aeruginosa and clarithromycin. Antimicrob. Agents Chemother 1993; 37:1749-1755. 5. Ichimiya T, Yamasaki T, Nasu M,In-vitro effects of antimicrobial agents on Pseudomonas aeruginosa biofilm production. J AntimicrobChemother 1994; 34~331-341. 6. Hoiby N, Olling S. Pseudomonas aeruginosa infection in cystic fibrosis.Acta Pathol Microbiol Scand C 1977; 85:197-114. 7. Govan JRW,Hams GS.Pseudomonas aeruginosa and cystic fibrosis: unusual bacterial adaptation and pathogenesis. MicrobiolSci 1986; 3:302-308. 8. Lam JS, Chan R, Lam K, Costerton JW. The production of mucoidmicrocolonies by Pseudomonas aeruginosa within infected lungs in cystic fibrosis. Infect Immunoll980: 28546-546-556. 9. Koch C, Hoiby N. Pathogenesis of cyctic fibrosis. Lancet 1993; 341:1065-1069. 10. Brown DFJ, BrownL. Evaluation of the E test, a novel methodof quantifying antimicrobial activity. J Antimicrob Chemother 1991;27:185-190. 11. Terry JM, Pina SE, Mattingly SJ. Environmental conditions which influence mucoid conversion in Pseudomonas aeruginosa PAOl. Infect Immunol 59 1991; 59:471-477. 12. Govan JRW. Mucoid strains of Pseudomonas aeruginosa. The influence of culture medium on the stability of mucus production. J Med Microbiol 1975; 8513-522. 13. Livermore DM. Penicillin-bindingproteins, porins and outer membrane permeability of carbenicillin-resistant and-susceptible strains of Pseudomonas aeruginosa. J Med Microbioll984; 18:261-270.
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14. Lorian V. In vitro simulation of in vivo conditions: physicalstate of the culture medium. J Clin Microbioll989; 27:2403-2406. 15. National Committee for Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, 3rd ed; Approved Standard M7-A3. Villanova, PA: NCCLS, 1993. 16. Costerton JW, Lewandowski Z, DeBeer D, Caldwell D, Korber D, James G. Biofilms, the customized microniche. JBacterioll994; 176:2137-2141. 17. Magnusson KF. Physiochemical properties of bacterial surfaces. Biochem SOC Trans 1989; 454-458. 18. Costerton JW, LamK, Chan R.The role of the microcolony inthe pathogenesis of Pseudomonas aeruginosa. Rev Infect Dis1983; 5 (suppl):S867-S873. 19. Simpson JA, Smith SF, Dean RT. Alginate inhibition of the uptake of Pseudomonas aeruginosa by macrophages. J Gen Microbioll988; 34:29-36. 20. Meluleni GJ,Grout M, EvansDJ, Pier GB. Mucoid Pseudomonas aeruginosa growing in a biofilm in vitro are killed by opsonic antibodies to the mucoid exopolysaccharide capsule but not by antibodies produced during chronic lung infection in cystic fibrosis patients. J Immunol 1985; 155:2029-2038. 21. Doring G, Goldstein A, Roll A, Schiotz PO, Hoiby N, Botzenhart K. Role of Pseudomonas aeruginosa exoenzyme in lung infectionsof patients with cystic fibrosis. Infect Immunoll985; 4937-562. 22. Giwercman B, Jensen ET, Haoiby N, Kharazmi A, Costerton JW. Induction of p-lactamase production in Pseudomonas aeruginosa biofilm. Antimicrob Agents Chemother 1991; 35:1008-1010. 23. Vishwanath S, Ramphal R. Adherence of Pseudomonas aeruginosa to human tracheobronchial mucin,Infect. Zmmun. 45:197-202 (1984). 24. Davies DG, Chakrabarty AM, Geesey GG. Exopolysaccharideproduction in biofilms: substratum activation of alginate gene expression by Pseudomonas aeruginosa. Appl Environ Microbiol1993; 59:1181-1186. 25. Davies DG, Geesey GG. Regulation of the alginate biosynthesis gene algC in Pseudomonas aeruginosa during biofilm development in continuous culture. Appl EnvironMicrobioll995; 61:860-867. 26. Evans LR, Linker A. Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa. J Bacterioll973; 116:915-924. 27. Sherbrook-Cox V, Russell NJ, Graceas P. The purification and chemicalcharacteristics of the alginate presentin extracellular material produced by mucoid strains of Pseudomonas aeruginosa. Carbohydr Res 1984; 135:147-154. Robertson JA, Trulear MG, Characklis WG. Cellular reproduction and extracellular polymer formationby Pseudomonas aeruginosa in continuous culture. Biotechnol Bioeng1984; 26:1409-1417. structure of lipopolysaccharides from Pseudo29. Wilkinson SG. Composition and monas aeruginosa. Rev Infect Dis1983; 5 (suppl5):S941-S947. 30. Cryz SJ, Pitt TL, Furer E, et al. Role of lipopolysaccharide in virulence of Pseudomonas aeruginosa. Infect Immunoll996; 44508-513. 31. Day DF, Marceau-Day ML. Lipopolysaccharide variability inPseudomonas aeruginosa. Curr Microbioll982; 7:93-98.
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Stratton Gilbert P, Collier PJ, Brown MRW. Influence of growth rate on susceptibility to antimicrobial agents:biofilms, cell cycle, dormancy and stringent response. Antimicrob AgentsChemother Roszak DB, Colwell RR. Survival strategiesof bacteria in the natural environment. Microb Rev Moyer CL, Morita RY. Effect of growth rate and starvation-survival on the viabilityandstability of a psychrophilicmarinebacterium. Appl Environ Microbioll989; 55: WrangstadhM,Conway PL, Kjelleberg S. The role of an extracellular polysaccharide producedby the marine Pseudomonas sp. in cellulardetachment duringstarvation. Can J Microbio Kita E, Sawaki M, Nishikawa F, et al. Suppression of virulence factors of Pseudomonas aeruginosa by erythromycin. J Antimicrob Chemother Molinari G, Guzmhn A, Pesce A, Schito GC. Inhibition of Pseudomonas aeruginosa virulence factors by subinhibitory concentrations of azithromycin and other macrolide antibiotics. J Antimicrob Chemother Mizukane R, Hirakata Y, Kaku M, et al. Comparative in vitro exoenzymesuppressing activitiesof azithromycin andother macrolides antibiotics against Pseudomonas aeruginosa. Antimicrob Agents Chemother Mizukane R, Hirakata Y, Kaku M, et al. Comparative in vitro exoenzymesuppressing activitiesof azithromycin andother macrolides antibiotics against Pseudomonas aeruginosa. Antimicrob Agents Chemother Haight "H, Finland M. Observations on mode of action of erythromycin. Proc SOC Exp Biol Med
8 Effects of Macrolides on Leukocytes and Inflammation Marie-ThCr&seLabro INSERM U294 CHU X.Bichat Park, France
INTRODUCTION Macrolide antibiotics, azalides (a subgroup of macrolides), and streptogramins (MAS) are all avidly concentrated by host cells, a property crucial to their intracellular bioactivity. Another consequence of this cell traping is the possibility to modify host cell functions and transport to the site of infection or inflammation by mobile cells (the basis for the concept of tissue-targeted pharmacokinetics). This chapter will review an important aspect of MAS interaction with host cell functions: their modulatory effects on the inflammatory response.As no data are yet availableon streptogramins in this context, only macrolides will be considered here. After a schematic overviewof the inflammatory response (beneficial anddetrimentalaspects)andrecognizedanti-inflammatoryactivities of various antibacterial drugs, the anti-inflammatory activity of macrolides will be approached on the basis of in vivo models (therapeutic efficacy in humans and animals) andthe underlying mechanisms identified in ex vivo and in vitro studies (e.g., cellular uptake mechanisms, effects on phagocyte functions, cytokine production and other interference with the immune system). A prospective conclusion will be drawn by examining the place of 101
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macrolides in the treatment of inflammatory diseases and looking atthese molecules in the extended continuum of macrocyclic structures which express dual effectson host cells and microorganisms.
INFLAMMATION: FROM THE GREEKS TO THECYTOKINES FromCelsus(30 B.C.-A.D.38), Rubor, Dolor, Calor, Tumor the four cardinalsigns ofinflammation-towhich Galen (or Wirchow?)added Functiolaesa-havestood the test of time.But the fundamentalapproach to the humoraYcellular factors involved in this phenomenon only began in the 19th century with the work of so many illustrious scientists as Cohnheim, Metchnikoff, Ehrlich, Von Behring, and Landsteiner. day, our visionof the scenario of eventswhichoccurseachtimehost integrity is altered by exogenous (mechanical, chemical, biological) factors or endogenous (tumoral, immune) disturbances has reached an extreme degree of complexity,parallel to our better understanding of pathophysiology at the level of cell-to-cell and intracellular communication pathways. It is outside of the scope of this chapter to analyze the successive (sometimes intricate) cascadesof humoral mediators and cellular activities involved in the inflammatory response, which may otherwise be found in various excellent books and papers(1-7). Here, I only will summarizethe cascade of events that may be possible targets for the anti-inflammatory activity of macrolides andother antimicrobials. When microorganisms have succeeded in overcoming the natural host barriers, a localized and beneficial inflammatory response is initiated to prevent tissue damage, destroy the infective pathogen, and activate repair processes. This well-orchestrated sequence of events is known the as acute-phase response. It involves the coagulation system in case of vascular breeches,the complement system (either activated by antigen-antibody complexes,or, in the alternative pathway, by the lipopolysaccharides present on bacterial surfaces) which leads to the formation of various chemoattractantsfor blood cells and mast cell activators (e.g., C3a, C5a) and opsonins (C3b,theiC3b); vasogenic response (increased dilatation and vascular permeability) mediated by histamine released from mast cells andthe kinin cascade; and the cytokine cascade initiated by resident macrophages activated by microbial or altered-tissueby-products. The early“alarm”cytokinestumornecrosis factor-a (TNF-a) and interleukin-l (IL-1) act both locally and distally by initiating “second wave” cytokines suchI Las8 and other chemokines that are highly chemotactic for blood phagocytes, and induce endothelial cell changes such as expression of adhesion receptors.
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All these changes rapidly promote (within minutes) the firm adhesion of neutrophils to the endothelium, followedby diapedesis and migration toward the infected site, where they perform their crucial function in infection (phagocytosis and bacterial killingby highly destructive oxidants, enzymes and antibacterial proteins, and peptides). Also, leukocytes synthesize and release their set of cytokines withinthe target tissue, aswell asmetabolites of arachidonic acid (prostaglandins, leukotrienes, and thromboxan M ) responsible for vasoconstrictionand bronchoconstrictioddilatation and secondary activationof leukocytes. In parallel, systemic inflammation mediated mainly by cytokines (IL-1, IL-6, TNF) alters the temperature setpoint in the hypothalamus (febrile response) and that of metabolism and gene regulation in the liver with production of acute-phase response proteins (a,-acid glycoprotein, C-reactive protein, complement component C3, etc.) and increased leukocytosis (mainly neutrophil leukocytosis). The involvement of blood monocytes and monocyte-derived tissue macrophages in bacterial eradication occurs later. However, these cells are potent producers of cytokine and other inflammatory mediators, which perpetuate the inflammatoryresponse.Exaggeratedinflammatoryreactions seen in the first stage of infection (when the excess of bacterial and cell products upregulate the host response) involve excessive production of reactive oxygen species targeting host cells and tissues, and cytokines which lead to septic shock, the systemic inflammatory response syndrome, and multiorgan failure.At a later stage, phagocytes initiate the second phaseof the immune response by digesting microbial antigens and transmittingthe messages to T cells, which become involved both in the specific immune response and the inflammatory reaction. Cell-mediated immunity is a major mechanism to terminate infection. In summary, the multifactorial nature of the inflammatory process offers an enormous potential range of therapeutic manipulation for its control. Based on the ultimate fundamental knowledge of the transduction pathways involved in the production, activity, and regulation of the various humorallcellular effectors, research is ongoingto widen our antiinflammatory armamentarium.
ANTIBACTERIAL AGENTS AND INF'LAMMATION It is now recognized that antibacterial agents, whose main target is the microorganism, may interfere with host cell functions (8). The many studies which have been performedvitro, in ex vivo, and in vivo have raised the possibility that some antibacterial drugs may indeed alter the inflammatory response. It is well documented that ampicillin, penicillin G and various aminothiazolyl-cephalosporinspossess oxidant-scavengingproper-
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tiesinvitro(9-11),althoughinvivop-lactam treatment may, on the contrary,enhance the inflammatoryresponseinmeningitis or lead to inflammatory sequelae, particularly in otitis. Recently, we demonstrated that OH-iminocephems directly inhibit myeloperoxidase in vitro (12,13), but no data areavailable on the in vivo effects of these drugs in inflammatory settings. Dapsone, clofazimine, and isoniazid also inhibit the myeloperoxidase system in vitro (14-15), and several authors have suggested is linked to that part of their therapeutic activity in mycobacterial diseases their anti-inflammatorypotential.Finally, the ansamycins and the cyclines have proved beneficial in various inflammatory settings not directly linked to infections. For both kinds of drugs, the hypothesis underlying clinical trials was their cellular uptake property and bioactivity on several microorganisms suggested to play a role in the pathogenesis of rheumatoid arthritis. This hypothesis has not been substantiated, but clinical trials have shown that intraarticular rifamycin SV was beneficial in rheumatoid arthritis, ankylosing spondylitis, and juvenile rheumatoid arthritis (16-18). Similarly, cyclines (particularly minocycline) have shown therapeutic benefit in reactive arthritis and mild to moderate rheumatoid arthritis (19-21). It must be noted that outside of their antibacterial activity these drugs have widely acknowledged effectson various inflammation mediators/effectors suchas inhibition of neutrophil functions and oxidant-scavenging properties (ansamycins, cyclines) and inhibition of metalloproteinases and prostaglandin synthesis (cyclines).The question as to whether macrolides could attenuate inflammatoryresponseswereraisedabout 25 yearsago (22-24) (e.g., erythromycin and troleandomycin in asthma, chronic bronchitis, and other inflammatory settings).In the last 5 years, an explosion of clinical trials and fundamental research in vitro and in animal models has again put this new potential for macrolideson center stage.
MACROLIDES AS ANTI-INFLAMMATORY AGENTS? In Vivo Studies Low-dose, long-term erythromycin treatment has beenreported effective in patients with chronic lower respiratorytract disease including diffuse panbronchiolitis (25-27). Diffuse panbronchiolitis is characterizedby chronic inflammation of the respiratory bronchioles and infiltrationof chronic inflammatory cells, accompanied by repeated episodes of respiratory infections, especially due to Pseudomonas aeruginosa, which finally result in respiratory failure. The clinical effectivenessof erythromycin in such pa-
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tients has been suggested to rely not on its antibacterial but on its antiinflammatory properties. In particular, in those patients receiving erythromycin mg/dayfor months), the number of neutrophils and the neutrophil-derived elastolytic-like activity in broncho alveolar lavage (BAL) fluid decreased andthe number of alveolar macrophages increased significantly compared tothe values obtained beforetreatment. Ampicillin (1 g/day) did not modify these parameters (28). A decreased number of neutrophils in BAL fluid of erythromycin-treated patients with diffuse panbronchiolitis was also observed another in study The authors found that neutrophil chemotactic activity of BAL fluidwasalsosignificantly decreased after a 4-week treatment with erythromycin. In addition, the intrapulmonary influx of neutrophils triggeredby intratracheal injection of IL-8 or lipopolysaccharide (LPS)was suppressed in mice given erythromycin mg/animal) h beforethe intratracheal challenge. Recently, Shirai et al. have reported that roxithromycin or mg) and clarithromycin or mg) given daily for at least months are as effective as erythromycin or mg) in diffuse panbronchiolitis (efficacy in and respectively) and in bronchiectasis (efficacy about Furthermore, chemotaxis of blood neutrophils from patients with diffuse panbroncholitis was significantly reduced after roxithromycin therapy. It has also been shownthat erythromycin and troleandomycin favorably affect the clinical status of patients with severe steroid-dependent asthma An effect of macrolides on theophylline or corticosteroid clearance has been postulated to contribute to their beneficial actions. Macrolide interactionswith various processess involved the in pathogenesis of asthma [e.g., bronchial inflammation and bronchial hyperreactivity is an area of active investigation (see below). The anti-inflammatory activity of macrolides has also been investigated in animal models. Erythromycin proved beneficial in various acute mouse ear inflammation models Similarly, inrats, roxithromycin (one dose of or m a g ) wasaseffective as indomethacin (5 m a g ) in reducing hind-paw edema induced by poly-L-arginine, but slightly less effective in-the L-carrageenan hind-paw edema model and in ear edema induced by croton oil in contrast, this macrolide mg/kg/day for days) was not active in a chronic inflammation model (polyester sponge implantation) Other animalmodelshavebeenused to study the protection afforded by erythromycin and roxithromycin mg/kg/day for days) in a model of endotoxin-induced vascular leakage and neutrophil accumulation inthe rat trachea The mechanism(s) involved in these anti-inflammatory activities have not been elucidated. According to Agen et al. the anti-inflammatory
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activity of roxithromycincouldbedue to a preferential action on the vasogenic component ofthe inflammatory response. However, Tamaokiet al. (37) suggested an effect of macrolides on neutrophil chemotaxis and/or activation. Thiswas also proposed in the model of zymosan-induced (aseptic) peritonitisof mice, where long-term administration of erythromycin (10 mgkg for 28 days) was as effective as dexamethasone (40pg for2 days) in reducing intraperitoneal extravasation and leukocyte (mainly neutrophil) accumulation, as well as prostaglandin E, (PGE,) production (38). That macrolides may modify immuneparameters relatedto inflammation was also suggested by various animalhuman models in which their administration resultedin the ability of immune cells to synthesize inflammatory/anti-inflammatory mediators, particularly cytokines, ex vivo. The mononuclear cells of volunteers receiving azithromycin 500 mg/day for 3 days released an increased amount of soluble IL-2 receptor after phylohemagglutinin (PHA) and phorbol myristateacetate (PMA) costimulation (39). The blood mononuclear cells of volunteers receiving erythromycin (600 mgfor 5 days) had an enhanced capacity to produce IL-la and TNF-a after stimulation with PMAor interferon-y (IFN-y), whereas IFN-yproduction was reduced (40). Ex vivo modification of cytokine production has also been reported in mice. Erythromycin stearate (10 mgkg/day for 28 days) increasedthe production of IL-1 by peritoneal macrophages and IL-2 by spleen cells (41). Similarly, in mice receiving roxithromycin mgkg for 28 days), IL-1and TNF-a production by LPS-stimulatedperitoneal exudate cellswasincreased, aswas that of IL-2, IL-4, andIFN-y by mitogen-stimulated spleen cells (42). Konnoet al. (43,44) observedthat LPS-stimulated resident peritoneal macrophages of mice receiving roxithromycin (5 mg/day) produced more IL-1 than controls on days 7-14 and 28, but on day 42, the production was decreased. Similarly IL-2 production by Con A-stimulated spleen cells or mesenteric lymph node cells was significantly increasedon day 14 and reduced on day 42. IL-5 production was significantly reduced throughout the test period (days 7 to 28). Finally Hirakata et al. (45) observed that peritoneal macrophages from mice receiving erythromycin, mgkg 250 for 7 days, produced significantly greater amounts of thymocyte-activating factors, possibly IL-1 and IL-6. Another immune parametermodified ex vivo by macrolide treatment is the motility of human neutrophils,which was increased after erythromycin treatment in healthy volunteers and in patients with defective chemotaxis (46-47). On the other hand, we have observedthat roxithromycin (300-mg single dose) increased various functions of human neutrophils90 min after ingestion (48), but multiple administrations (300 mg/day for 7 days) resul in a decreased oxidant production in fiveout of six neutrophil samples (49).
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In Vitro Studies Although some data indicate that certain macrolides may interfere with various pathophysiologic processes [for instance, inhibition of cholinergic neuroeffector transmission inthe human airway smooth muscle (50)], I will focus on the numerous aspects of macrolide interference with phagocytes which are corner stones inthe cascade of events associated with beneficial and detrimental aspects of inflammation. Because mostof the macrolideinduced modifications are thought to rely on their phagocyticuptake, this important property will be discussed first in relation with new findings made inmy laboratory. In Vitro Uptake of Macrolides All macrolides display intracellular (phagocytic) accumulation, with cellular to extracellular ratios greater than 20 (51). The precise mechanism underlying this cellular uptake is poorly understood. Trapping by protonation of these weakly basic molecules may explain their intragranular accumulation, particularly in the case of dibasic compounds (azithromycin, dirithromycin). We have recently comparedthe uptake of erythromycin A derivatives for their accumulation in human neutrophils and proposed a preliminary classificationof these compounds basedon their cellular kinetics, efflux from loaded cells, intracellular location, and activation energy (52) (Table 1).We also demonstratedthat extracellular Ca” channel regulating intracellular calcium homeostasis(53). This exchanger is unlikely to Table l Preliminary Classification of Erythromycin A Derivatives Accordingto Uptake by Polymorphonuclear Neutrophils (PMN) Groupkompounds
I:
Azithromycin Dirithromycin Erythromycylamine
11: Erythromycin A
Roxithromycin Clarithromycin
Characte Dibasic drugs Nonsaturable uptake Preferential intragranular location8 AG > kJ/mole Moderate efflux from loadedcells Monobasic drugs Rapid uptake + plateau Bimodal (granulekytoplasm) location AG 60-70 kJ/mole Rapid efflux
-
Erythromycylamine is located in cytoplasm and granules (poor liposolubility hinders membrane permeability).
a
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be the macrolide channel. Preliminary data from our laboratory suggest that PKA-mediated phosphorylation is critical for neutrophil uptake of erythromycin A-derived macrolides (54). Mod$c&n
of Phagocyte Functions by Macrolidees
The possibility that macrolidesdirectlymodifyphagocytefunctions,as suggested byinvivoandexvivo studies, has been widely investigated (reviewed in Ref. 55). In particular, many data suggest that erythromycin A derivativesimpairoxidantproduction by phagocytes(e.g., neutrophils) in a time-dependent and concentration-dependent manner and that thiseffectmaybeobtainedattherapeuticallyrelevantconcentrations (56-58). The mechanism has not been elucidated. Although Mitsuyama et al. (58) suggested that erythromycin-induced inhibition of the neutrophiloxidativeburstismediatedthroughPKAactivation,theydidnot check the importance of PKA for macrolideuptake.Perry et al.(59) have clearly shown the importance of the phospholipase D and phosphatidate-phosphohydrolase transduction pathway in roxithromycin-induced inhibition of neutrophil NADPH oxidase, the crucial enzyme in oxidant production by phagocytes. Parallel to their inhibitory effect onthe oxidative burst, erythromycin A derivatives have a stimulating effecton neutrophil exocytosis (60-62). This activity may serveto explain, partlyat least, the intracellular bioactivity of macrolides on some microorganisms which inhibit phagolysosome fusion; it does not ruleout potential anti-inflammatory activity, as various anti-inflammatory drugs (chloroquint, amodiaquine, staurosporine, etc.) impair oxidant generation while inducing neutrophil exocytosis. It must be noted that 16-membered-ring macrolides do not display these two neutrophil-modulating activities; onthe contrary, josamycin has been reported to enhance oxidant production by phagocytes, an activity suggested to underly its inhibitory effect on antibody production (63). The in vitro effect of macrolides on cytokine production has been also demonstrated. Spiramycin at the high concentrations of10-50 mgL enhanced IG6 production by LPS-stimulated human blood mononuclear cells (64) and josamycin > midecamycin > clarithromycin decreased IL-2 production in these cells (65). This effect was additive withthat of FK 506 and cyclosporin A. Clarithromycin > erythromycin decreased IL-1 production by LPS-stimulated peritoneal macrophages of mice (66), and roxithromycin (2-10 m&) decreased that of TFN-a bymouse spleencells and macrophages (42). This latter effect was suggested to involve increased IL-4 production. Recently, Takizawa et al. (67) demonstrated that erythromycin was also able to suppress IL-6 expression by human bronchial epithelial cells.
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Other interactions of macrolideswithvariousparameters of the inflammatory response have been described. Briefly, erythromycin (1-10 m&) has been reported to shorten neutrophil survival by accelerating apoptosis (68), to promote monocyte to macrophage differentiation and to inhibit respiratory glycoconjugate secretion from human airways (70),whereasroxithromycinhasbeenshown to inhibitcell cycle progressionin HL cells (71) and T-lymphocyte transformation induced by mitogenstimulation(72).Furthermoreithasbeenshown that erythromycin may also alter various virulence factors of Pseudomonas aeruginosa, apathogenofteninvolvedininfectionsinpatientswithpanbronchiolitis (45).
CONCLUDING REMARKS Whatever the in vivo mechanisms(effectson phagocyte numbers and functions, cytokine production, or other inflammatory parameters) there is a worldwide consensusthat in additionto their antibacterialproperties, some macrolide antibiotics are also endowed with anti-inflammatory activity. The host target in most studies isthe phagocyte, which is a cornerstone in host defenses and inflammation, whereasthere is no concern for a beneficial anti-inflammatory activityof macrolides in the case of acute infection with exaggerated destructive inflammatory responses. What wouldbe the consequences of long-term use of these drugs in inflammatory diseases (asthma or panbronchiolitis)? The specter of increasing microbial resistance is a major limitation. Second, anti-inflammatory drugsare also potentially immunosuppressive. What might be the consequences on host natural defences themselvesof uncontrolled useof macrolides in inflammatory diseases? Indeed, there are experimental data showing that erythromycin may induce the suppression of pulmonary antibacterial defences, a potential mechanism of superadded infection (73).It is not easyto answer these questions, but I advocate careful management of clinical trials and extensive cooperation between clinicians and fundamentalists to elaborate new prospects. In particular, elucidation of the structures and mechanism underlying macrolide anti-inflammatory activity will be crucial to develop new therapeutic drugs. The extended classificationof macrolides fromthe classical definition of Woodward (Fig. 1) presents a vast continuum of macrocyclic lactonic structures in which some molecules are mainly antibacterial (true macrolides), whereasothers possess mainly immunosuppressant activity (FK 506, rapamycin) or even antifungal activity with host cell inhibitoryproperties (bafilomycin, concanamycins).
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Macrolides True .
Lankacidins (17)
FK 506 (23)
Rapamycin (311
No Sugars Immunosuppressants
Antibiotics Antifungals
(Antifungals Immunosuppressants) Antibiotics
+
(MIP-producing organisms) Inflammation
Figure I “True” macrolidesare characterized by a 12-to 16-membered ring macrocyclic lactone nucleus with few (if any) double bonds and no nitrogen atom, substituted by several aminoand/or neutral sugars. Recently, azalides were proposed as a new subgroup of macrolides andare characterized by the presence of a nitrogen in the lactonic ring. All these molecules display a homogeneous antimicrobial spectrum. Another subgroup in the macrolide family corresponds to the bafilomycins and concanamycins, which are not therapeutically used, owing to their strong immunodemessant potential related to the property of inhibitingmammalian V-typeH+ATPases, which are crucialin regulating intracellular pH. An extended definition of macrolides will include lankacidins (which are active against microorganisms but do not possess glucidic substituents) and the macrocycliccompounds, FK 506, rapamycin,and their derivatives (which are mainly immunosuppressants). New therapeutic potentials are looked for throughout this vast continuum of macrocyclic compounds with the development of new antimicrobial drugs in the group of rapamycin derivatives and that of the antiinflammatory properties of the macrolide derivatices. The number atoms the lactonic ring given inparentheses. Abbreviations:MIP: Macrophage infectivity potentiating factor (immunophih);organisms which produce these factorsinclude Neisseria, Chlamydia, and Legionella.
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Understandingthe mechanisms that confer on these chemically related molecules different degrees of immunosuppressantlantimicrobial activity will help to define the optimal structures for both therapeutic properties.
Z like the dreams of the f i t w e better than the history of the past. Thomas Jefferson
(letter to J. Adams).
ACKNOWLEDGMENT The author thanks Miss Fr. Breton for her expert secretarial assistance.
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57. Anderson R. Erythromycin and roxithromycin potentiate human neutrophil locomotion in vitroby inhibition of leukoattractant-activatedsuperoxide generation and auto-oxidation.J Infect Dis1989; 159:966-973. 58. Mitsuyama T, Tanaka T, Hidaka K, Abe M, Hara N. Inhibition by erythromycin of superoxide anion production by human polymorphonuclear leukocytes through the action ofcyclic AMP-dependentproteinkinase. Respiration 1995; 62:269-273. 59. Peny DK, Hand WL, Edmonson DE, Lambeth JD. Role of phospholipase D derived diradyl glycerolin the activation of the human neutrophil respiratory burst oxidase.J Immunoll992; 149:2749-2758. 60. Abdelghaffar H, Mtairag EM, Labro MT. Effect of dirithromycinand erythromycylamine on human neutrophil degranulation. Antimicrob Agents Chemother 1994; 39:1548-1554. 61. Labro MT, Abdelghaffar H, BryskierA.Effect of macrolides on human neutrophil degranulation. 33d ICAAC, 1993; abstr 309. 62. Labro MT, Abdelghaffar H, Douhet C, Bryskier A. Investigation of the mechanism underlyingthe stimulation of neutrophil exocytosisby macrolides. 35th ICAAC, 1995; abstr G39. 63. Villa ML, ValentiF, Mantovani M, ScaglioneF, Clerici E. Macrolidic antibiotics: effects on primary in vitro antibody responses.Int. J. Immunopharmacol. 1988; 10~919-924. Bailly S, Pocidalo JJ, Fay M, Gougerot-Pocidalo MA. Differential modulation of cytokineproduction by macrolide:interleukin-6productionisincreased by spiramycinanderythromycin.Antimicrob Agents Chemother 1991; 35~2016-2019. 65. Morikawa K, Oseko F, Mirikawa S, Iwamoto K. Immunomodulatory effects of three macrolides, midecamycin acetate, josamycin and clarithromycin on human T-lymphocyte function in vitro. AntimicrobAgents Chemother 1994; 38~2643-2647. 66. Takeshita K, Yamagishi I, Harada M, Otomo S, Nakagawa T, Mizushima Y. Immunological and anti-inflammatory effectsof clarithromycin: inhibitionof interleukin 1 production of murine peritoneal macrophages. Drugs Exp Clin Res 1989; 15527-533. 67. Takizawa H, Desaki M, OhtoshiT, Kikutani T, Okazaki H, Sat0 M, Akiyama N, Shoji S, Hiramatsu K, Ito K. Erythromycin suppressesinterleukin-6expression by human bronchial epithelial cells: a potential mechanism of its antiinflammatory action. Biochem BiophysRes Commun 1995; 210:781-786. 68. Aoshiba K, Nagai A, Konno K. Erythromycin shortens neutrophil survival by accelerating apoptosis. Antimicrob Agents Chemother 1995; 392372477. 69. Keicho N, Kudosh S, Yotsumoto H, Akagawa KS. Erythromycin promotes monocyte to macrophage differentiation. Antibiot 1994; 47:80-89. Erythromycin inhibits respiratory glyco70. Goswami SK, Kivity S , Marom conjugate secretion from human airways in vitro.Am Rev Respir Dis 1990; 141:72-78. 71. Nagai M, YamadaH, Nakada S, Ochi K, NemotoT, Takahara S, Hoshima S, Horiguchi-Yamada J. Amacrolideantibiotic,roxithromycininhibits the
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growth of human myeloid leukemia HL 60 cells by producing multinucleate cells. Mol Cell Biochem 1995; 144:191-195. 72. Konno S, Adachi M, Asano K, Okamoto K, Takahashi T. Inhibition of human T-lymphocyte activation by a macrolide antibiotic, roxithromycin. Life Sci 1992; 51~231-236. 73. Nelson S , Summer W, Terry PB, Warr GA, Jakab GJ. Erythromycin-induced suppression of pulmonary antibacterial defenses. Am Rev Respir Dis 1987; 136~1207-1212.
Animal Usesof Macrolides and Related Antibiotics Jean-Pierre Lafont
Elisabeth Chaslus-Dancla
Institut Nationalde la RechercheAgronomique Nourilly ,France
Jean-Louis Martel Centre National d'Etudes Vktkrinaires Alimentaires-LYON et Lyon, France
ANIMAL USES OF MACROLIDES AND RELATED ANTIBIOTICS Macrolides and related antibiotics (MRAs) are widely used in the various animal sectors, fortwo main purposes: therapy and growth promotion.
Growth Promotion This particular useof antibiotics originates in experiments dating from the early fifties (1) demonstrating that the addition of very low doses in feed has stimulating effects on animal growth and, more generally, on animal production (e.g., eggs,milk).Thiseffectwasshown to result from increased feed efficiency,due to modifications of bacterial metabolism in the presence of subinhibitory concentrationsof antimicrobials inthe intestinal tract. Despite a sizable amount of research done by animal nutritionists, the mechanisms of this growth promotion have not been clearly elucidated
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and the bacterial targetsare still unidentified.The best documented mechanisms are reduction of the microbialproduction of growthdepressing metabolites, in the microbial destruction of essential nutrients such as amino acids in the intestinal tract, enhanced efficiency of absorption of nutrients due to a thinner intestinal wall,and sparing of nutrients for the host because of a lower turnover of the intestinal epithelium in treated animals (2). It should be noted in this respect that germ-free animals frequently exceed conventional controls in growth rate This nontherapeutic use of antibiotics in animal husbandry became widespread and controversial (4). Public health concerns led the agreement commissions of the European Union (EU) to restrict the list of substances authorized in this respectto specific families not (e.g., flavophospholipol) or scarcely (e.g., bacitracin) employed in animal or human therapy and unlikely to select for transferable drug resistance of medical importance. Tetracyclines and penicillins, for instance, were banned from this use in the EU, whereas certain MRAsare among the few exceptions to this rule. In France, spiramycin, tylosin, and virginiamycin are therefore used as growth- (and production-) promoting feed additives in birds, pigs, and ruminants. The doses (usually5-50 grams per ton of feed) are well below therapeutic doses. The selective effectof such low doses of MRAs hasnot been conclusivelydemonstrated, but this question remains open.Another undesirable consequence of long-term growth promoting supplementation of feed with MRAs could be the impairment of the “barrier effects” (“resistance to colonization,” “competitive exclusion”) exerted by the normal intestinal microflora of animals, especially poultry, againstSalmonella (5). The most dependable experimentson this topic suggestthat virginiamycin does not increase Salmonella shedding in animals, whereasan increase in shedding canbe occasionally observed with tylosin (6-9).
Therapy Macrolides and related antibioticsare invaluable therapeutic agents in animals. This results from their activity against prominent bacterial pathogens of animals, chiefly Mycoplasma (e.g., M . gallisepticum, M . iowae, M . meleagridis, and M. synoviae in birds, M. hyosynoviae and M. hyorhinis in pigs, M . agalactiae, M. capricolum, M . dispar, and M . mycoides in ruminants, etc.)but also Pasteurella, the trepanematous agentSerpulina hyodysenteriae, gram-positive cocci, and forth. The relative absenceof toxicity or adverse effectsof most MRA drugs in animals [except in horsesand in rabbits (lo)] and their low prices, at least for the older molecules of the family,which are the most extensively used (erythromycin, spiramycin, tylosin), have also fostered their use in veterinary medicine. Macrolides
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and related antibiotics are thus used under veterinary prescriptionto treat respiratory, intestinal, systemic, genital, and skin infections in most animal species (11). The molecules used in France and in European countries belong to the macrolide group with both 14-membered ring (erythromycin, oleandomycin)and16-memberedring(spiramycin,tylosin,tilmicosin, josamicin) molecules, to the lincosamides (lincomycin, and clindamycin in dogs andcats), and to the pleuromutilin group (tiamulin). This last group is specific to veterinary medicine andis tentatively presentedhere along with the MRAs because. its therapeutic uses are similar, targeted at the same infectious microorganisms(Actinobacillus, Mycoplasma, Pasteurella, Serpulino), but its structure with a cyclopentacyclooctene ring differs noticeably from that of the other MRAs. Streptogramins are no longer intherapeutic useandazalides are absentfrom the veterinarymarket.Tilmicosin, a semisynthetic derivative of demyracosyltylosin with an improved activity against Pasteurella species responsible for pneumonia cattle in and pigs, has been recently introduced asa new veterinary antibiotic (12). Depending on the drug, administration is by the oral andor parenteral routes with, in addition, pharmaceutical preparations proper to veterinary practice: intramammary suspensions used to treat or prevent mastitis, and medicated feed madeby mixing concentrated “premixes”to the other feed components. Medicated feed is distributed for curative purposes but also for prevention in large populations of intensively reared farm animals exposed to a risk of infection (e.g., at the onset of the first symptoms ina few individuals);the concentrations are in the therapeutic range (e.g., 400 grams/ton or ppm). Some preparations associate a MRA with another antibiotic substance; this takes into consideration the frequent involvement of severalpathogens inmanybacterialdiseasesofanimalsofwhich colisepticaemiaandchronicrespiratorydisease of poultry,associating Mycoplasma gallisepticum and pathogenic strains of Escherichia coli, are typical examples. Antibiotic use in animals is ruled by a vast array of national and European regulations (13). In particular, these regulations impose withdrawal periods before marketing, to avoid the presence of residuesin human food products coming fromtreated animals. The administration to laying hens of drugs liable to be excreted in eggs (14) is banned.
MICROBIAL RESISTANCE Evolution of Susceptibility in Bacterial Pathogens The evolution of susceptibility to MRAs has not been the subject of extensive work in veterinary medicine. Therapeutic failures are not commonly
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reported by fieldpractitioners, andthe major speciesof Mycoplasma which cause diseases frequently requiring MRAs treatments appear as remaining clinically susceptibleto the drugs Resistant mutantsof M . mycoides can be obtained in several steps in vitro However, although minimal inhibitory concentrations (MICs) of the MRAs can be higher for recent avian isolates than for reference strains isolated a long the time microorago, (18). ganisms still can often be considered as susceptible This situation concerning the MRAs is in contrast with an increase in resistance to the tetracyclines reported in MRA-susceptible bovineMycoplasma species The existence of resistant isolates has been reported in many other bacterial pathogensof animals such as Actinobacillus (formerly Haemophilus) pleuropneumoniae Actinomyces (formerly Corynebacterium) pyogenes Campylobacter coli Clostridium per,fringens various coagulase-negativeStaphylococcus species Staphylococaureus Staphylococcus epidermidis Staphylococcus hyicus Staphylococcus intermedius Streptococcus suis Streptococagalactiae, S . dysgalactiae, S. uberis and Rhodococcus .equi but not in Corynebacteriumpseudotuberculosis Serpulina hyodysenteriae appears as intrinsically resistant to macrolides and lincosamides but susceptible to tiamulin and Haemophilus somnus and Renibacterium salmoninarum appear as generally susceptible species. If the existence of resistant strains is well established for many species of pathogenic microorganisms of animals, the evolution of this resistance has not been systematically surveyed. Few specific studies have been devoted to this topic, and they have yielded somewhat discrepant results which need further clarification by more systematic investigations. Mostof these studies deal with a limited number of isolates, and the samples of strains have not been collected in a systematicway over the years to avoid biases in comparing these samples and in concluding about the evolution of the bacterial species considered. Although resistant strains do exist, gram-positive cocci involved in bovine (and small ruminant) mastitis remain generally susceptible whereas resistance is frequently observedStaphylococcus in hyicus or Streptococcus suis frompigs A possible evolution toward increased resistance has been reported for Campylobacter, by comparing pig and human isolatesof C . coli It should be notedhere that MRAs are not commonly used to treat animal Campylobacter infections. In Pasteurella, a 4-year survey of antimicrobial susceptibilitytrends of bovine isolates from the United States and Canada ledto the conclusion that the proportion of resistant strains had increased, but wide variation in the year-to-year results was observed By contrast, results obtained in France by the
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network monitoring antibiotic resistance in bovine pathogens (40,41) does not point to such atrend, with samplesof strains of similar size. The selective effect of treatments in animals can be easily expected and has been reported in field conditions (42). However, experimental studies in controlled conditions are generally lacking and it is difficult to appreciate the actual contributionof selective pressure to the evolution of resistance to the MRAs. Thus, it is presently impossible to establish whether resistance to these drugs in animals constitutes a public health problem. One has to recall in this respect that resistance to tylosin, a macrolide widely used in animal production, is very rare in human isolatesof Staphylococcus aureus, Streptococcus pneumoniae, and Campylobacter coli in many countries (43). A staphylococcal isolateof possible human origin was isolated fromthe udder skinof cows byPereira and Siqueira-Junior(38), and Saikiaet al. (44)reported the isolation of macrolide-resistantandlincosamide-resistantstaphylococci from cowsreared in a closed flock (taking no replacement other fromfarms) in whichthere was no useof any antibiotic, in a country (India) where none of the MRAs is usedfor treatment or growth promotion. Exchange of resistant strains between man and animals is, thus, also highly probable, but its significance for veterinary medicine is still unknown.
Genetic Supportof Resistance Few studies have been conducted on the genetic support of MRA resistance in veterinary medicine.Such studies are required for a better understanding of the public (and veterinary) health significance of this resistance: among pathogenic microorganisms causing diseases treated by MRAs in animals, none is a prominent zoonotic agent. Species of gram-positive cocci are generallyhost-specificand the critical :risk here is the selection of resistance determinants liable to be exchanged between animal and human pathogenic bacteria. Although MRAs are not first-choice antibiotics for the treatment of human listeriosis, the appearance of macrolide-resistant strains of Listeria monocytogenes may be of particular concern in this respect. These strainsare multiresistant and their multiresistance is encoded by plasmids bearing the ermB gene in addition to resistance genes for tetracycline/minocycline, streptomycin,andchloramphenicol (45). The presence of similar plasmids, showing extensive homology with plasmids from Streptococcus agalactiae, is established in L. monocytogenes strains from different countries (46). Although such strains are infrequently encountered (47), it is not known whether they were selected in animals, and particular attention should be paid to resistance in veterinary isolates of Listeria: resistance plasmids can be acquired readily from Enterococcus or
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Streptococcus donors in this species (47,48). Resistance to the macrolidelincosamide-streptogramin ( M U ) in gram-positive cocci from animals is generally constitutive and can be plasmid mediated (29,49,50). Some of theseplasmidsfrom Staphylococcus hyicus are close to plasmidsfrom strains of human origin (49). However, interspecific conjugal transfer of the resistance is often not obtained from staphylococcal strains isolated from bovine mastitis (51). Medical concerns arise here mostly from results suggesting that resistant Enterococci from animal sources could colonize humans (52,53) and thereby exchange transferable determinants of resistance in this occasional host.
Mechanisms and Determinants of Resistance Several mechanisms of resistance to the MRAs are known in various species of bacteria (54-56). All have been initially described from medical microorganisms of human origin. Whereas intrinsic resistance due to outer membrane impermeability is a general characteristic of gram-negative bacteria, three main mechanisms of acquired resistance have been reported: posttranscriptionalmethylation of. ribosomalRNA(target protection), modification inactivatingthe drug(s), and active.efflux. All have been documented in bacteriaof animal origin,by hybridization with geneor oligonucleotideprobes,obtention of PCRproducts withspecificprimers, or biochemical identificationof the mechanism. Here again, one can deplore the absence of systematic studies in veterinary medicine.The distribution of r-RNA methylase genes in bacteria causing disease in animals has received the attention of severalgroups:mostdeterminantsidentifiedin gram-positive cocci correspond to genes belonging to hybridization classes ermB and ermC (30,31,57,58). In contrast to human medicine, genes belonging to hybridization class..ermAappear as very rare in animal strains being far reported from one Staphylococcus strain only(S. intermedius), isolated from a dog (the samestrainalsohybridizedwith ermB) (58). Sequences homologous to Tn917, a well-characterized transposon from a human Enterococcusfaecalis strain, are present on plasmids of Enterococisolated from healthy chickens and pigs, which suggests that ermBrelated genes can spread by transposition in fecal streptococci of animals (59). Homologies with Tn916, the prototype of which does not bear MLS resistance, have also been recorded in macrolide-resistant plasmids from veterinary gram-positive cocci (50).The ermP determinant ( e m A M class) has been detected in animal erythromycin-resistant isolatesof the toxinogenic anaerobic speciesClostridium perfringens (60). The linA gene encoding 4-lincosamide-O-nucleotidyltransferase has been identified inStreptococcus uberis strains from dairy cows (61), andthe
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msrA gene encoding an active efflux system has been detected Staphyloin coccus strains from dogs and pigs (58). It should be stressed that these studies rest on a limited number of strains and can be considered only as occasional and welcome forays intothe question. As yet unidentified erm determinants encoding potentiallynew methylases are probably present in animal strains (27,28,58).
SPECIFIC VETERINARY PROBLEMS In veterinary aswell as in human medicine, the interpretation antibioticsusceptibility testing follows rules edicted by specific ad hoc scientific committees such asthe CA-SFM in France orthe NCCLS in the United States. This interpretation, categorizing strains as susceptible, intermediate, or resistant, is based on bacteriological, pharmacological, and clinical considerations. Veterinary laboratories carryout antibiograms and in vitro methods of susceptibility testing according to these rules and are subjected to qualitycontrolswithreference strains, usually E. coli and S. aureus (40,41). However, some specific veterinary pathogens such as Pasteurella or Actinomyces pyogenes (21) require the addition to culture media of growth factorswhich mayinterfere with diffusion.International agreement about well-characterized reference strains of these particular species would be needed for more accurate interpretation susceptibility testing. The pharmacological data used for interpretation should be specific to the target animal species for new molecules proper to the veterinary market such tilmicosin (62). But for old antibiotics nowin common use and no longer covered by patents, no pharmaceutical firm is willing to embark on costly pharmacological studies in several animal species, and the data used, as recommended by the official committees, are frequently those obtained in man. Thesedata may not be appropriate for veterinary medicine, which may explain some discrepancies between the results of susceptibility testing inthe laboratory and clinical results inthe field. The use breakpoints recommended by the official committees may not be relevant to veterinary situations, even withdrugsforwhichveterinary pharmacological data are available. This is especiallytrue with macrolides such as spiramycin for which, asfor azalides the use of blood levels as breakpoints for susceptibilitywould appear to be inappropriate: levels of drugs at the tissue site of infection could be a better guide to predicting efficacy. In all the animal species studied, including man, spiramycin concentrations reachvery high levels, far in excess of serum levels, in particular organs such as the lung (64,65). In bovine medicine, most Pasteurella strains should be considered as resistant according to the recommended breakpoints. However, lung levels in calves are very high, exceeding for
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long periods of time the MICs of most strains, and good clinical results are obtained with this antibiotic in treating infections the lower respiratory Better tissue penetration could also tract of calves due to Pasteurella be responsible for better in vivo activity in the female genital tract (67). This is moreor less true of other macrolide antibiotics, and the definition of specific veterinary breakpoints should be considered here
CONCLUSION AND PROSPECTS In veterinary medicine, MRAs have probably not received the attention they deserve, especially as far as bacterial resistance is concerned. Thisis probably due to the generally satisfactory results reported by field therapists with the use of these antibiotics in most animal species. Although resistant strains are known, the investigation of resistance has not been given a high priority by the small number of veterinary microbiologists involved in this field of research. MRAs were considered as raising no particular problem concerning public health: no prominent “life-saving” molecule usedin hospitals belongedto this class, and most target veterinary microorganisms did not represent a direct threat to human health. Their rational use could thus simply derive from the general recommendations and codes for good practices about antimicrobial use in animals expressed by registration expert groups(68). The situation may change with the new molecules this class introduced in human medicine, suchthe asazalides, requiringmore attention aboutveterinaryuseanditspotentialconsequences. In this respect, several questions would have to be answered: 1. Does the prolongeduse oflow doses of MRAs asgrowthpromoting feed additives contribute to the selection of resistant strains? Even if they are not the mostcontroversialgroupof antibiotics in this respect, MRAs require specific experimental studies here. 2. Is there a definite trendof certain veterinary pathogens toward increased resistanceto the MRAs? Veterinary surveillance networks (40) could be focused on this question, and pharmaceutical firms should work on this problem, especially regarding post marketing surveillance of molecules which have recently come the intoveterinary market.It would be of specific veterinaryinterest to consider the situation in major Mycoplasma species, and Campylobacter should be specifically surveyed with particular attention (69). Are intestinal gram-positive cocciof animal origin liable to carry transferable resistance determinants, ableto colonize the intestinal tract of humans, and transfer their resistance to indigenous
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strains or to cause human infection directly? Both experimental and genetic studies using the all modem tools of genomic analysis are needed to establish this most important point. 4. Forparticularclinicalsituations(e.g.,therapy of bovinepasteurellosis), should we use levels of drugs at the tissue site of infection as guidesto predicting efficacy rather than breakpoints for susceptibility deducedfrom blood levels?
ACKNOWLEDGMENTS The authors thank R. Leclercqforcriticalreading of the manuscript. Thanks are also due to G . Menou and P. Maillard for their help in the search for the relevant literature, and to F. Tardo-Din0 for typing.
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62. Gourlay RN, Thomas LH, Wyld SG. Effect of a new macrolide antibiotic (tilmicosin) on pneumonia experimentally induced in calvesby Mycoplasma bovis and Pasteurella haemolytica. Res Vet Sci 1989; 47:84-89. 63. Neu HC. Clinical microbiology of azithromycin. Am J Med 1991; 91 (suppl A):12S-l8S. Osono T, Umezawa H. Pharmacokinetics of macrolide$, lincosamides and streptogramins. J Antimicrob Chemother1985; 16 (supplA):151-166. 65. Rolin 0, Bouanchaud DH. ActivitB antipneumococcique de l’drythromycine et de la spiramycine dans deuxmodtles expdrimentaux chez la souris. Pathol Biol 1987; 35742-745. B, Van Goo1 F,Bayle R, Libersa M, Espinasse 66. Alzieu JP, Bichet HJ, Levrier Efficacy and long-lasting activity of spiramycin in young beef cattle with infections enzootic broncho-pneumonia. Bovine Practitioner 1989; 24:38-41. 67. Cester CC, Laurentie MP, Garcia-Villar P, Toutain PL. Spiramycinconcentrations in plasma and genital-tract secretions after intravenous administration in the ewe. J Vet Pharmacol Therap 1990; 13:7-14. 68. Espinasse J. Responsible useof antimicrobialsin veterinary medicine: perspectives in France.Vet Microbioll993; 35:289-301. 69. Kerr R, Moore JE. Potential for erythromycin resistancein porcine Campylobacter species. Trends Microbiol 1995; 3:440.
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l0 The New Macrolidesfor Treatment of Pediatric Infections: Roundtable Discussion George H.McCracken, Jr. University of Texas SouthwesternMedical Center at Dallas Dallas, Texas
Urs B. Schaad University of Basel Basel, Switzerland
M.J. Tarlow Birmingham HeartlandsHospital Birmingham, United Kingdom
Dr. McCracken: In this discussion we will present various aspects of the use of the macrolides in pediatrics. At the outset, wewill not mention every study of every drug, although some reference will be madeto the studies presented inthe poster sessions. As a general dictum,what applies to one macrolide appliesto the others as well, althoughthere are very specific differencesthat distinguish them. I would liketo provide a brief introduction on these drugs, reviewing their in vitro susceptibilities as they apply to pediatrics and, then, their pharmacokinetics in infants and children. 131
I32 Table l
McCracken et al. Potential Uses of New Macrolides in Pediatrics
1. Alternative when erythromycin is not tolerated 2. RespiratoryInfections Otitis media (shortened regimens) Sinusitis Bronchitis (pertussis in pediatrics) Pneumonia, especially in children and adolescents 3. Nontuberculous mycobacterialand helicobacter infections
The macrolides have enjoyed tremendous success in pediatrics, and pediatricians worldwide are very familiar and very confident inthe use of these drugs (Table 1).They have been used for a number of indications from microbiological standpoint, although several of these agents represent new developments for the use the new macrolides and azalides such as Bordetella pertussis, but they do not yet have an indication for this organism. My ownbias with‘regard to the new macrolides in pediatricsthat is they will find their greatest usefulnessin the therapy of respiratory infectionspharyngitis, acute otitis media, acute sinusitis, bronchitis (which really not is a disease in children, except for pertussis), and pneumonia. The drugs shown in Table2, erythromycin, clarithromycin, roxithromycin, and azithromycin, are active against the common organisms of the respiratory tract in pediatric patients. ForStreptococcus pneumoniae, they all are active, although azithromycin may be a little less active than the others. It’s very difficult to make any conclusion about relative activity, especially in light of Dr. Craig’s discussion on how to correlate in vitro Table 2 In Vitro Activity of Newer Macrolides
Erythromycin Clarithromycin Roxithromycin S. pneumoniae Staph. aureus H.infruenzae M.catarrhalis Legionella M . pneumoniae C. pneumoniae C. trachomatis MAC a+
(least) ++
++ + ++ + ++++ ++++ ++ 0
++ + (most) activity.
++++ ++ ++ ++ ++ ++++ ++++ ++++ +++
Azithromycin
+++ + ++ ++ ++ ++++ ++++ +++ ++
++ + +++ +++ + ++++ ++ ++ +
New Macrolides Pediatric and Infections: Discussion Table 3 Susceptibilities pneumoniae
133
Penicillin-Resistant Isolates of Streptococcus Intermediate 1.0 pg/ml)
MIGO
Macrolide Erythromycina Clarithromycina Azithromycina of isolates had MIC
High (> Pg/ml)
0.03 0.016
8 2 4
MGO
MIGO
> 16 > 16 > 16
1 pglml (data fromTrudy Murphy).
Source: Antimicrob Agents Chemother 1995; 39:533.
activity with in vivo success, when dealing with the pharmacokinetics and intracellular concentrationsof these drugs. The other two respiratory pathogens of importance are Chlamydia pneumoniae and Mycoplasma pneumoniae and allof these agents appear to be active for these organisms. The technique usedto test MIC valuesfor S. pneumoniae is critical as Michael Jacobs has pointed out. Not only is it important whether they are penicillin resistant (i.e., intermediate versus high resistant) but also whether they are erythromycin resistant, as crossresistance isthe rule among macrolides. The MIC, and MIC, values for the intermediately penicillin-resistant and highly resistant strainsare presented in Table When we compare the MIC, values, especially for thosethat are highly penicillin resistant,these agents have minimal effect, although in some instances they may be clinically effective, depending on thesite andthe type of infection. The footnote in the table presents Dr. Murphy’sdata on strains isolated in Dallaswhich show that 90% of the isolates tested from patients in Dallas County had MIC values for clarithromycinandazithromycinless than 1 pg/ml by Mueller-Hinton broth dilution testing. One of the issues of concern is middle ear infection caused by Streptococcus pneumoniae. Table 4 presents the representative peak concentrations of erythromycin estolate, clarithromycin, and azithromycin in the middle ear fluid versus those of amoxicillin givento pediatric patients.The dosages are given in milligrams per kilogram. The achievable concentrations in middle ear fluid exceedthe MIC, value for intermediate penicillinresistant organisms in most instances, and these values be should successful for management. The MIC, values are not necessarily representative of usual values encountered in practice. Whether you determine bacteriologic response by peak concentration over MICor time over MIC is an issue that hasn’t been resolved, although
lrg)
McCracken et al.
134 Table 4 Macrolide Therapy for Acute Otitis Media
MC, values
MEP
Drug 8
4-5 10-12 8-10 4-6
Erythromycin (est.) (15) Clarithromycin (7.5) Azithromycin (10) Amoxicillin (15)
2 4 0.5
>l6 >l6 >l6 2-8
'Middle ear fluid. Concentration inpg/ml. bIPR Intermediate penicillin resistant;HPR highly penicillin resistant.
Bill Craig has shown in the March 1996 issue of The Pediatric Infectious Diseuse Journal that bacteriologicsuccesscan be predicted on either pharmacodynamic marker (concentration over MIC or time over MIC) for most of the commonly used antimicrobial agents. Finally, Table 5 reviews some of the pharmacokinetic profilesof the macrolides in pediatric patients. Weknow that witherythromycin one expects peak serum concentrations the in range of 4-6 pglml; with clarithromycin 3-5 pglml; and for azithromycin, less than1 pglml. The half-life values in childrenare shown inthe table. Clarithromycinand azithromycin have very long half-life values, particularly azithromycin. The dosage schedule is based principally on the half-life values.The shorter the half-life, the more frequentlythe agent mustbe administered. Also, we might addressthe issue of the effect of food on theabsorption or bioavailability of thesedrugs. There appears to be no adverse effect; in fact, there may even be an enhancing effectof coadministration of food with the macrolides to pediatric patients. It has been known for years that erythromycin suspensions are better absorbed if taken with food, and it appears that there is no interference or diminished bioavailability with the ingestion of food withthe other macrolide suspensionsas well. In Table 5 Pharmacokinetic Profile of Macrolide Antibiotics
mycin rithromycin Erythromycin Indices Serum conc. (pg/ml) Half-life (h) Vol. Dist. (L) Urin. Exc. (%) Dosage (mgflrg)
4-5
1.5 50 5
10.5 q &12h
3-5 5 250 35 7.5 q12h
~0.4 12 2100 15 5-10 q24h
New MacrolidesPediatric and Infections: Table
Discussion
135
General Aspects of Respiratory Infections
Respiratory tract infections (RTI)-most common infectious disease worldwide Majority of RTIs-viral Principal challenge-promptly diagnose bacterial RTIs
fact, this could be an advantage because children sometimes don’t like antibiotic suspensions, but this is not a problem because they can be disguised in a food substance. Dr. Schaad: Mr. Chairman, ladies and gentlemen. For the next couple of minutes, I will include some general remarks with regard to respiratory tract infections in children, then some specific comments to the group A streptococcal tonsillopharyngitis, and finally, some pharmacokinetic and pharmacodynamic considerations andthe results of clinical studies with the macrolides forthat indication. Respiratory tract infections in childrenare the most common infectiousdiseasesworldwide(Table 6). This isalso true for adults. A big problem is that the majority of these respiratory tract infectionsare viral; therefore, the principal challenge for all of us to promptly diagnose the bacterial infections because neither the new oral cephalosporins nor the new macrolides have antiviral activity. We also know the bacterial respiratory tract infections can be either primary or secondary (Table 7). Secondary refers to superinfections to initially viral or even allergic disease. One more fact we all know is that antibiotics are effective for bacterial respiratory tract infections. The diagnosis is often difficult because it is principally based on clinical findings (Table 8) and, especially in pediatric patients, on only a few additional investigations (Table9). The therapeutic problems with groupA streptococcal tonsillopharyngitis are quite remarkable. The goalsare more or less clear:We would like to cure the sick patient of the sore throat; we would liketo stopcontagion, and then, of course, prevent bacterial and nonbacterial complications. Tabk 7 GeneralAspects
ndary orPrimary RTIs: Bacterial Antibiotics are effective Clinical findings Diagnosis on: based Few investigations (microbiological, radiological, laboratory)
al.
136
McCracken et
Table 8 Group A StreptococcalTonsillopharyngitis-Diagnostic Problems
Incidence of group A streptococcal (GAS) pharyngitis: (age, season) “Typical” clinics of GAS pharyngitis +headache, fever +sore throat, t tonsils + pharyngeal erythema and exudation + tender and t cervical nodes + absence of rhinorrhea, cough, and hoarseness The choice of drug is also a hotly debated issue. Is there any reasonto delay treatment in order to allow the patient to mount a protective immune response, or is this not necessary? Also, is the appropriate length of treatment really days or is it 5 days? Another big problem isthe frequency of recurrence that we all see. It is hardto decide if this is reinfection, which is probably much more common thantrue relapse. The recommendations for the treatment of group A streptococcal tonsillopharyngitis are presented in Table10. I think that even for this very simple disease, it is essentialto have a proper diagnostic workup.Another point is that the 40-year-old dominance of penicillin is clearly being challenged. It is not that penicillin is no longer a good drug, but there are conditions or situations whereother therapeutic modalitiesare indicated. I believe, and we hope to confirm this in our upcoming new study, that a delay of approximately 48 h after the start of symptoms is a reasonable approach to allow the patient to mount a protective immune response.Of course, there are indications where individualized management is necessary to adequately control recurrence epidemicsor outbreaks of rheumatic fever. The clinical efficacy of treatment can be predicted either by pharmacokinetic factors or, as is more fashionable, by pharmacodynamic factors. Table 9 Group A StreptococcalTonsillopharyngitis-Diagnostic Problems ~~~
~
~
Throat culture= gold standard +false
+:
+false -:
Carriers of group A streptococcal (GAS) swabbing Faulty Inadequate bacteriology Occult antibioticRx
Rapid antigen detection tests +false +: +false -:
Carriers of GAS Validated by culture
Microbiologic search for GAS requires proper clinical indication
New Macrolides and Pediatric Infections: Discussion
137
Table l 0 Recommendations for GAS Tonsillopharyngitis
Proper diagnostic workup >40-year-old dominance of penicillin is clearly challenged (oral cephalosporins: 5d; azithromycin: Rx delay of 48 hoften reasonable Individual management of recurrence, epidemics, and outbreaks ofARF
Especially for the macrolides, the time above the minimal inhibitory concentration or the MBC is most important. These drugs also produce a clinically relevant postantibiotic effect. In Table 11, the ratios between drug concentrations achieved in the extracellular space (serumor interstitial fluid) comparedto intracellular or tissue concentrationsare presented for the macrolides and penicillins.The first column represents the peak concentrations achievable with normal dosing dividedby the MIC, value for groupA streptococci. The drugs that accumulate the most will have a significantly higher value. It is not clear where the relevant multiplicationof the group A streptococci takes place in this simple disease. Based on the analysis of histopathological findings, I believe that both intracellular and extracellular organismsare important. The results of clinical studies with group A streptococcal tonsillopharyngitis are summarized in Table 12. The clinical responses with penicillin, erythromycin, roxithromycin, clarithromycin, and azithromycin are usually very satisfactory and there is no clear difference. The microbiologic responses in these older and newer studies appears to be slightly less with penicillin than the macrolides; however, there is some indication that in some studies,the bacteriologic response to azithromycin is somewhat lower. In arecentlycompleted Swissstudy, 170 pediatricpatientswith proven groupA streptococcal tonsillopharyngitis were treated with azithromycin for days, and a similar sized cohortwas randomized to penicillin Table I l
Drug ConcentratiodMIC, for Group A Beta-Hemolytic Streptococci Serum, interstitial, intracellular Tissue, extracellular ~
Erythromycin Roxithromycin Clarithromycin Azithromycin Penicillin
=
= =
0.410.03 = =
50
~~
4-8
138 Table 12
McCracken et al. Results of Clinical Studies in Streptococcal Tonsillopharyngitis Clinical response Bacteriologic response
Penicillin Erythromycin Roxithromycin Clarithromycin Azithromycin
90%
twice daily for 10 days. The clinical response was 93% and respectively.However,microbiologicresponseswerelower for azithromycin (65%) than with penicillin(82%). What is the explanation for this relatively low group A streptococcal eradication in our study? Although wewere quite strict withinclusion criteria that included really ill patients,we may have had an unusual number of asymptomatic carriers.There were only very few cases of reinfection with discordant strains. Mostof the strains isolated atthe end of treatment in these children were the same as the initial isolate, according to serotyping. We did not find resistance, andwe monitored compliance withthe measurement of antibacterial activity in the urine, but we cannot exclude some reinfections with identical strains. Both drugs were well tolerated, and at 6-month follow-up examinations, there were no cases of rheumatic fever.There was only one case of post streptococcal glomerulonephritisin the penicillin group, andrecurrent episodes of documentedgroup A streptococcaltonsillopharyngitisoccurred at a similar frequency in both groups (17% with azithromycin and 20% with penicillin). Even though it is a very simple disease, streptococcal pharyngitis remains a quite attractive issue. Thank you.
Dr. McCracken: Dr. Tarlow will now talk on acute otitis media and the study withthe macrolides.
Dr.Tarlow:
Thank you very much. In the next few minutes,I will discuss the bacterial etiology of acute otitis media in children and the specific pharmacokinetics of the new macrolides as they particularly affect middle ear infections in children. I will outline in general terms the results of comparative studies of the new macrolides in acute otitis media in childhood and consider overalltreatment regimens. The first table (Table 13) shows the typical frequency of bacterial isolates by tympanocentesis in acute otitis media. A caveat of these data is that tympanocentesis in otitis media is not practiced in all countries: it is
New Macrolides and Pediatric Infections:Discussion
139
Table 13 Acute Otitis Media-Etiology Streptococcus pneumoniae 4 0 % Haemophilus influenzae catawhalis Moraxella 10% Others-e.g., Strep. pyogenes,Staph, aureus, etc. of minor importance
not performed in Britain and Switzerland. These data are largely but not entirely from the United States and I am not sure whether they will have general geographic relevance. About 40% of the cases are associated with Streptococcus pneumoniae; the proportion of penicillin-resistant, intermediate, andsensitive strains will vary with the geographical area. About 25% will be associated with Haemophilus influenzae, and it is importantto mention that these are largely nontypeable strains;therefore, immunization against withthe new HIB vaccine is not likely to have a significant impact. About 10% are associated with Moraxella catarrhalis. Also, a few percent may be associated with chlamydial infection. Other bacteria, except inthe newborn and the immunocompromised, are really of minor importance in contributing to the overall pattern. In about 30% of cases, no bacterial etiology can be determined, and these caseswe think are viral. Table 14 summarizes the approximate MICsof azithromycinand clarithromycin against these organisms has been mentioned repeatedly, azithromycin is rather more active against hemophilus and clarithromycin may be slightly more active againstthe pneumococcus. The concentrations of clarithromycin and its active metabolite, 14 hydroxyclarithromycin, inthe middle ear fluid and in plasmaare shown in Table 15. Plasma peaks at around 2-4 h after the dose, whereas these antibiotics will continue increasing in middle ear effusion fluid in acute otitis media upto about 12 h when concentrations approaching8 pg/g can be found. Table 14 Approximate MICs of Principal Pathogens ~~~
~~
Azithromycin Clarithromycin (Pdd
Pneumococci Haemophilus Moraxella Source: Khurana, 1995.
(Pdd
ErythromycidSulfisox
140
McCrucken et al.
Table IS Antibiotic Concentrations in Middle Ear Fluid-Clarithromycin
Multiple doses (7.5 mgkg bid) After dose 6: Plasma conc.: 2.9 pglg after 2 h; 0.7 pglg after 12 h MEEa conc.: 3.0 pg/g after 2 h;7.4 pglg after 12 h 14-OH C 2.5 pglg after 2 h; 3.8 pglg after 12 h MEE conc. WEE = Middle ear effusion. Source: Cam, 1996.
The high levels foundare probably associated withthe accumulation of both clarithromycin and its metabolites in polymorphs which migrate into middle ear fluidwith the inflammation.Similarly,azithromycinis taken up and is concentrated to an even greater extent in the middle ear fluid (Table 16). After a single 10-mg dose, azithromycin is still present in the middle ear fluid in a concentrationof almost pg/g after 24 h and still has a significant concentration at 48 h. After multiple doses of azithromycin given daily, around 8.61 pg/g are present 24 h after the dose. After the end of a courseof azithromycin, effective tissue concentrations are still present for several days. These concentrations are effective against the majority of bacterial pathogens in acute otitis media. Specifically, theyare effective againstthe vast majority of penicillin-sensitive and penicillin intermediately resistant strain of S. pneumoniue, although there is a significant minorityof strains which are resistant to macrolides, including azithromycin and clarithromycin. Some of the reasons for in vitro and in vivo efficacy differ, and we cannot just extrapolate from these in vitro studies to assume that an antibiotic will or will not have an appreciable clinical effect.We need to look at Table 16 Antibiotic Concentrations in Middle Ear Fluid-Azithromycin
Single dose (10 mgkg) 3.97 pg/g after 24 h;
1.42 pglg after 48 h
Multiple doses 8.61 pg/g after 24 h;
9.43 pglg after 48 h
Middle ear effusion (MEE) conc. lOOX greater than serumconc. Source: Pukander, 1994.
New MacrolidesPediatn'c and Infections: Discussion
141
clinical trialsto see how these newer macrolides compare with established treatment in the management of otitis media in childhood. First, clarithromycin. Urs Schaad has presented inthe past an overview of seven trials of clarithromycin in which days of clarithromycin were compared with amoxicillin or with amoxicillin and clavulanic acid, with really equivalent success in both groups but rather fewer adverse effects inthe groups given clarithromycin than in the others. More recently, a multicenter blinded trial has been published by McCarty showing, again, that clarithromycin isas good as amoxicillin clavulanate withrather fewer side effects inthe clarithromycin-treated group.Another trial comparingit with cefaclor showedthat 10 days of clarithromycin wereas effective as days of cefaclor with moreor less equal incidence side effects. As far as azithromycin is concerned-I'm just picking out heresome of the clarithromycin and azithromycin studies-there are three comparative UnitedStates studies published by McMin last year covering over 1200 children in which 5 days of azithromycin was compared with days of amoxicillidclavulanate. Again, equivalent efficacy was found in the two groups with fewer side effects in those treated with azithromycin. Because of the long action of azithromycinafter the last dose has been given and because even very short courses have been shown to be effective, Urs Schaad presented a study where only days of azithromycin were compared with 10 days of amoxicillidclavulanate, again showingsuccess with azithromycin and fewer side effects than the amoxicillidclavulanate in group. A group in Belgium has recently reduced the course of clarithromycin in a slightly smaller study in which only 5 daysof clarithromycin were compared with azithromycin; both appeared successful, with very similar levels of adverse effects. what can we conclude from thisand where do we go from here? First all, both azithromycin and clarithromycinare useful drugs in the management of childhood otitis media. Both have a more effective spectrum of antibacterial activity than erythromycin. They can be given less frequently than many conventional antibiotics, clarithromycin twice daily and azithromycin once daily, and they are both well accepted and tolerated. Most, but not all, studies show a significantly lower incidence of side effects in children with otitis media given these drugs compared with traditional antibiotictreatment. In most studies, clarithromycin has been given for 10 days, but it seems that 5 days may possiblybe sufficient. Azithromycin is given 5 days in the United States, but outside the United States, the course has been cutto days and it still seemsto very effective. Couldthe course of azithromycin in otitis media be cut any further? Maybe 2 days or even 1 day of treatment might be possible. I think I will stop there and pass overto you again, George.
142
et
McCracken
al.
Dr. McCracken: Actually, I can add one interesting sidelight. There are only twocenters in the world that are doing two tympanocenteses to demonstrate bacteriologic cure: the one in Galveston which has done it for years with Dr. Howie and colleagues and the other with Ron Dagan in Israel. These are studies that are very hardto accomplish but probably provide the most enlightening information about bacteriologic responses. In the last moments we’re goingto talk about pneumonia, which is a very large subject, but fortunately there is only one published study that I think addresses this best. This is by Stan Block and a numberofcollaborators in a multicenter study published in the Pediatric Infections Disease Journal last year.There are several studies here presentedon azithromycin by Gail Cassel, Maggie Hammerschlaag, and others that look almost identical in termsof bacteriologic and clinical responses. In ambulatory pneumonia in the age groupof 3-12 years, the etiology was detected in 50% by culture, PCR, serology, or a combinationof these. Interestingly, M . pneumoniae was detected in 27% of the individuals by culture or PCR; with positive cultureor PCR had serologic response. This represents an interesting question in that, although found by culture, only half had a serologic response, and this makes it difficult to determine whether this, the isolated organism, is a true pathogen or a colonizer. C . pneumoniae infection was detected in 28%,essentially the same percentage of children hadeither M. pneumoniae or C. pneumoniae. l7venty-three percent of these with positive cultures had serologic responses. Was therapy started early enough to interfere with this response or are our serologic techniques not refined or sensitive enoughto demonstrate a rise intiter? Coinfection was seen in 37patients, 14% of all patients and 22% of those with etiology defined. In some instances, bothC . pneumoniae and M.pneumoniae were recovered. Coinfection doesn’t necessarily mean that something went wrong because we’ve known for yearsthat viral infections predispose to bacterial disease of the lower respiratory tract. It is possible that either M . pneumoniae or C . pneumoniae infection maydo the same thingor predispose one to the other. The data of Stan Block and colleagues on the bacteriologic response in the children with pneumonia and a documented etiologic agent show that for C . pneumoniae and M . pneumoniae, the microbiological response to clarithromycin was similar to that of erythromycin, as was the clinical response. Clarithromycin appeared to be comparable to erythromycin in this study. They are both well tolerated. In studies that are presented in posters ofICMAS 111, azithromycinappeared to be comparable to its comparator, and both clarithromycin and azithromycin appear to be effective for ambulatory infections in children of 3-12 years of age, perhaps younger. One of the interesting aspects of Dr. Cassels’ study is that M.
New Macrolides and Pediatric Infections: Discussion
143
pneumoniue was found in a certain percentageof children youngerthan 5 years, which isagainsttraditionalthinking. We don’tusuallythink of mycoplasma as a pathogen in the 2-3 or 4-year-old’ but in her study this was the case. Well, we’ve gonethrough agreat deal of information. I think wecan summarize by sayingthat the new macrolidesare interesting and important to pediatrics. Hopefully they will be used carefully. I have some concern that the overuse of these agents, as has been shown in Finland and Japan with erythromycin, can lead to resistance. I hope that this will not be the case. I do not think for otitis media, for example, that the macrolides represent the drugs of choice. In the United States, we stillprefer amoxicillin andthen use the macrolidesor otheragents for those failuresor when amoxicillin should notbe given. They are well tolerated and appear to be very safe. Thank you.
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l1 The Molecular Mechanismof Action of Streptogramins and Related Antibiotics C. Cocito, M. Di Giambattista, E. Nyssen, and P. Vannuffel University of Louvain Medical School Brussels, Belgium
INTRODUCTION Proteins are the main polymers endowed with catalytic function and constitute the basic structural and superstructural components of the cell. Inhibition of protein synthesis.is, thus, a prominent intervention in the chemotherapy of infectiousdiseases, for bacterialmultiplicationisprevented when formation of these essential polymers is halted. Protein structure is highly specific. It is the amino acid sequence of single proteins that provides this specificity, which is precisely recognized by the immune system and by the immunoglobulins. Instructionsfor the synthesis of the specific protein structure, which is encoded in the cell genome, is transmitted to the translating machinery of the cell (the ribosome), by vector molecules [messengerRNA (mRNA)]. Fidelityof translation of the genetic message is providedby a complex mechanism, wherein ribosomes faithfully hang amino acids in a sequence collinear with that of the corresponding gene. Such a ribosome-catalyzed template-dependent polymerization is a multistep process, to which a series of cytoplasmic protein factors participates. 145
Cocito et al.
I46
Antibiotics inhibit protein synthesis by interfering withthe metabolic pathway leadingto the assembly of these polymers. Essentially, each step of this path is blocked by a specific inhibitor and, in fact, antibiotics have been invaluable reagents for dissecting this pathway and identifyingits components. Only a limited number of the in vitro active compounds, however, has found clinical applications in chemotherapy of infectious diseases. We will briefly sketch the scheme of protein synthesis in bacteria before introducing the antibiotics that interfere with single steps of this pathway and analyzing the molecular bases. Special emphasis willbe cast on streptogramins and related antibiotics of the macrolide-lincosamidestreptogramin. (MM)group.
THE METABOLIC PATHWAY FOR BACTERIAL PROTEIN SYNTHESIS
Specificity of protein synthesis relieson the order in which some 20 amino acids are hooked together, a specific order encoded in the nucleotide sequence of DNA. Ribosomes ensurethe fidelity oftranslation of the genetic message. Bacterial ribosomesare ribonucleoprotein particleswith a sedimentation coefficientof 70S,a mass of 2.5 lo6Da and a size of about 200 A (1,2). There are about 2 lo4ribosomes per bacterial cell, representing a substantial fractionof total cell proteins (10%) and RNA (80%). Some ribosomes are free in the cytoplasm, whereas others are attached to the membrane. Ribosomes (70s) contain two subunits (Fig. 1):a large subunit including 2rRNA molecules (5s and 23s) and some 31 rProteins, and a small subunit made of 16s rRNA and 21 rProteins (1,2) (Table 1). During the course of protein synthesis,the two subunits separate after completion of the polypeptidechainandjoinagainwhenaminoacidconcatenation starts (ribosomal cycle). A series of cytoplasmic protein factors promote some steps of this process by transiently binding to the ribosome in an active form. From a chemical pointof view, protein synthesis consists in establishing a covalent link between the COOH group of one amino acid and the cr-NH2 group of the next. In reality, the activated forms of amino acids undergo the polymerization reaction on linkage with a specific aminoacyl transfer RNA (AA-tRNA) whose anticodon triplet recognized the codon triplet of mRNA. Initiation takes place on the subunit, which binds mRNA and the first AA-tRNA containingformylmethionine (Met-tRNA).The three initiation factors ( E l , IF2, IF3) which promote the binding reaction recycle when the large subunit joins to form the initiation complex (Met-tRNA.70S.mRNA).
Molecular Mechanism of Action of Streptogramins
-,
,-Head
147
Head
(3 ‘len
Plateform
d
,-Central protuberance
a
I s e
I ,- Centralprotuberance
Figure Z Consensus models of ribosomes and subunits. Projections in dimensions the (A, B) and SOS (C, D) ribosomal subunits, and of ribosomes (E, F) of E. coli: front (A, C, E) and side (B, D, F) views of the particles.
Cocito et al.
148 Table l
Properties of Bacterial Ribosomes and Subunits Ribosome Small
Sedimentation coefficient Mass (kDa) Number of rRNA bases Mass of rRNA (kDa) Proportion (%) Number of polypeptides Total mass (kDa) Proportion (%) Dimensions (nm)
subunit
Large subunit
60
Elongation consists of three step that are sequentially repeated for each amino acid joining. The accepted model for this process is based on the presence on the subunit of two sites (A and P), to which two tRNA molecules bind. Peptide bond (-CO-NH-) formation occurs between the COOH group of the growing peptide chain (pep-tRNA) attached to the peptidyl P site(donorsite)and the AA-tRNA at the aminoacyl A site (acceptor site) (Fig. 2). Thiscrucialeventinprotein synthesis occurs inthree steps: (a) binding of AA-tRNA to the A site, (b) peptidyltransfer reaction from the P site to the A site, and (c) translocation
peptidyl transferase
center PTC
Figure 2 Peptide bond synthesis. Peptide bond synthesis, promoted by the catalytic PTC center of SOS subunits, consists in the transfer of a peptidyl strand (n
aminoacids) at the P site to the AA-tRNA at the A site; a one-unit longer peptide chain (n+l aminoacids) is thus formed.
Molecular Mechanism
Action
Streptogramins
149
reaction whereby the polypeptide chain is transposed from A to P, thus allowing the process to be repeated. These steps are catalyzedby the elongation factors EF'Ib and EFTS (step a) and EFG (step c), respectively. Peptide bond formation (stepb) is promoted by the catalytic locusof the subunit (peptidyltransferase centeror PTC). When the polypeptide chain is completed, it detaches from the ribosome togetherwith tRNA and mRNA, a reaction promoted by PTC as well as by the three termination factors(RF1, FW2, RF3). The overall pathway is depictedin Fig. including the interwound ribosomal cycle.
Figure 3 Metabolic pathway for protein synthesis and ribosomal cycle. I: initiation complex;11: initiation complex;111: binding (elongationstep I), IV:peptidization (elongation step 11); V: translocation (elongation step 111). The asterisk (*) repetition of cycle I11 to V.
150
Cocito et al. ,ANTIBIOTIC INHIBITORS OF PROTEIN SYNTHESIS
Numerous independently discovered substances, mostly produced by streptomycetes, were foundto block bacterial proliferation by inhibiting protein synthesis. According to their inhibitory action, antibiotics are classed in three groups: bacteriostatic (transient inhibition of growth limited to the presence of the inhibitor), bactericidal (irreversible bacterial inactivation), and bacteriolytic (lysisof the cell). Most inhibitors of protein synthesis are bacteriostatic; those interfering with nucleic acid metabolism are lethal, and those halting cell wall formation (which confers resistance to the bacterial envelope) have lytic properties. Protein synthesis inhibitors may act either at the ribosome level or on cytoplasmic factors. Antibiotics can interfere with particle functions by binding either to the small or to the large subunit(3-6). Because the unique functionof 30s particles isto start the first step of protein synthesis, antibiotics binding tothe small subunits primarilyinterfere with initiation. However,30s particles are also involved in elongation, being responsible forthe correct codon-anticodon pairingthat ensures the proper selection of the AA-tRNA that corresponds to a given mRNA triplet. Consequently, the aminoglycosides (a large antibiotic group including widely used therapeutic agents such as gentamicin, kanamycin, kasugamycin, neomycin, paramomycin, spectinomycin, streptomycin, and tobramycin) produce two kinds of effects. They interfere with initiation, thus halting protein synthesis completely, and they induce the formation of erroneous proteins (incorporation of amino acids differing in one of the anticodon nucleotides, a lesion known as misreading). W Oother antibiotic families, edeines and tetracyclines, interfere not only with initiation but also with elongation (the growing peptide chain is, in fact, located at the interface betweenthe two subunits). The main function of the large subunit is to promote peptide bond formation. The activecenter of the enzymaticactivityresponsible for amino acid polymerization (peptidyltransferase) is located at the base of the central protuberance. This enzyme is specifically inhibited by chloramphenicol, the firstbroad-spectrumantibioticproducedsynthetically andusedtherapeutically.Affinity-labeling of ribosomeswithchloramphenicol analogs has led to identification of proteins L2, L6, L16, L24, and L27 at the binding site of this antibiotic, henceat the catalytic center of 50s (6). Three other antibioticfamilies(macrolides,lincosamides,and streptogramins) are clustered within the M U group because of similarities in their mode of action and their common resistance patterns. The MLS are also peptidyltransferase inhibitors, thoughtheir binding sites do
Molecular Mechanism of Action of Streptogramins
151
not overlap that of chloramphenicol. From a functional viewpoint, the catalytic center of PTC is to bedistinguishedfrom the two substrate binding sites of the enzyme, which correspond to the A and P sites of 50s subunits. Macrolides include several subgroups differing in the number of atoms in the macrocyclic lactone rings: M12 (methymycin), M14 (erythromycin, oleandomycin), and M16 (carbomycin, lincomycin, spiramycin, and tylosin). Proteins U,L15, and L22, which were found to be altered in some MLS-resistant mutants, are located at the binding sites of these antibiotics 7-9). Puromycin is an antibiotic endowed with a peculiar action on 50s particles. Owing to its structure mimicking the OH-terminal end of PhetRNA, this antibiotic bindsto the ribosomal A site and triggers a peptidyltransfer reaction. The peptidyl-puromycin, which formsunder these conditions, is released from ribosome, thus causingthe premature interruption of peptide chains. This antibiotic, with no therapeutic application, has thus become an irreplaceable reagent for the assay of the peptidyltransfer reaction and its inhibitors (the aforementioned antibiotics, for instance). Proteins L11 and L23 have been located at the puromycin binding site of 50S, which corresponds to the A site of the particle (10). A last group of antibiotics interfere with the function protein factors. Neglecting the inhibitors acting onIF and RF factors, we restrict our interest to those that interfere with EFTu and EFG, which promote the AA-tRNA bindingreactionandtranslocation,respectively.Kirromycin attaches to the complex AA-tRNA.GTP.EFlb, which binds to the ribosomal A site; elongation is stopped at this level (11).W Oantibiotics, fusidic acid and thiostrepton, which bind to ribosomes in the proximity of the A site, inhibit the EFG-mediated translocation (driven by the energy of hydrolysisof the ribosome-bound EFG.GTP complex). Protein L11 has been located at the thiostrepton binding site (12).
BIOLOGICAL EFFECTS OF STRJWTOGRAMINS The unique property of streptogramins (synonyms: mikamycins, pristinamycins, synergimycins, synergistins, and virginiamycins) is that they contain two groups of components A and B), producing a synergistic inhibition of bacterial growth.W Oaspects of such a synergism should be mentioned. One is quantitative: the inhibitory power of B components undergoes a hundredfold increasein the presence of A components. The other is qualitative: single components, A or B, are bacteriostatic, whereastheir mixture is bactericidal. Consequently, protein synthesis is halted transiently by A or B, and permanentlyby A + B (13-17) (Table 2).
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Table 2 In Vivo Synergistic Actions of A and B Streptogramin Components Growth proteinviability inhibition (MIC)a Single components(A or B) Mixture of components (A+B)
100 1
cell
Inhibition of synthesis
Unchanged Lowered
Reversible Irreversible
*Minimalinhibitory concentration (pglml). bColony forming units.
Virginiamycin S
I h
I
/ \2
!
I l l
Thr
AmBut
Pm
PhGly
Pipec
Ma&/
/
\$' I
0
N-
{C
\
/
Virginiamycin
M
Figure 4 Chemical structure of streptogramins: virginiamycin M nent) and virginiamycinS (type B component).
A compo-
Molecular Mechanism of Action of Streptogramins
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The two streptogramin components are produced together by some strains of streptomycetes. Although chemically unrelated, both of them are macrocyclic lactone rings. Type B components are cyclic hexadepsipeptides of about 800 Da, whereas A compounds can be considered as highly modifiedcyclopeptides of about 500 Da, with multiple,conjugateddouble bonds (16) (Fig.4). Multiplication of most gram-positive bacteria is rapidly stopped upon incubationwith single streptogramin components. Proliferation is resumed, however, when cellsare transferred to antibiotic-free media, although growth resumption of cells treated with type A compounds occurs after a prolonged lag (bacteriopause). When bacteria are incubated with a mixture of A and B components, a viability drop of several logs within a single generation time can be witnessed (17). Most gram-negative bacteria are insensitive to streptogramins, owing to the impermeability barrier of LPS-containingenvelops.However,permeabilitymutants of gram-negative bacteria have been described which proved to be sensitiveto streptogramins and related antibiotics. Ribosomal RNA is metabolically stable under normal growth conditions,becauseassoonassynthesized, it becomesassociatedwith rProteins within newly formed ribosomes. Nonetheless, when protein synthesis is halted by streptogramins, the newly synthesized rRNA becomes metabolically unstable and undergoes turnover when protein formation resumes (16). Density gradient ultracentrifugation of cytoplasm samples from bacteria growing under physiological conditions invariably shows peaks of 70S, 50S, and S particles. An additional 60s peak appears under certain experimental conditions in bacteria treated with A streptogramins (Fig,5 ) . Such an unusual peak isdue to the occurrence of pressure-labile particles, which split at critical centrifugal speed and Mg concentration. No such alteration (whose molecular mechanism will be further discussed) appears in cells treated with type B components or other MLS antibiotics (17).
MECHANISM OF ACTION OF TYPE A STREPTOGRAMINS These antibiotics halt protein synthesis in intact bacteria as wellasin cell-free systems: They are powerful inhibitors of the poly(U)-directed poly(Phe) synthesis (Nirenberg system) (18). Type A compounds bind to free ribosomes in a monomolecular reaction, whose kinetic constants have not yet been determined with precision (19,20). It has been shownthat in the presence of type A streptogramins such as virginiamycinM (W),initiation complexesare assembled in anapparently normal fashion butare functionally inactive (Fig.6), thus pointingto
Cocito et al.
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SOS
1.37 1.36 1.35 1.37
-
1.36 1.35
z_
L
fr 1.37 1.36 1.35 1 1
1.35
1.34 10
20
30
FRACTIONS
Figure 5 Roduction of pressure-labileribosomesin the presence type A synergimycins. [3H]uracil-labeled bacteriaare incubated witheither type A (B), or type B (C) components and their mixture (D) prior to ribosome fractionation by density gradient centrifugation. Sample A is the control (no antibiotic) and sample E is glutaraldehyde-fixed sample B. Wherever cells are incubated with type A components, pressure-labile ribosomesappear unless they are fixed.
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Figure 6 Assembly and reactivityof initiation complexes in the presence of type A synergimycins. To initiationcomplexes (30s ribosomalsubunits,MS,-RNA, [3H]fMet-tRNA, and IF-1, IF-2, IF-3), 50s subunits wereadded, in the presence of either GTP [(A) and (B)] or its nonhydrolyzable analog G-PPCP(C); only sample (B) contained typeA synergimycin. Samples were splitinto two parts, one of which was incubated with puromycin (o----o = no puromycin) and fractioned by density gradient centrifugation. Radioactivity tracing shows that initiation complexes were formed in all cases, but those assembled inthe presence of the antibiotic (B) and those with G-PPCP (C) held unreactive Met-tRNA.
an inhibition of the elongation phase (21). Moreover, of the three elongation steps (AA-tRNA binding to the A site, peptidyltransfer from site P, and translocation from the A to the P site), the first two proved to be inhibited by type A compounds. The AA-tRNA binding reaction assayed at equilibrium was indeed found to be blocked by V M (18). However, this step can be further dissected by the use of an additional inhibitor, kirromycin, which binds to
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EJ3b. The overall reaction sequence is as follows: (a) formation of the AAtRNA.EFTh.GTP complex, (b) binding of the complex to the ribosomal A site, (c) release of ElTu.GDP, and (d) irreversible fixation AA-tRNA to the substrate acceptor site of peptidyltransferase (11).Kirromycin prevents reaction (c), irrespective of the presence V M , whose interference isthus restricted to step (d) (12). Indeed, in the presence of GTP and E m u , labeled AA-tRNA firmly binds to ribosomes, from which it cannot be released by an excess unlabeled AA-tRNA; this exchange reaction occurs whenV M is present (22,23) (Fig. 7A). In addition, the puromycin reaction with peptidyl-tRNA linked to the ribosomal P site was found to be inhibited bytype A streptogramins (18,21) (Fig. 6). Here again, the P-site-bound substrates (labeled acAA-tRNAfor
Time (mint
0
10 20 Time ( m i n )
Figure 7 (A)TheVM-inducedexchangebetweenfreeandA-sitebound
aminoacyl-tRNA. Control (R)-ribosomes and those transiently incubatedVM with (R*), both carrying (14C)Phe-tRNA at the A site, were incubated with unlabeled Phe-tRNA, GTP, and EF-Tb in the presence or absence ofVM; at various times, ribosome-bound radioactivity was measured. R ribosomes: no VM (o),plus VM (A); R* ribosomes:no VM plus VM (A). (B) Translocational ejection of AA-tRNA from Psite of ribosomes incubated with type A synergimycins. Poly(U).ribosomes complexes containing Ac(14C)Phe-tRNA at the P site were incubated with tRNA (A site filling) and then with EF-G and GTP (to promote translocation), in the presence (filled symbols) and in the absence (open symbols) of V M . Ribosomebound radioactivity was measured. Samples: complete system (A,A), minus tRNA (0, minus EF-G+GTP(o, Translocational ejection occurred in the presence of
Molecular Mechanism
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instance) cannotbe chased out by the corresponding unlabeled derivatives, a sign of irreversible linkage with ribosomes. However, this exchange does take place inthe presence of VM, which prevents a stable interaction of peptRNA with the donor site of peptidyltransferase (Fig. 7B). The overall conclusion is that type A streptogramins are specific inhibitors of both substrate binding sites of the enzyme. How can such an interference be reconciled with the well-known fact that only one VM molecule can bind to each 50s particle? The clue has been furnished by a new model, whereby the acceptor and donor substrate binding sites of peptidyltransferase exchange position, conformation, and function at each elongation step in a jigsaw-like motion (Fig. 8). 'Qpe A streptogramins can bind to both subunits and free 70s particles but notto ribosomes engaged in protein synthesis or topolysomes. In fact, translating particles carrying peptidyl-tRNA or AA-tRNA are proIn agreement withthe proposed model tected against these antibiotics of interchangeable peptidyltransferase sites, particlesare protected by Asite bound AA-tRNA, as well as by P-site-bound pep-tRNA, or by AAtRNA derivatives translocated from the A to the P site. These observations imply that type A components can only bindto the free arms of peptidyltransferase, and thusto particles at the termination-initiation phase (runoff ribosomes) As previously mentioned, in cells treated with type A streptogramins, pressure-sensitive ribosomes accumulate, yielding unusual ultracentrifugal patterns (Fig. 5). It has been shown that particles carrying uncharged tRNA are unstable in centrifugation at low Mg. This contrasts with the stability of ribosomes containingAA-tRNA and pep-tRNA linkedat the A and P sites, respectively. When the latter two types of complexes are assembled in the presence of type A compounds, they become pressure sensitive It is the stable interaction of the aminoacyl and peptidyl moietiesof tRNA derivatives with the substrate binding sites of peptidyltransferase that renders particles pressure-resistant.
THE MECHANISM OF ACTION OF TYPE B STREPTOGRAMINS Unliketype A components,type B components are ineffectivein the Nirenberg system This observation has promptedthe development of other cell-free systems with copolymers as messengers. In these models, polypeptides with diverging propertiesare synthesized: charged and hydrophilic, those primed by adenine polymers; and neutral and hydrophobic, those directed by uracil-containing messengers.Indeed, the system containing poly(A,C) copolymers as templatesare highly sensitiveto inhibition by
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translocation
recycle
I
I
aa-tRNA binding peptidebond formation
translocation
translocation aa-tRNA binding peptidebond formation
recycle
translocation
{tRNA
peptidyl-RNA
Figure 8 Models of peptidyltransferase function. Iko schemes for the events occumng at the peptidyltransferase centerof the SOS subunit during translocationare compared. According to the conventional model (A), the donor and acceptor sites of the enzyme have fixed positions, and translocation involves the detachment of peptidyl-tRNA from the acceptor site. The proposed model (B) postulates an exchange of position, conformation, and function of the two sitesof the enzyme at each elongation round, with no detachment of peptidyl-tRNA in translocation.
type B components which, in addition, produce a premature release of short polypeptides (28) (Fig. 9). The followingconclusionswere then drawn: (a) Streptogramins B inhibit the in vitro peptide bond synthesis; (b) the inhibition level is templatedependent; (c) these antibiotics also induce a detachmentof incomplete protein chains; and (d) the overall inhibition is proportional to the peptide length. This indicates that in the presence of these antibiotics, the elongation process becomes increasingly difficult as the protein chain extends, by a steric hindrance effectthat slows the poly-
Molecular Mechanism Action
Streptogramins
159
lime (min)
Figure 9 Inhibition of polypeptidesynthesisandprematurechainreleaseproduced byVS and Ery and in acell-free system. Poly(A, C)-directed protein synthesis was carried out in a cell-free system of Escherichia coli containing [4C]lysine,
[“Clproline, and unlabeled threonine, histidine, glutamine, and asparagine, in the absence (0)and in the presence of VS (A, type B synergimycin) and Ery (0,14membered macrolide). Amino acid incorporation into ribosome-bound peptides total peptides(B), and high-molecular-weight peptides (C) was measured.
merization process and promotes its interruption at the level of basic amino acids (points of physiological instabilityof the elongation complexes). Indeed, peptides isolated from cell-free systems incubated with B compotype nents werefar shorter than controls and bore basic amino acids in carboxylterminal position (29). Unlike type A streptogramins that bind exclusively to naked particles, type B compounds can link also ribosomes engaged in protein synthesisand polysomes. Note that comparable inhibition of poly(A, C) and poly(U, C) systems was observed in the presence ofvirginiamycin S (type B streptogramin) and erythromycin (M14 macrolide) (28,29)(Fig.9). The same mechanism of action canbe proposed for the two antibiotics: inhibitionof peptide bond synthesis and interruption of protein elongation (26). Although unfinished peptides were not previously detected in wild-type bacteria incubated with these inhibitors, an accumulation of peptidyl-tRNA was observed in a hydrolase-negative mutant incubated with erythromycin (this precursor being rapidly degraded by the enzymes of wild-type cells) (30). As type B streptogramins are peptidyltransferase inhibitors, identification of their binding site would give a hint of the, PTC location. This information was search by the useof the biophysical technique of non-
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radiant energy transfer between fluorophores.The 50s subunit holds two L7L12 dimers: a strong site located at the base of the central protuberance, and a weak site composing the stalk. Each dimerwas removed selectively, labeled by the addition of a fluorescent coumaryl group, and replaced in its original location. Energy transfer measurements between each coumaryl-17Ll2 dimer and virginiamycin S (a type B streptogramin endowed with intrinsic fluorescence) on the same particle yielded the distances between the fluorophore couples. the result of this triangulation process, the VS binding site corresponding to the PTC was located at the base of the central protuberance (31) (Fig. 10). The proteins at the binding site of type B streptogramins were identified by an affinity-labeling approach. This relies on the synthesis of an antibiotic derivative that carries a reactive arm susceptible to forming a covalent linkage withthe receptor protein under certain experimental con-
Figure Localization of the binding site for type B synergimycins on the large ribosomal subunits.The distances between ribosome-boundVS (a type A synergimycin withinherent fluorescence) and coumaryl fluorophores located on the strong and weak L7L12 sites and on the L10 site were measured by nonradiant energy transfer. The topology of the VS binding site (hatched surface)was made by triangulation with respectto the known position of L7L12 dimers.
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Figure ZZ Topological model of the binding site for MLS antibiotics on the 50s ribosomalsubunit.Ribosomalproteins (LE,L18, L22,andL27),whichwere affinity-labeled by different MLS antibioticsandlocated by immuneelectronmicroscopy, and bases in domains I1 and V of rRNA, which were altered by M L S mutations (A) are depicted. The dottedarea in the insert indicates the consensus topological situation of the PTC domain, whichis overlapped by the MLS binding site.
ditions. The hydroxysuccinimide ester of a VS oximation derivativewas the chosen reagent; it was able to reversibly bind to particles at and to attach irreversibly at 30°C. After dissociation of the affinity-labeled particles and protein fractionation by two-dimensional electrophoresis, proteins L18 and. L22 wereidentified (32). These rProteins, in addition to the previously recognized L15 and L27, components of PTC (Fig. 11). The presence at the base of the central protuberance, of the four aforementioned proteins, and of L2 and L16 (a conformation protein) has been confirmed by the immune electron microscopy approach(33-35).
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THE MOLECULAR BASIS OF THE SYNERGISM BETWEEN A AND B STREPTOGRAMINS The synergistic inhibitory action exerted by the two streptogramin components, a biological phenomenon unique innature, was reproduced in vitro inabiophysicalmodelmeasuring the antibiotic-ribosomecomplex at equilibrium. Q p e B streptogramins,suchasvirginiamycin S (VS), are endowed with intrinsic fluorescencedue to thepicolinyl component at the ribosome binding portion of the molecule. When these antibiotics bind to the 50s subunits, there isanincrease of fluorescenceintensity that is proportional to the concentration of the formed complex; such an increase provides a valuation of the affinity of the particle for the bound inhibitor (36). In the presence of type A components,such as virginiamycin M (VM), the fluorescence intensityof the ribosome.VS complex undergoes afurther increase. Indeed, the association constantof this complex formation is about tenfold higher in the presence of VM (K,= 2.5 lo6 M" without W, and 15 lo6 M" with VM) (37) (Fig. 12).
B
of synergimycinsAand B to ribosomes. (A) Scatchard plot of the binding of type A component to ribosomes, in the presence (A----A) and inthe absence (o----o)of type B components. (B) Scatchard plotof the binding of a type B component to ribosomes in the presence (o----o) and in the absence of type Acomponents. The two binding reactions were monomolecular; although bindingwas not modified by their partners, the K , of the binding reaction of components Bwas strongly increased inthe presence of synergimycin A.
Figure 12 Bindingreactions
Molecular Mechanism Action
Streptogramins
163
0 Z 3
g
50
0.2 0.4 0.6 08 100
0
I
0
0.4
ERYI50 S
Figure 13 Competitive displacement of type B components by macrolides in the presence of synergimycin A. 50s ribosomal subunits were incubated with synergimycin B (VS), in the presence (e----*) or in the absence (o----o) of type A synergimycin (W).Increasing amounts of erythromycin were added, and ribosomebound VS was measured. Erythromycin completely displaced bound VS but was ineffective in the presenceof W.
Fast kinetics measurements (stopped-flow techniques) have provided evidence for a decrease of the dissociation rate constant of the VS.ribosome complex, which is inducedby VM Further structural details were gathered with fluorescence attenuationmeasurements.Accordingly, the VSbinding site on the ribosome surface was viewed as having the shape of an open well, undergoing a transannularconstrictionin the presence of VM The abovementioned spectrofluorometric model and fast kinetics techniques have also allowed the delineation of the synergistic and antagonistic interactions existing among ribosome-binding antibiotics. Thus, there is antagonism between erythromycin (ERY, M14 macrolide) and virginiamycin S (VS, type B streptogramin) for bindingto Because these antibioticshavesimilarmechanisms of action and overlapping binding sites, their ribosome fixation is mutually exclusive. The association constant of ERY (K,= 2 lo7 M-'),being higher than that of VS (2.5 lo6 " l ) , ERY addition to VS.ribosome complexes induces VS detachment and its replacement by ERY. Such an exchange does not occur, however, in the presence of type A streptogramins (Fig. 13). The following succession of events has been identified by stopped-flow studies.Attachment of V M to ERY.ribosomes complexes induces a conformational change reducing the ribosome affinity for ERY (which is thus released) (Fig. 14) and
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0
5 TIME, (min)
10
Figure 14 Displacement of ribosome-bound erythromycin by virginiamycin M. A ribosome suspension was incubated with [14C]erythromycin (Ery,a 14-membered macrolide) and splitinto three parts which were incubated (zero time) respectively with V M (a type A synergimycin) (a), unlabeled Ery (O), none (A); ribosomebound radioactivity was measured.
Tabk Kinetic Parameters forthe Binding Reactionsof Some Antibiotics .to Ribosomes
Rate constants Antibiotics Type A synergimycins 1.4 (virginiamycin M) Type B synergimycins (virginiamycin S) 14-Membered macrolides 3.2 (erythromycin) 16-membered macrolides (leucomycin A3) Lincosamides 4.5 (lincomycin)
k+ (M-' s-l)
Dissociation constant
k- (s-l)
X
104
-
X X X
l6 l6 16
0.042 0.008 4.0 0.0039
1.5 x
lo4
0.00032 1.5
R 2.8 2.1 R*
x l@ 0.25
Ribosomes nonincubated (R) or incubated (R') with type
Kd(M)
3.1 X
(2.0 X
1.8 X 107
x
lo8
1.4.x X 1O"l
3 X 10-~ synergimycins.
.
< .
Molecular Mechanism
of Action
165
Streptogramins
Antibiotic
Peptidyl-transferase domains
I
II
I
111
vs
Type B streptogramins
ERY 14-membered macrolides
Type A streptogramins Lincosamides
I
Figure 15 Topological model of the PTC domain on the large ribosomal subunit. The binding sites five antibiotic families inhibiting peptidyltransferase have been located according to competitive (overlapping) and noncompetitive (nonoverlapping) interactions with ribosomes.
increasing the affinity for VS (which binds with a tenfold higherstrength) (26,37). This approach has allowed the kinetic constantsof all the members of the MLS group of antibiotics to be determined (40) (Table 3). These constants define the behavior of these inhibitors, their affinity for the particles, as well as synergistic and antagonistic interactions withthe other antibiotics; By these works, a topographic mapof PTC has been sketched, which contains three sections with partly overlapping antibiotic binding sites. One section comprises the sites of type A streptogramins andM16 macrolides, another those of lincomycin plus M14 and M16 macrolides, and a third the sites of B streptogramins, M14 and M16 macrolides (26,40) (Fig. 15).The first section might correspondto the catalytic center of PTC, whose function could also be altered by antibiotics binding to the nearby sections I1 and 111. In conclusion, typeA streptogramins induce a conformational change of 50S, leading to increased affinity for typeB components and decreased affinity for macrolides. Additional confirmationof such aninterference has beengathered byanalysisof the patterns of protectionafforded by ribosome-bound streptogramins against chemical reagents altering rRNA structure.
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THE PEPTIDYLTRANSFERASE CENTER: THE TARGET OF MOST 50s SUBUNIT INHIBITORS Ribosomalsubunits are notsimplesuperpositionsof protein shells on RNA nuclei. Although singlestranded, rRNA molecules acquire complex tridimensional configurations due to the occurrence of double-stranded regions (base pairing of RNA segments with complementary sequences) (41).WhereasrProteins adhere tospecificpartsof the folded rRNA structures, portions of the latter molecules protrude on the particle surface, hence their interaction with cytoplasmic nucleic acids and proteins (42). In fact, incubation of ribosomes with RNAse causes a splitting ofthe particles and the separation of their components. A series of chemical reagents specifically attacking nucleotide bases proved to be able to alter discrete portionsof rRNA within intact particles,the corresponding RNA alterations beingrevealed by sequencing(43). One of the approaches used to identify the ribosomal receptors for antibiotics is based onthe use of these nucleic acids reagents: Ribosome-bound antibiotics would protect against chemical attack the RNA segment present at the corresponding binding site As the result of these studies,two regions of the 23s rRNA molecule lying at the PTC locus have been identified:the central loop of domain V with adjacent sequences, and a stem of domain 11. Within these regions, type A streptogramins protected basesA2037, A2042, G2049, and C2050 (stem adjacent to the central loop of domain V) (49, whereas type B streptogramins protected basesA2062 and G2505 (central loop of domain V) plus base A752 (stemof domain 11, presumably folding over domain V) (44,45) (Fig. 16). Note that macrolides and chloramphenicol also protect bases within this region. In fact, A2058, A2059, and G2505 are protected by erythromycinandcarbomycin, the latter alsoshieldingA2062and A2451, and chloramphenicol segment2497-2507 This approachalsohasprovidedevidence for the conformational ribosomal changes inducedby type A streptogramins, which is responsible for synergism between compounds of type A and B. Indeed, base A2062, which is protectedby ribosome-bound VS, becomes unshielded whenVM is addedto the system (46). Moreover, typeA streptogramins increasethe reactivity of C2073, U2086, and U2092 toward RNA reagents, other indications of conformational particle changes (47). Notethat although protection could be due to different alternative effects (direct shielding, steric hindrance, or conformational change),the increased accessibility of a base (A2062 in our case) is only compatible with a conformational change. An-additional observation inthiscontextis the lasting ribosomal alteration produced by the attachment of type A streptogramins. Ribosomes isolated from VM-treated cells or incubated in vitro with this antibi-
Molecular Mechanism of Action of Streptogramins
167
.
A U
mo~
2
m
2460
m
I
I
o^ t
d
0
V V A
h
tl$
: :: l Figure 16 The protection patterns of rRNA bases inducedby V M , other antibiotics, andtRNA derivatives. In this illustrationof the central loop of domain V of 23s rRNA and related stems, the bases displaying analtered reactivity toward chemical reagents, in the presence of ribosome binding substances (tRNA derivatives and antibiotics) are indicated as follows: (a) bases protected by VM (D),(b) bases protected by other antibiotics are encircled; (c) basedprotected by aminoacyl-(A) and peptidyl-(P)tRNA; and (d) base A2062, while protected by VS alone, became accessible inthe presence of VM+VS (filled arrow).
Cocito et al.
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otic, after purification by sedimentation or bycolumn chromatography (procedures supposedly removing all the bound inhibitor), were found to (48). Analysis be inactive in directing protein synthesis in cell-free systems of these particles has shownthe presence of noncovalently bound residual VM, removable by anti-VM immunoglobulins. However, VM-free particles, although able to catalyze peptide-bound formation, did still bindVS with increased affinity(49). The overall conclusions are as follows: (a) type A streptogramins bind to ribosomes with an affinity level higher than previously reported (Kd = 3.1 (b) residual V M rendersparticlesunsuitable to catalyze peptide bond formation; (c) after complete V M removal, ribosomes still hold a lasting conformational change, conferning higher affinity for type B streptogramins; and (d) the latter alteration can only be removed by particles dissociation and reassociation(50).
CONCLUSIONS AND SUMMARY Bacterial protein synthesis is a multistep process catalyzed by ribosomes and cytoplasmic protein factors; initiation, elongation, and termination are the phases of this path. In parallel, ribosomes undergo an associationdissociation cycle, whereby 30s and subunits separate at termination and rejoin at initiation, started by 30s. Most antibiotics transiently inhibit protein synthesis (bacteriostatic action) by binding either to ribosomes or to cytoplasmic factors. Only aminoglycosides (pleiotropic inhibitors of and streptogramins have a bactericidal (i.e., permanent action). The unique trait of streptogramins isthe presence of A and B components, which are separately bacteriostatic and jointly bactericidal. Both components bind to SOS subunits with monomolecular reactions,the affinity of ribosomes for B components being tenfold increased by their partners. The two-site model for protein synthesis is basedon the presence at the SOS surface of two binding sites,P and A, for two moleculesof precursors AA-tRNA, which are also linkedto mRNA by codon-anticodon pairing. Elongation occurs in three steps: AA-tRNA binding to the A site (promoted by E m ) , peptidization between pep-tRNA at the P site and AAtRNA at the A site (promoted by the PTC of subunits), and translocation of pep-tRNA from P to A site (promoted by EFG). Most of the best known therapeutically active antibiotics that act on elongation interfere with PTC function. Within the PTC domain, a catalytic center (inhibited by chloramphenicol) is to be distinguished from the substrate donor and acceptor sites (corresponding to the P and A sites of ribosomes), to which the aminoacyl or peptidyl portions of the two reacting substrates are bound. Type A streptogramins specifically inhibit the substrate interac-
Molecular Mechanism
of Action of Streptogramins
I69
tion with the corresponding PTC sites: A release of precursors and a halt of polypeptide synthesis followthe attachment 'of these inhibitors to the 50s subunits. 'T)lpe B streptogramins and macrolidesslow the elongation process and cause a premature halt at the level of basic amino acids, hencethe release of incomplete polypeptide threads. Thus, type A compounds can only bind to naked particles (i.e., to the free substrate binding arms of peptidyltransferase), whereas type B streptogramins can attach to polysomes in a pointof the 50s surface situated at a certain distance from the catalytic center,of PTC (and yet withinthe domain of the enzyme). A streptogramins cause a conformational change of 50s subunits, which is also responsible for the synergistic actionof A and B components. Several proofs for suchachangehavebeenprovided: (a) after contact with A streptogramins, ribosome affinity for type B components undergoes a tenfold increase,high a affinitystate that lasts after removal of A components; (b) a decreased ribosome affinityfor erythromycin is also producedundertheseconditions;(c) the accessibilityoffluorescence quenchers to the ribosomal bindingsite of B streptogramins is decreased in the presence of type A compounds; and (d) protection of rRNA bases against nucleic acids reagents, which is provided by type B components, is modified by the binding of type A streptogramins to particles. In conclusion, antibiotics may interfere in three ways with ribosome functions and protein synthesis: (a) direct functional block (the case of chloramphenicol which binds to the catalytic sector of PTC); (b) steric hindrance (typeB streptogramins and macrolides which interfere with elongation, by binding at adistancefromcatalytic center); and (c) conformational changeof ribosome triggered by antibiotics which bind in the proximity of the catalytic center (typeA streptogramins). Conformational PTC changes are responsible for the synergistic and antagonistic interactions among different antibiotic families and also for the synergism between A and B streptogramins.
REFERENCES 1. Wittman HG. Components of bacterial ribosomes. Annu RevMicrobioll982; 51:155-183. 2. Wittman HG. Architecture of prokarioticribosomes.Annu Rev Microbiol 1983; 52~35-65. 3. Pestka S. Inhibitors of ribosome functions. AnnuRev Biochem 1971; 40:697710. 4. Vasquez D. Inhibitors of proteins synthesis.FEBS Lett 1974; 40:S63-S84. 5. Pestka S. Insightsintoproteinbiosynthesis and ribosomefunctionthrough inhibitors. Prog NucleicAcid Res Mol Bioll976; 17:217-245.
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6. Vhzquez D. Inhibitors of Protein Synthesis. Berlin: Springer-Verlag,1979. 7. Teraoka H, Nierhaus KH. Proteins from Escherichia coli ribosomes involved in the binding of erythromycin. JMol Bioll978; 126:185-193. 8. Tejedor F, Ballesta JPG. Reaction of some macrolide antibiotics with the ribosome. Labeling of the binding site components. Biochemistry 1986; 25: 7725-7731. 9. ArCvalo MA, Tejefor F, Polo F, Ballesta JPG. Protein components of the erythromycin binding site in bacterial ribosomes. J Biol Chem 1988;263: 58-63. 10. Olsen HM, NicholsonAW, Cooperman BS, Glitz DG. Localization of sites of photoaffinity labelingof the large subunitof E . coli ribosomes by an arylazide derivative of puromycin. J Biol Chem 1985; 260:10326-10331. 11. Parmeggiani A, Swart GWM. Mechanism of action of kirromycin-likeantibiotics. Annu Rev Microbiol1985;3937-577. 12. Thomson Y, Cundliffe E, Stark M. Binding of thiostrepton to a complex of 23s rRNA with ribosomal protein L11. Eur J Biochem 1979; 98:261-265. 13. VAsquez D. The streptogramin family of antibiotics. In: Gottlieb D, Shaw PD, eds. Antibiotics, Vol. I. Berlin: Springer-Verlag, 1967:387-403. 14. Vhzques D. The streptogramin family of antibiotics. In: Corcoran J W , Hahn FE, eds. Antibiotics, Vol 111. Berlin: Springer-Verlag 1975:521-534. 15. Tanaka N.Mikamycin. In: Corcoran JW, Hahn FE, eds. Berlin: SpringerVerlag, 1975521-534. 16. Cocito C. Antibiotics of the virginiamycin family, inhibitors which contain synergistic components. Microbiol Rev 1979; 43:145-198. 17. Cocito C. Properties of virginiamycin-like antibiotics (synergimycins), inhibitors containingsynergisticcomponents.In:CorcoranJW, Hahn FE, eds. Antibiotics, Vol. IV. Berlin: Springer-Verlag, 296-332. 18. Cocito C, Kaji A. Virginiamycin M-A specific inhibitor of the acceptor site of ribosomes. Biochimie 1971;53:763-770. . 19. Cocito C, Di Giambattista M. The in vitro binding of virginiamycin M to bacterial ribosomesandribosomalsubunits. Mol Gen Genet 1978; 166: 53-59. 20. Aumercier M, BouhalladS, Capmau ML, Le Goffic F. Irreversible binding of pristinamycin IIA (streptogramin A) to ribosomes explains its “lasting damage” effect.J Antibiot 1986; 39:1322-1328. 21. Cocito C, Voorma H, Bosch L. Interference of virginiamycin M with the initiation and the elongation of peptide chains in cell-free systems. Biochim Biophys Acta 1974; 340:285-298. 22. Chinali G. Moureau Ph, Cocito C.The mechanism of action of virginiamycin M on the binding of aminoacyl-tRNA to ribosomes directed by elongation factor Tu. Eur J Biochem 1981; 118577-583. 23. Cocito C, Chinali G. Molecular mechanism of action of virginiamycin-like antibiotics (synergimycins)on protein synthesis in bacterial cell-free systems. J Antimicrob Chemother 1985; 16 (supplA):35-52. 24. Chinali G, Moureau Ph, Cocito C. The action of virginiamycin M on the
Molecular Mechanism
Action
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acceptor, donor and catalytic sitesof peptidyltransferase. J BiolChem,
25.
Chinali G, Di Giambattista M, Cocito C. Ribosome protection by tRNA derivatives against inactivation by virginiamycin M. Evidence for types of interaction of tRNA with the donor site of the peptidyltransferase. Biochemistry Di Giambattista M., Chinali G, Cocito C. The molecular basis of the inhibitory activities of type A and type B synergimycins and related antibiotics on ribosomes J. Antimicrob Chemother Chinali G, Vanlinden F, Cocito C. Actionof virginiamycin Mon the stability of differentribosomalcomplexes to ultracentrifugation.BiochimBiophys Acta Chinali G, Nyssen E, Di Giambattista M, Cocito C. Inhibitionof polypeptide synthesis in cell-free systems by virginiamycin S and erythromycin. Evidence for a common mode of action of type B synergimycins and 14-membered macrolides. Biochim Biophys Acta Chinali G, Nyssen E, Di Giambattista M, Cocito C. Action of erythromycin and virginiamycin S on polypeptides synthesis in cell-free systems. Biochim Biophys Acta Menniger JR, Otto DP. Erythromycin, carbomycin, and spiramycin inhibit protein synthesis by stimulating the dissociation of peptidyl-tRNA from ribosomes. Antimicrob AgentsChemother Di Giambattista M, Thielen A, Maassen J, Moller W, Cocito C. Localisation of virginiamycin S binding site on bacterial ribosome by fluorescence energy transfer. Biochemistry Di Giambattista M, Nyssen E, Pecher A, Cocito C. Affinity labeling of the virginiamycin S binding site on bacterial ribosome. Biochemistry Stoffler G, and Stoffler-Meilicke M. Immuno electron microscopy of ribosomes. Annu Rev Biophys Bioeng Stoffler G, and Stoffler-Meilicke M. Immuno electron microscopyon Escherichia coli ribosomes In: Hardesty B, KramerG, eds. Structure, function and genetics of ribosomes. New York: Springer-Verlag Lake JA. Evolving ribosome structure: domains in archaebacteria, eubacteria, eocytes and eukaryotes.Annu Rev Biochem Parfait R, de BCthuneMP, Cocito C. Aspectrofluorimetric study of the interaction between virginiamycin S and bacterial ribosomes.Mol Gen Genet Moureau Ph, Engelborghs Y, Di Giambattista M, Cocito C. Fluorescence stopped flowanalysisof the interaction of virginiamycin components and erythromycin with bacterial ribosomes. J Biol Chem Di Giambattista M, Ide G, Engelborghs Y , Cocito C. Analysis of fluorescence quenchingof ribosome-bound virginiamycinS. J Biol Chem Cocito C, Di Giambattista M, Nyssen E, Vannuffel P. Inhibition of protein
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synthesis by streptogramins and related antibiotics. J Antimicrob Chemother suppl A, in press. Di Giambattista M, Nyssen E, Engelborghs Y, Cocito C. Kinetics of,binding of macrolides, lincosamides and synergimycins to ribosomes. J Biol-Chem Noller HF. Structure of ribosomalRNA.AnnuRevMicrobiol Noller HF. Ribosomal RNA and translation. Annu Rev Biochem
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Stem S , Moazed D, Noller HF. In: Noller HF, Moldave K. eds. Methods in Enzymology NewYork: AcademicPress, . .. 44. Moazed D, Noller Chloramphenicol, erythromycin, carbomycinand vernamycin B protect overlapping sites inthe peptidyl transferase region of . rRNA. Biochimie Vannuffel P, Di Giambattista M, Cocito C. Chemical probing of virginiamycin M-promoted conformational changeof the peptidyltransferase domain. Nucl 22 Ac. Res. 46. Vannuffel P, Di Giambattista M, Cocito C. The role of rRNA bases in the interaction of peptidyltransferase inhibitors with bacterial ribosomes. J Biol Chem Vannuffel P. L'interactiondesinhibiteurs de la peptidyltransferase, avec YARN Le r61e des bases du domainV. PhD thesis. Faculty of Sciences, UniversitB catholique de Louvain, pp. 48. Parfait R, CocitoC.Lastingdamage to bacterialribosomes by reversiblybound virginiamycinM. Proc Natl Acad SciUSA Nyssen E, Di Giambattista M, Cocito C. Analysisof the reversible bindingof virginiamycin Mto ribosome and particle functionsafter removal of the antibiotic. Biochim Biophys Acta 50. Moureau Ph, Di Giambattista M, Cocito C. The lasting ribosome alteration produced by virginiamycin M disappears upon removal of certain ribosomal proteins. Biochim Biophys Acta
€€F.
12 Early Clinical Results with Quinupristid Dalfopristin for the Therapy of Bacteremia Due to Resistant Gram-Positive Bacteria C. Moellering, Jr. Beth Israel Deaconess Medical Center Boston, Massachusetts
Sharon L. Cerwinka RhSne-Poulenc Rorer Collegeville, Pennsylvania
Quinupristiddalfopristin (Synercide) is a novel streptogramin antibiotic with potent activity against gram-positive organisms, including methicillinresistantstaphylococciandvancomycin-resistant Enterococcus fuecium (VREF) Even thoughthe drug has yetto be released for clinical use, it has attracted widespread attention inthe infectious diseases community, as it is often the only or one of the few agents with useful activity against multiresistant staphylococci and enterococci. Because of this, an emergency use program was initiated in June and as of December patients had been enrolled in this program.Of the patients treated with the drug thus far, 608 have been infected with vancomycin-resistant Enterococcus fuecium, with other vancomycin-resistantenterococci, with staphylococci, and with streptococci or Corynebacteria. Criteria for enrollment in this study include the,following:
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and
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1. Positive culture for a pathogen presumed susceptible to quinupristiddalfopristin 2. Signs and symptoms of infection 3. Infecting organism resistant to all clinically appropriate antibiotics or patient was a documentedtreatment failure for all clinically appropriate antibiotics or patient was intolerant of all available clinically appropriate antibiotics The majority of patients enrolled in the study thus far (613 patients) have come from the United States. Includedin the 234 patients for whom completed data are currently available are 126 patients with bacteremia due to VREF or staphylococci who form the basis for this interim report: 115 patients (111 adults and 4 children) with Enterococcusfueciurn bacteremia and 11 with staphylococcal bacteremia (one or more positive blood cultures within 7 days prior to quinupristiddalfopristin). Of the 115 patients with Enterococcus fueciurn bacteremia treated with the study drug, 76% were immunosuppressed, 22% were leukopenic (total white blood cell count with a maximal plasma concentration four to six times higher than inthe control period. Erythromycin increases bromocriptine bioavailability through alterations in its absorption or hepatic first pass.In patients receiving erythromycin,the dose of bromocriptine shouldbe reduced to a thirdor a quarter (23).
Drug Interactions of Macrolides and Azalides
+II
II
185
II
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Methylprednisolone On the basis of a favorable response of asthma patients to erythromycin therapy a kinetic study was done on the interaction of erythromycin with methylprednisolone. It was found that erythromycin reducedthe clearance of methylprednisolone by and increasedthe half-life from to h Troleandomycin also impairs significantly the elimination of methylprednisolone, reducing clearanceby Reduced tissue binding or increased plasma protein binding of methylprednisolone by troleandomycin have been reported to increase the steady state of the hormone. Similarly, the metabolism of estrogen- and progesterone containing contraceptives were inhibitedby troleandomycin giving riseto cholestatic jaundice in female patients who received troleandomycin.
Ergot Alkaloids Ergotism described in a patient who took concomitantly ergotamine tartarate andtroleandomycin was the firstdescribedinteractionbetween macrolide and another agent The phenomenon has been repeatedly described not only with troleandomycin but also with erythromycin and is presumably due to inhibition of the ergot compound’s metabolism in the liver. The occurrence of ergotism is unpredictable; therefore, this drug combination should not be used(8).
Alfentanil Alfentanil is a synthetic opioid usedin anesthesia; inhibition of alfentanil elimination by erythromycin was studied in volunteers.The average clearance decreased from to ml/h/kg and T,, increased from to min following erythromycin mg bid orally for days Triazolam elimination was reduced by troleandomycin threefold (from to giving riseto psychomotor disturbances
Disopyramide Disopyramide is an antiarrhythmic agent that undergoes oxidative metabolism inthe liver. Concomitant administration of this agentwith erythromycin caused prolongationof the QT interval on electrocardiogram and intraventricular conduction disturbances as a signof increased plasma level of disopyramide Amiodarone, another antiarrythmicagenthasalso caused increasedQT intervals in patients who receivedit with erythromycin concomitantly.
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Terfenidine Erythromycin alters the metabolism of terfenidine an antihistaminic agent which resulted in altered cardiac repolarization disturbances(29). In summary, the newer macrolides seem to offer a far greater degree of safety from drug-drug interactions, mainly because of lower degree of interaction with the hepatic cyctochrome P-450 enzyme complex. At the presenttime,azithromycin,roxythromycin,anddirithromycinhave no clinically recognizable significant interactions.
REFERENCES 1. Ludden TM. Pharmacokinetic interactionsof the macrolide antibiotics. Clin. Pharmacokineti 1985; 10:63. 2. Periti P, Mazzei T,Mini E, Novelli A. Pharmacokinetic drug interactions of macrolidesstudies. Clin. Pharmacokineti1993; 24:70. 3. Pessayre D, Larrey D, Funck-Brentano C, Benhamou JP. Drug interactions and hepatitis producedby some macrolide antibiotics. J Antimicrob Chemother 1985; 16 (suppl A):181. 4. Ortiz de Montellano PR. Cytochrome P-450: Structure, Mechanism and Biochemistry. 1986. New York: Plenum Press 5. Delaforge M, JaouenM, Mansuy D. Dual effects of macrolide antibioticson rat liver cytochromeP450 induction and formationofmetabolite complexes. A structure activity relationship. BiochemPharmacoll983; 32:2309. 6. Cummins D, Kozak PP, Jr. Gillman SA. Erythromycin effecton theophylline blood levels. Pediatrics 1977; 59:144. 7. Renton KW, Gray JD, Hung OR. Depression of theophylline eliminationby erythromycin. Clin Pharm Ther 1981; 30:422. Used. 8. Ludwig M. In:Macrolides,Chemistry,PharmacologyandClinical Bryskier AJ, Butzler J- P,Neu HC, "kens PM,eds.Oxford: AmetteBlackwell, 1993:495. 9. Hildebrandt R, Moller H, Gundert-Remy U. Influence of theophylline on renal clearance of erythromycin. Int J Clin Pharmacol Toxicol Therap 1987; 25:601. 10. Weinberger M, Hudgel D, Spector S, Chidsey C. Inhibition of theophylline clearance by troleandomycin. J AllergyClin Immunoll977; 59:228. 11. Saint-Salvi B, TremblayD, Surjis A, Lefebvre M. A study of the interaction J Antimicrob Chemoof roxithromycin with theophylline and carbamazepine. ther 1987; 20 (suppl B):121. 12. Mesdjian E, Dravet C, Cenraud B, Roger E. Carbamazepine intoxicationdue to triacetyl-oleandomycin administration in epilepticpatients. Epilepsia 1980; 21:489. 13. Wong W, Ludden TM, Bell RD. Effect of erythromycin on carbamazepine kinetics. Clin Pharmacol Ther 1983; 33:460-464.
Rubinstein and Segev 14. Dravet C, Mesdjian E, Cenraud B, Roger J. Interaction between carbamazepine and triacetyloleandomycin (Correspondence). Lancet1977; 1:810. 15. Ben-Ari J, Eistenstein B, Davidovits M, Shmueli D, Shapira Effect of erythromycin on blood cyclosporin concentrations in kidney transplant patients. Pediatrics 1988; 112:992 16. Martell R, Heinrichs D, Stiller CR, Jenner M, Keown PA, Dupre J. The effects of erythromycin in patients treated with erythromycin. Ann. Intern Med 1986; 104:660. 17. Freeman DJ, Martell R, Carruthers SG, Heinrichs D, Keown PA, Stiller Cr. Cyclosporin-erythromycin interaction in normal subjects.Br J Clin Pharmacol 1987; 23:776. 18. Gupta SK, Bakran A, Johnson RWG, Rowland M, Cyclosporin-erythromycin interaction in renal transplant patients. Br J Clin Phrmacol 1989; 27:475. 19. Lindenbaum, Rund DG, Butler W, Tse-Eng D, Saha JR. Inactivation of digoxin by the gut flora: reversalby antibiotic therapy. New Engl JMed 1981; 305789. 20. Maxwell DL,Gilmour-White SK, HallMR. Digoxin toxicity due to interaction of digoxin with erythromycin. Br Med J 1989; 198572. 21. Husserl FE. Erythromycin-warfarin interaction (Correspondence). Arch Intern Med 1983; 143:183. 22. Bachmann K, Schwartz JI, Fornet R, Frogameni A, Jaregui LE. The effect of erythromycin on the dispositionkinetics of warfarin.Pharmacology1984; 28: 171. 23. Nelson MV, Berchou RC, Kareti D, LeWitt PA. Pharmacokinetic evaluation of erythromycin and caffeine administered with bromocriptine. Clin Pharmacol Ther 1990; 47:694. 24. LaForce CF, SzeflerSJ,Miller MF, Ebling W, Brenner M. Inhibition of methyl-prednisolone elimination in the presence of erythromycin therapy. Allergy Clin Immunoll983; 72:34. Heyton A. Precipitation of acute ergotism by triacetyloleandomycin (Correspondence). Med 1969; 69:42. 26. Bartkowski RR, McDonnell "E. Inhibition of alfentanilmetabolism by erythromycin. Clin Pharmacol Ther 1989; 86:465. 27. Warot D, Bergougnan L, Lamiable D, Berlin D, Bensimon G, Danjou P, Puech N.Troleandomycin-trialam interaction in healthy volunteers:pharmacokinetic and psychometric evaluation. Eur J Clin Pharmacoll987; 32:389. 28. Ragosta M, Weihl AC, Rosenfield LE. Potentially fatal interaction between erythromycin and disopyramide.Am J Med 1989; 86:465. 29. Honig PK, Zamani K, WoosleyRL, Conner DP, Cantilena LR, Jr. Erythromycin changes terfenidine pharmacokinetics and electrocardiographic pharmacodynamics. Clin PharmacolTher 1992; 51:156. 30. Szefler SJ, Brenner M, Jusko WJ, Spector SL, Flesher KA, Ellis EF. Doseand time-related effectof troleandomycin on methylprednisolone elimination Clin Pharmacol Ther 1982; 32:166.
Macrolides, halides, and Streptogramins in Treatment of Opportunistic Infections in Immunocompromised Patients Jack S . Remington Stanford UniversitySchool of Medicine, Stanford, and Palo Alto Medical Foundation, Palo Alto, California
INTRODUCTION The purpose of thismeetingwas to reviewnewerinformation on the macrolides, azalides, and streptogramins. For this reason, we shall attempt to confine our remarks to certain newer observationson these drugs as they relate to the treatment and prevention of infections in immunocompromised individuals. Much of the available data presented at the meeting were incomplete;many of the studies are still in progress.The usefulness of the newer macrolide and azalide antibiotics has only begun to be appreciated (Table 1). Thisisreminiscent of the many antibiotics licensed by governmental drug regulatory bodies that initially were consideredto have a relatively narrow spectrumof activity. Withfurther study andneed, a far broader spectrum was often discovered that turned out to be lifesaving. Here, we refer to such antimicrobial agents as trimethopridsulfamethoxazole (a “urinary tract antibiotic”) with its extended spectrum against Pneumocystis carinii and Toxoplasmagondii, the extended useof clindamycin (a “gram-positive antibiotic”) to include not only anaerobes but also 189
190 Table I
Remington Newer Macrolide Antimicrobials
Advantages compared with erythromycin Improved bioavailability Fewer adverse effects Fewer drug interactions Improved antibacterial spectrum Activity against MAC Disadvantages compared with erythromycin No parenteral form available in the United States Cost
Toxoplasma gondii and Pneumocystis carinii. It is likely that the full antimicrobial spectrum of the newer macrolides, azalides, and streptogramins and their usefulness for treatment of opportunistic infectionsin immunocompromised patients will be evengreater than now appreciated.
LEGIONELLA Many of the newer macrolides and the azalide azithromycin have significant advantages when compared with erythromycin (see Table 1). These include improved bioavailability, fewer adverse effects, fewer drug interactions,andimprovedantibacterialspectrum. One disadvantagein the United States is that parenteral forms of clarithromycin and azithromycin are not available. Depending on the geographical locale, there may be a significant cost differential between the use of erythromycin andthe newer macrolides that must be evaluated in light ofthe features mentioned above. Because of the appreciable untoward side effects of high-dose oral and intravenous erythromycin, many physicians who treat Legionnaire’s disease frequentlynow use clarithromycinor azithromycin in preferenceto erythromycin. Although each of these drugs is active against a variety of strains of legionella, meaningful differences in clinicalor bacteriologic response and outcomein humans have not been demonstrated. number of studies have been performed to assess the in vitro activity of different macrolides on intracellular Legionella. Considerable differences in activity were obtained by different investigators, likely reflectingthe lack of standardization of the procedure. Thus, greater or lesser activity of a given macrolide or group of macrolides against intracellular Legionella should not necessarily be interpreted as a reflection of their in vivo activity or extrapolated to what might be their potential usefulness in humans. Any significant differences inthe clinical efficacy of these drugs against Legion-
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naires’ disease will only come from appropriately designed clinicalTotrials. date, almost all such trials have been retrospective. One recently published trial by Kuzman et al. (1) was a retrospective, noncomparative study in 16 patients in whom the diagnosis was serologically confirmed. Azithromycin days. was administered orallyfor a total dose of 1.5 g over a periodof All of the patients were stated to have been cured. Stuart and Yu have recently stated that because of gastrointestinal intolerance, the requirement for large fluid volume administration andthe toxicity the 4-g dose of erythromycin, the newer macrolides will likely displace erythromycin as the drugs of choice for the treatment of legionellosis (2).
PNEUMOCYSTIS CARINII The AIDS epidemic in the United States was heralded by Pneumocystis carinii pneumonia (PCP). Inthe early yearsof the epidemic, diseasedue to this organism accounted for more than 60% of AIDS-defining illnessesand occurred in80% of persons with AIDS. Prophylacticregimens,primarily trimethoprim/sulfamethoxazole (TMP/SMZ) or pentamidine have dramatically reduced the incidence of this opportunistic disease. Unfortunately, many patients suffer significant untoward effects when given TMPMSZ propylaxis, and pentamidine is less effective. Thus, there is a needfor effective and less toxic drugs for prophylaxis of thisinfectionin AIDS patients.Whether the macrolidesand azalides or streptogramins will play any role in such preventive measures remains to be shown. The activity of clarithromycin alone and in combination with sulfamethoxazole inrata model would suggestthat studies will be conducted in humansto clarify this. An interesting observationin this regard was presentedat this meeting by Crampton and colleagues In a study similar to that described below for giardiasis, these authors assessed whether clarithromycin as administered in the multinational clarithromycin prophylaxis trial described abovehadanyeffecton the occurrence ofPCP andconcluded that clarithromycin prophylaxis formycobacterium aviumcomplex (MAC) provided additional prophylaxis for PCP. It should be notedthat these patients were allowedto continue on whatever prophylaxisfor PCP their physicians considered appropriate. Whether there were differences in prophylaxis, duration of prophylaxis, dosing, and forth, during the trial was not stated. Thus,whether the remarkableandstatisticallysignificantresult between the clarithromycin recipients and placebo group (p = .021) was indeed due to clarithromycin or to some other factor(s) will hopefully be clarified whenthe results are analyzed andthe data published.
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GIARDIASIS
Giardia lamblia, the most common parasitic cause of traveler’s diarrhea (4), has been described in HIV-infected patients with diarrhea and was classically associated with the “gay bowel syndrome..” In other patients classified as immunocompromised,it is those with dysgammaglobulinemia in whom giardiasis is most closely associated with an immune defect (5). Whether diseasedue to this parasite is more severe in the immunologically impaired patient is unclear. Craft and his colleagues presented some intrigu ing data on the effect of clarithromycin on giardiasis inAIDS patients (6). In the large prospective randomized multicenter, multinational study of clarithromycin versus placebo for preventionof MAC described above, Giardia lambia wasfoundin10(2.9%) of the patients who had been randomized to the placebogroup,whereas it wasidentifiedinonly (0.9%) of patients randomized to receive clarithromycin (p = .048). Of interest isthe observation that less diarrhea was experienced bypatients in the clarithromycin group than inthe placebo group. Althoughthe author’s conclusion of their data was that clarithromycin prophylaxisfor MAC infections also prevents development of giardiasis in patients with AIDS who have CD4 cell countsof < 100 cells/pl, there was insufficient information in the abstract to allow one to determine whetherother differences, including other therapy and/or disease entities, influenced the presence or absence of giardiasis and whether all patients in the study were screened for this pathogen if they developed diarrhea.
TOXOPLASMOSIS Toxoplasma gondii, a cause of life-threatening infectionsin immunocompromised patients including those with hematologic malignancies, organ transplants (especially bone marrow and heart), and AIDS (7-g), is susceptible to the newer macrolides-roxithromycin, clarithromycin, and azithromycin. The latter twohavebeenstudiedinclinicaltrials. In 1991, Fernandez-Martin and colleagues (10) reported the first resultsof a trial in which clarithromycin was usedfor the treatment of toxoplasmic encephalitis. These investigators obtained promising results in their pilot study performed in11AIDS patients with toxoplasmic encephalitistreated with the combination pyrimethamine (75 mg/day) and clarithromycin (1 g q12h). The protocol was completed in eight patients and discontinued in five patients (2 = voluntary withdrawal,2 = deterioration of neurological condition and thrombocytopenia,1 = suspicion of liver toxicity). Sixty-twopercent of these patients had a complete and23% a partial clinical response; 15% diedby week of therapy. Of the surviving patients,27% had clarith-
Use of MAS in Immunocompromised Patients
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romycin discontinued because of drug toxicitythat resulted in thrombocytopenia and/or liver enzyme abnormalities. Clinical and CT scan responses at 6 weeks of treatment were 80% and 50%, respectively. l b o patients died, one of toxoplasmic encephalitis and the other of cerebral bleeding considered to becausedbypyrimethamine-inducedthrombocytopenia. lbenty-four percent of the patients experienced significant increases in liver enzymes and hearing loss was noted in 15%; 31% experienced severe hematologic abnormalities. The authors concluded that their results with clarithromycidpyrimethaminewere comparable to the results of previous separate studies inwhich pyrimethamine/sulfathiazine or pyrimethamineklindamycin were used for therapy of acute toxoplasmic encephalitis in patients with AIDS. They alsostated that the optimal doseof clarithromycin is yet to be defined. Further studies examining the efficacy of clarithromycinisyet to bedefined. Further studiesexamining the efficacy of clarithromycin have not been reported and, to the best of my knowledge, none are in progress. In another report in which onlya single case is described, there was a dramatic responseto azithromycin. The patient had toxoplasmic encephalitis, was allergic to sulfonamide and pyrimethamine, and had beentreated unsuccessfully with clindamycin and doxycycline (11). In a recent trial (ACTG 156; data and information provided by Dr. Benjamin Luft) designed to characterize the clinicalandradiologicresponse rates in the treatment of toxoplasmic encephalitis by cohort, patients were treated with azithromycin in combination with pyrimethamine as shown in Table 2. The numbers of patients in each cohort were relatively small, making analysisof the data more difficult. The data presented here are froman initial analysis. Classificationof the 32 evaluable patients was as follows: 10 (31%) responders; 3 (9%) potential responders; 7 (22%) relapse failures; 9 (28%) induction failures; 3 (9%) indeterminate. There was no significant difference in theserates by cohort. When the figures for induction and relapse failures were combined, they yielded an overall fail-
Table 2 AzithromycinPyrimethamine for Treatment of Toxoplasmic Encephalitis inAIDS (ACTG Trial 156) Drug regimen
Pyrimethamine Cohort
I I1
111
Azithromycin mg qd mg qd mg qd
25-50 mg qd mg qd mg qd
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ure rate of 73% (8/11), 43% (6/14), and50% (2/4) respectivelyfor cohorts I, 11, and 111, without a significant difference between cohort groups. The percentage of patients respondingby week 6 was 64% (7/11), 79% (11/14), and 50% (2/4) respectively for cohortsI, 11, and I11 and, again, no significant differences betweenthe cohorts were noted. Untilthe data analysis is completed, a final conclusion about the effectiveness of the azithromycin/ pyrimethamine regimen cannot be made. What is evident from this study i that azithromycin is effective (when used in combination with pyrimethamine) for treatment of some cases of toxoplasmic encephalitis in AIDS patients.
CRYPTOSPORIDIUM
Cryptosporidia, a protozoan parasite, may cause severe and persistent diarrhea in immunologically impaired patients including the neutropenic patient, bonemarrowandrenaltransplantrecipients,thosewith certain immunoglobulin deficiencystates, and those with AIDS (12-14). Severe, unremitting infectionof the gall bladder and gastrointestinal tract occurs in such settings. Unfortunately, no single therapeutic regimen has been described that dependably eradicated cryptosporidia fromthe intestine and gall bladder in sufficient numbers of patients to allow for afirm recommendation regarding optimal specifictreatment for the parasite. In a trialof roxithromycin, mg orally bidfor 4 consecutive weeks, for therapy of AIDS-related diarrhea caused by cryptosporidium, Sprinz reported his initial results in 21 patients in Port0 Alegre, Brazil (15). In 11 (52.4%), remission in symptoms was complete and in 6 (28.6%), the response was partial with a decrease in numbers of diarrheic evacuationsfor 1 month of follow-up;4(19%)failed to respond to the treatment. This investigator concluded that roxithromycin treatment resulted in an overall favorable outcomein more than 75%of their cases andthat the antibiotic was relatively safe and well tolerated. Their study is ongoing. In a similar study from Sao Paulo, Brazil, Ulp and his colleagues (16) studied 22 adults with AIDS who were infected with cryptosporidia; 17 had significant diarrhea. The treatment regimenwas the same as described above forthe study by Sprinz. In this study,the investigators stated that good results [absence of cryptosporidium on fecal examination or colonoscopy after the end of treatment (50%), or improvement defined as persistence of cryptosporidium inthe feces but an increase in weight of the patient and/or a decrease in number of evacuations/day (27%)] with roxithromycin were obtained in 72.7% of their patients and that the drug was well tolerated. Amsden and Bessette from Worcester, Massachusetts described their results with azithromycin in the treatment of an HIV-negativepatient with
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severe diarrhea caused by cryptosporidium (17). This case,in an otherwise normal individual, andthe therapy used appear relevant to theproblem of cryptosporidiosis in immunocompromised patients. The patient was a 42year-old male whose diarrhea continued despite treatment with loperamide and by the time of hospitalization for severe diarrhea and dehydration; he had lost 24 pounds. He was placed on azithromycin 1250 mg/day by mouth and within 48h he had resolution ofhis diarrhea and subsequently was asymptomatic. He was treated for 14 days and his stools became negative for cryptosporidia.The investigators feltthat they had ruledout spontaneousresolutionas a cause of the excellentresults. Further studies of azithromycin seem warranted for this disease inAIDS patients and other immunocompromised patients as well as in otherwise normal individuals. One wonders why further studies as follow-up to the promising results of Soave and her colleagues (18) have not been published to clarify where this drug belongs inour armamenarium fortreatment of this disease.A trial of intravenous azithromycin is presently ongoing and hopefully will answer the question as to whether this antimicrobial willeradicate cryptosporidia from extraintestinal sites such as the biliarytractandtherebyprevent recurrence. MYCOBACTERIUM AVIUMCOMPLEX The mycobacterium avium complex (MAC) is the most common cause of systemic bacterial infection inAIDS patients in the United States, where the lifetime incidenceof this disease ranges from18% to 43%. Individuals at greatest risk for disseminated disease are those with CD4+ cell counts of less than75 cellslpl andwho have hada prior AIDS-defining opportunistic infection. The portals of entry appear to be both the gastrointestinal and respiratory tracts. Disseminated MAC predisposes to earlier death than occurs in AIDS patients without this disseminated infection (19). Because of its frequency andthe severe debility it causes, it has been a prime focus for drug discovery for both prevention and treatment. Optimally, it would be ideal were a single drug, or even two drugs, identified that would prevent multiple opportunistic infections in AIDS patients. Becauseof their broad antimicrobial spectrum,that includes both bacterial and protozoal pathogens, the newer macrolide/azalide drugs have the potential for preventing a number of such infections. Inthe past several years, giant steps have been made in both prevention and treatment of MAC infections in AIDS patients. These advances have extended to and benefit non-AIDS patients who acquire this infection and for whom we previously had littleto offer inthe way of effective treatment. A number of interesting studies and observations were presented at this meeting.
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Prophylaxis Chrithromycin In a recent prospective, randomized, double-blind, multicenter, multinational,placebo-controlledtrial(M91-561; Abbott) in682 patients of clarithromycin for prophylaxis of disseminated MAC infection in AIDS patients with CD4 countsof < 100 cells/mm3, this macrolide was foundto provide effective and safe prophylaxis andwas associated with longer survival than observed in untreated patients (data provided by Dr. J Carl Craft, Abbott Laboratories). There were 341 patients in each arm of the study. Clarithromycin was given at a dose of 500 mg PO bid. The overall beneficial effectswereclassifiedas either bacteriologic or clinical. The primary bacteriologic benefit was a 69% reduction inofrisk developing MAC infection. The primary clinical benefit was improved survival. A significantly higher proportionof clarithromycin patients than placebo patients reported one or more adverse events; these were attributable to higher rates of digestive events andtaste perversion. Patients inthe clarithromycin group had a statistically significant reduction in mortality of 28%, compared to the plaof MACprophycebo group(p C .OS). This is the first well-controlled study laxis to reveal such a significant survival benefit. Of interest, patients in the clarithromycin arm experienced a statistically significant(p < .05) reduced riskof hospitalization and of developing symptoms associated with MAC bacteremia. Risk of hospitalization was reduced by 23% and the risk of bacteremia accompanied by weight loss, pyrexia, night sweats, and anemia were each reduced by more than 80% when comparedto the placebo group. Greater than 50% of MAC strains isolated from the clarithromycin recipients with breakthrough bacteremia were resistantto clarithromycin, whereas noneof the MAC isolates fromthe placebo recipients were resistant to clarithromycin might have been due to a previously existing (prior to clarithromycin prophylaxis) active (without bacteremia) MAC infection in these patients. In regard to breakthrough bacteremias, it is interestingto note that when such breakthroughs have occurredin studies in which patients were receiving rifabutin, the breakthrough usually involved rifabutin-susceptible strains,whereasinthosereceivingclarithromycin it usuallywaswith clarithromycin-resistantstrains. Azithromycin Following the report of promising results with azithromycin treatment of MAC infection in patients with AIDS by Young and his colleagues from San Francisco, California (20),there has been increased interest infurther
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evaluation of this drug for both prevention andtreatment of this infection in AIDS patients. In two recent randomized, double-blind comparative trials, azithromycin, 1200 mg once weekly, was evaluated to determine its role in prevention of disseminated MAC inAIDS patients withCD4 counts of < 100 cells/mm3 (see Ref. 21 and data provided by Dr. Michael Dunne, Pfizer Central Research). Azithromycin alone was compared with rifabutin alone or with azithromycin in combination with rifabutin.The mean duration of therapy was 11months. The incidence of disseminated MAC inthe 3 groupsof patients in an intent-to-treat analysis was: azithromycin alone, 13.9%; rifabutin alone, 23.3%; azithromycin plus rifabutin, 8.3%. The difference betweenthe azithromycin and rifabutin groups was significant (p = 0.11). The percentages of side effects in the three groups of patients at any time were azithromycin 78.1% rifabutin 59.7%; and azithromycin plus rifabutin 83.5%. Side effects were primarily (approximately 60%) diarrhea, mostly mild to moderate. In approximately 5% of patients, the diarrhea was stated to have been severe. The percentage of discontinuations were azithromycin 13.5%, rifabutin 15.9%, and azithromycin plus rifabutin 22.7%. In the second study, azithromycin was evaluated against placebo in 180 HIV-infected patients with CD-4 counts of < 100 cells/mm3.The mean duration of therapy was 1year. The incidence of disseminated MAC inthe two groups of patients was azithromycin 15.3% and placebo 30.3% (p = The percentage of side effects were azithromycin 79.8% and placebo 31.9%; of discontinuations, azithromycin 8.2% and placebo 2.3%.
Treatment Chrithromycin
Chaisson and his colleagues (22) presented data obtained in a controlled trial of clarithromycin plus ethambutol with or without clofazamine for treatment of MAC bacteremia in AIDS patients. The doses given were clarithromycin 500 mg bid orally and ethambutol 15 mg/kg/day with randomization to receive either clofazimine 100 mg/day or no clofazimine.The median time to obtaining negative cultures was 58 days for the two-drug regimen and 63 days the for three-drug regimen.The proportion of patients that became culture negativewas in the two-drug group and 54% in the three-drug group. Fever and night sweats improvedin approximately the same number of patients in each group, whereas the proportion of patients that had to be withdrawn from therapy was higher in the threedrug group (22% versus 13% in the two-drug group). The authors concluded that clarithromycin is effective in treating MAC bacteremia, that it prevents emergenceof resistance, andthat the addition of clofazimine does
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not contribute to the clinical response [it was actually associated with a higher mortality (p = .03)]. In another study, Craft and his colleagues (23) reported that the addition of ethambutol or ethambutol plus clofazimine to clarithromycin significantly reduced developmentof clarithromycin-resistantMAC in patientsbeing treated for disseminatedMACinfection. The dosages of clarithromycin, ethambutol, and clofazimine were essentially the same as in the study reported by Chaisson et al. In another study by Craft and his colleagues (24), clarithromycin-resistant MAC was noted during prophylaxis only in patients with CD4 counts of 25 cells/mm3and CD8 counts of 780 cells/mm3. The investigators stated that their results suggest that clarithromycin-resistant MAC reflect treatment failures in patients who had disseminated nonbacteremic MAC infections at the start of prophylaxis. The latter seems likely. Of potential importance to the non-AIDS immunocompromised patient with MAC isthe report at this meeting by Wallaceand his colleagues from the University of Texas Health Center in Qler, Texas (25). These investigators employed clarithromycin in treatment regimens for pulmonary infections caused by MAC. Non-AIDS patients were treated until culture negative for 1 year with clarithromycin, 500 mg bid (as initial monotherapy in some of them), ethambutol, rifabutin or rifampin and, initially, with streptomycin.In 6 of 39 patients, the organism became resistant to clarithromycin, most likely related to thefact that they had received clarithromycin as monotherapy. None of the 25 patients who completed their therapy relapsed during a 19-month period of follow-up. Despite reduced levels of clarithromycin serum levels caused by their rifampin, of 13 patients that received these two drugs together in their treatment regimens were treated successfully. The authors concluded that the clarithromycin-containingtreatment regimen is superior to those in which clarithromycinwas not included. patients were randomized In a studyby Dube and his colleagues to receive clarithromycin1gm bid plusclofazamine withor without ethambutol inan attempt to prevent microbiologic relapse in AIDS patients with MAC bacteremia.The two groupsof patients were generally comparable. The response rate was similar in both groups. The risk of relapse was remarkably and significantly greater in those patients who were receiving the two-drug regimen. Thus, the three-drug regimen of clarithromycin/ clofazamine/ethambutol wasmoreeffective than the two-drugregimen clarithromycin/clofazamine in preventing microbiologic relapse in AIDS patients with MAC bacteremia. Pierce et al. demonstrated in a prospective trial that MAC was a predictor of mortality. In the multicenter prophylaxis trial mentioned above, the data support their hypothesis that
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Table 3 Manifestations of Bartonella (Rochalimaea) Infection in Immunocompromised Patients
Peliosis hepatitis Bacillary angiomatosis Endocarditis Chronic lymphadenopathy Meningitis
the favorable effect of clarithromycin on survival in advanced AIDS was due primarily to prevention of MAC.
BARTONELLA Bartonella species are fastidious gram-negative bacilli that recently have been recognized asthe cause of significant and often life-threatening infections inimmunocompromisedpatients.Certainmanifestations of these infections are shown in Table 3. Macrolide antibiotics, includingerythromycin,clarithromycin,azithromycin,roxithromycin,anddirithromycin, have been shown to have excellent in vitro activity against B. visionii, B. hemelae, and B. quinfana (Table 4) (28,29). A number of antibiotics have been used successfully to treat various manifestationsof bartonellosis, including erythromycin and doxycycline, with resulting resolution of skin lesions and improvementin constitutional symptoms(30). Recently, Guerra and colleagues reported a case of AIDS-related bacillary angiomatosis ina 28-year-old male witha CD4 count of 100/mm3 who presented with fever, hepatomegaly, weight loss, and multiple polypoid, angiomatous lesionson his facethat were found on biopsyto be due to bacillary angiomatosis.He was treated with oral azithromycin, 1g daily as a single dose, and experienced rapid resolutionof skin lesions. After 1 Table 4 MICs (pg/ml) for 14 Strains of Bartonella
Erythromycin Clarithromycin Azithromycin
0.06-0.12 0.003-0.03 0.006-0.03
Note: Similar M 0 I for B. visionii, B. henselae, B. quintana. Source: Maurin et al. (1995).
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week of therapy, there was adiminution in size of his liverand spleen noted on computed tomography with resolution of the attenuation lesions in these organs. At 10-month follow-up, there was no trace of skin lesions suggestive of bacillary angiomatosis and neither liver nor spleen were pal ble. Blood cultures remained negative. At this meeting, Paucar and his colleagues reported a case inwhich there was coexistence of bacillary angiomatosis and Kaposi’s sarcoma in a black African woman who was treated successfully forher Bartonella infection with azithromycin, mg/ day for days and, thereafter, clarithromycin (500 mg/day) was administered for months. The in vitro results along with recent clinical experienc in limited numbers of cases suggestthat these new macrolides hold promise for treatment of this infectionin immunocompromised patients.
COMMUNITY-ACQUIRED PNEUMONIA
As is true for immunologically competent patients, those who are immunologically compromisedare susceptibleto organismsthat arecommon causes of community-acquired pneumonia. Of particular interest in this regard is that the macrolides and azalides are active against five of the common pathogens (Table Thus, althoughthese organisms do not always have a particular predilection for the immunocompromisedpatient, those individuals whoare not severely immunocompromised ill will likely do well when treated with the oral preparations of these antimicrobials. The more severely ill immunocompromised patients with community-acquired pneumonia will likely require hospitalization and intravenous antibiotic therapy. Recently completed is a multicenter trial of erythromycin plus cefuroxime versus azithromycin alone (500mg IV for days followed by 500 mg PO for days for a total duration of azithromycin of days) in community-acquired pneumonia. This five-center trial in the United States has foundthat azithromycinwas as efficaciousas erythromycidcefuroxime. Furthermore, azithromycin was better tolerated than erythromycin. (Personal communication from Dr. Victor Yu, Table MacrolidedAzalides are Active Against Five Common Pathogensof Community-Acquired Pneumonia
S. pneumoniae H . infiuenzae M . pneumoniae C. pneumoniae L. pneumophila
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VANCOMYCIN-RESISTANT E . FAECZUM The streptogramin, quinupristiddalfopriistin is being used in multiple trials for treatment of gram-positive infections, including those caused by vancomycin-resistant E. faecium. Undoubtedly, this latter organism will prove a significant problem in immunocompromised patients. W Osuch cases of life-threatening infectionsin the setting of severe immunosuppression, one in a patient with chronic renal failureon peritoneal dialysis who developed vancomycin-resistant E. faecium peritonitis and the other in a severely neutropenic patient with lymphoma who was receiving chemotherapy were seenat the consulting service at Stanford. The latter patient was a 44-year-old female with large cell lymphoma and recentCHAD therapy. On November 6,1995, she developed fever in a setting of severe neutropenia. Blood culturesgrew E. faecium sensitive to chloramphenicol, without high-level resistance to streptomycin but with resistance to penicillidampicillin, vancomycin, gentamicin, ciprofloxacin, tetracycline, andtrimethopridsulfamethoxazole.It had intermediate sensitivity to rifampin. On November 10, chloramphenicol was begun for the vancomycin-resistantE. faecium isolated from multiple blood cultures taken fromNovember6 to November10. On November 11, quinupristid dalfopristin 7.5 mgq8hwas begun. All subsequent blood cultures were negative. It cannot be stated with assurance that quinupristiddalfopriistin resulted in the negativebloodculturessinceshehadbeenplaced on chloramphenicol the daybefore. Quinupristiddalfopristin hadbeenrequested on a compassionate plea basis because the patient’s infection was life-threatening and in a situation in which chloramphenicol has not been proven in sufficient numbers of cases to be effective when usedalone.
ACKNOWLEDGMENT This work was supported by National Institutes of Public Health Services Grants A130230 and AI04717.
REFERENCES 1. Kuzman I, Soldo I, Schonwald S, Culig J. Azithromycin for treatment of community acquired pneumonia cased by Legionella pneumophila: A retrospective study. Scand J Infect Dis 1995; 27503-505. 2. Stuart JE, Yu, VL. Current concepts: Legionellosis. N Engl J Med 1996; Cramptom S, Craft JC, Notario G , Henry D. Prevention of Pneumocytstis carinii pneumonia in A I D S patients by clarithromycin prophylaxisfor Mycobacterium avium complex (MAC).The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon,1996; abstr. 7.09.
Remington 4. Connolly GM, Forbes A, Gazzard BG. Investigation of seeminglypathogennegative diarrhoea in patients infected with HIV-1. Gut 1990; 31:886-889. Ament ME, Ochs HD, Davis SD. Structure and function of gastrointestinal tract in primary immunodeficiency syndrome: A study of 39 patients. Medicine 1973; 52:227-248. 6. Craft JC, Henry D. Giardia lamblia prevention during MAC prophylaxis with clarithromycin 500 mg BID in AIDS patients with CD4 counts < 100 cells/ mm3. The Third International Conference on the Macrolides, halides and Streptogramins, Lisbon, 1996: abstr 7.28. 7. Israelski DM, Remington JS. Toxoplasmosis in the non-AIDS immunocompromised host. In: Current Clinical Topics in Infectious Diseases, Vol.13. Remington JS and SwartzMN, eds. Boston: Blackwell Scientific Publications, 1993~322-256. 8. Israelski DM, Remington JS. Toxoplasmosisin patients with cancer. Clin Infect Dis 1993; 17:S423-S435. 9. Wong SY, Remington JS.Toxoplasmosis in the setting of AIDS In: Broder, S, Merigan TC, Bolognesi D, eds.Textbook of AIDS Medicine. Baltimore: Williams and Wilkins,1994:223-257. 10. Fernandez-Martin J, Leport C, Morlat P, Meyohas MC, Chauvin JP, Vilde JL. Pyrimethamine-clarithromycin combination for therapy of acute Toxoplasma encephalitisin patients with AIDS.AntimicrobAgents Chemother 1991; 35~2049-2052. 11. Farthing C, Rendel M, Cume B, SeidlinM,Azithromycin for cerebral toxoplasmosis. Lancet 1992; 339:437-438. 12. Current WL, Reese NC, Ernst J V , et al. Human cryptosporidiosis in irnmunocompetent and immunodeficient persons: Studiesof an outbreak and experimental transmission. N Engl J Med 1983; 308:1252-1257. 13. DuPont HL. Cryptosporidiosis and the healthy host. N Engl J Med1985; 312:1312-1320. 14. Weisburger W R , Hutcheon DF, Yardley JH, et al. Cryptosporidiosis in an immunosuppressed renal-transplant recipientwith IgA deficiency. J Clin Patholl979; 72:473-478. 15. Sprinz E, AIDS-related cryptosporidiumdiarrhea: A pilot study with roxithromycin. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 7.26. 16. Ulp DE, Lima, ALLM Amato, VS, Neto, VA. The use of roxithromycin in the diarrhea caused by cryptosporidium sp associated with A I D S . The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 7.27. 17. Amsden GW, Bessette RE, Treatment of non-HIV cryptosporidial diarrhea with azithromycin. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon,1996; abstr 8.12. 18. Soave R, Havlir D, Lancaster D, Joseph P, Leedom J, Clough W, Geisler Dunne M. Azithromycin (U) therapy of AIDS-related cryptosporidial diarrhea (CD): A multi-center, placebo-controlled, double blind study. Program
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and Abstracts of the 33rd Interscience Conferenceon Antimicrobial Agents and Chemotherapy, American Societyfor Microbiology, New Orleans, abstr Benson C. Disseminated Mycobacterium avium complex disease in patients with AIDS. AIDS Res Hum Retroviruses Young LS, Wiviott L, Wu M, KolonoskiP, Bolan R, Inderlied CB. Azithromycin for treatment of Mycobacterium aviumintracellulare complex infection in patients with AIDS. Lancet 1991; 338:1107-1109. Havlir DV, McCutchan JA, Bozzette SA, Dunne M, CCTG/MOPPS Study Investigators. University of California, San Diego, and Pfizer Center Research, Groton, CT. A double-blind randomized studyof weekly azithromycin, daily rifabutin, and combination azithromycin and rifabutinfor the prevention of Mycobacterim aviumcomplex (MAC) inAIDS patients. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, abstr Chaisson R E , Keiser P, Pierce M, Fessel WJ, Ruskin J, Lahart C, Meek K. Controlled trial of clarithromycidethambutolwith or without clofaziminefor the treatment of mycobacterium avium complex bacteremia Ain I D S patients. The Third International Conference on the Macrolides, Azalidesand Streptogramins, Lisbon, abstr Craft JC, Siepman N, Notario G, Gupta S. The addition of ethambutol with or without clofazimineto clarithromycin for disseminated Mycobacterium avium complex (MAC) treatment. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon abstr Craft JC, Henry D, Olson CA, Notario G, Hom R, Crampton S, Pierce M. Prevention of resistance (R) to clarithromycin (C) during prophylaxis for disseminated Mycobacterium aviumcomplex (MAC).The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, abstr 25. Wallace RJ Jr, Brown BA, Griffith DE, Girard W, Murphy D. Clarithromycin (CLARI) regimens for pulmonary Mycobacteriumavium-intracellulare (MAI) in non-AIDS: longterm followup of the first 50 patients. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, abstr Dub6 M, Sattler F, Tomani F, See D, Havlir D, Kemper C, Dezfuli M, Bozzette S , Bartok A, Leedom J, TillesJ, McCutchan J. Clarithromycin plus clofazimine withor without ethambutol for the prevention of relapse of MAC bacteremia inAIDS. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, abstr Pierce M, CramptonS , Henry D, Craft C, Notario G. The effect of MAC and its prevention on survival inpatients with advanced HIV infection. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, abstr Maurin M, Raoult D. Antimicrobial susceptibilityof Rochalimaea quintana, Rochalimaea vinsonii, and the newly recognized Rochalimaea henselae. Br SOC Antimicrobi Chemother
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29.Ives TJ, Regenery R L , ManzewitschKebedeM. In vitro evaluation of macrolide antibiotics against bartonela (rochafimaea)hemselae, B. quintana and B. elizabethae via immunofluorescent antibody testing. The Third International Conference on the Macrolides, halides and Streptogramins, Lisbon, 1996; abstr 30. Guerra LG, Neira C Y , Boman D, H0 H, Casner PR, Zuckerman M, Verghese A. Rapid response of AIDS-related bacillary angiomatosis to azithromycin. Clin Infect Dis 1993; 17:264-266. 31. Paucar D, Kabeya K, HermansP, Van Laethem Y,Clumeck N. Coexistence of bacilaryangiomatosis (BA) and kaposi's sarcoma (KS) in black African woman: First description and treatment with macrolides. The Third International Conference on the Macrolides, halides and Streptogramins, Lisbon, 1996; abstr 7.25.
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15 Legionella and Spirochetes I. M. Hoepelman University Hospital Utrecht, The Netherlands
Daniel M. Musher Baylor College of Medicine and Veterans’ Affairs Medical Center Houston, Texas
LEGIONELLA Depending on the seasonandlocation, about 2-10% of communityacquired pneumonias (CAPS)are caused by Legionella spp. Most cases of Legionnaires’ diseaseare caused by Legionella pneumophila, although infections by other species are well documented. Legionella spp. belong to the group of supposedly “atypical” organisms, together with Chlamydia pneumoniae, Coxiella burnetti, and Mycoplasma pneumoniae. The formerly sharp distinction between typical pneumonia (usually caused by S. pneumoniae and said to be characterized by an abrupt onset, high fever, chest pain, tachycardia, and consolidation, together with the production of purulent sputum) and atypical pneumonia, caused by a different set of microorganisms and causing a different clinical picture (vide infra) is probably of historical interest. Recognitionof substantial clinical overlap,better diagnostic tools,the adoption of broad-spectrum regimensfor empiric cov207
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erage of CAP, and the introduction of newer antimicrobial agents that are equally effective against typical and atypical pathogens (1) have all ledto the blurring of this distinction.
Clinical Picture The clinical syndromeof Legionnaires’ diseaseis characterized by a nonproductive cough, pulse-temperature dissociation, abnormal liver function tests, rise in serum creatinine, diarrhea, hyponatremia, hypophosphatemia,myalgia,confusion,andrespiratoryfailure(2,3).However,ithas been shown that none of these signs, symptoms,or laboratory abnormalities is characteristic for legionellosis (2). In several prospective andretrospective comparative studies, chest roentgenographic findings have been found to benonspecific(3).Moreover,oneshouldnotforget that extrapulmonary forms (without initial radiology abnormalities) also and exist may present as peritonitis, pericarditis, and endocarditis.
Laboratory Diagnosis Laboratory diagnosis of Legionnaires’ disease may be done by antibody detection with a sensitivity of 75% and specificity of 95-99%, provided samples are taken during the acute phase and 6-9 weeks later (3). However, this is not usually helpful in caring for patients. Moreover, seroconversion typically occurs with type1L. pneumophila. Detection of L.pneumophila antigenuria is a convenient and sensitive method of diagnosing the disease, although at present this test only detects disease caused by serogroup I (which causes 70-90% of all cases). Direct fluorescent antibody (DFA) testing is an excellent method for the detection of L. pneumophila in sputum and tissue fluids, but the laboratory should have experience with this technique andthe monoclonal antibodies have a short shelf life. DNA probes and polymerase chain reaction (PCR) are currently in their developmental stages. Culture remainsthe gold standard; special media and selective techniquesare needed for optimal yields.
Therapy
Currently, the only FDA-approved therapy for Legionnaires’ disease is erythromycin 500mg-1 gqid, with or without rifampin 600 mg orally or intravenously bid, for 2-3 weeks. However, one should realize that’noprospective, controlled clinical trials of Legionnaires’ disease exist, and all available treatment information is based on retrospective analyses and clinical anecdotes (4). On average, the fatality rates associatedwith erythromycintreated or tetracycline-treatedLegionnaires’disease are about twofold
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lower than those associated with nonerythromycin-treated Legionnaires’ disease (4). In a recent study(5) of severe community-acquired Legionella pneumonia, patients (30%) who received erythromycin died compared to 5 of (33%) who received rifampin in addition. Fourpatients received no antibiotics and survived. This study highlights the difficulty in treating severe Legionella pneumonia in which mortality rates can approach 3342% for patients who require intensive supportive therapy (4), as well as difficulty in evaluating efficacyof new therapies. Antimicrobial therapy for infections caused by Legionella should be dictated by what is known from retrospective clinicaldata, in vitro susceptibility testing (taking into account the facultative intracellularnature of this agent), and animal studies (using intratracheal challenge). Extracellular susceptibility testing of L. pneumophila shows that macrolides, azalides, streptogramins, fluoroquinolones, and p-lactam antibiotics are all highly effective (6). However, neither direct minimal inhibitory concentrations (MICs)nortime-killcurveassaysaccuratelypredictintracellular L. pneumophilu susceptibility In vitro methodsusing intracellular models show that the newfluoroquinolonesandrifampin are highly effective, generally somewhat more active than the new macrolides and azalides and more activethan erythromycin (6-8). Some data suggest that azithromycin andciprofloxacin are bactericidalintracellularly,whereaserythromycin and clarithromycin are bacteriostatic (3’7). Regarding the newer macrolides and azalides, animal studies on experimental Legionella pneumonia show that clarithromycin and azithromycinare consistently more active than erythromycin (9). Josamycin, tested in animal models, is almost as active as erythromycin(8). already stated, the only drug currently approved for Legionnaires’ disease is erythromycin. In patients using immunosuppressive drugs, in other patients with impaired host defenses(HIV), and patients developing ARDS, because of mortality ratesthat approach 50% even withtreatment, it is advised to add rifampicin 600 mg bid. Animal studies show that the addition of rifampicin reduces lung bacterial cell counts and diminishes lung damage (9). In one preliminary report, spiramycin was studied in patients who required intensive care admission and the mortality was 30%. The compares favorably to data from patients with severe Legionnaires’ disease treated with erythromycin(4).
Role of the Newer Macrolides and halides Clinical data on clarithromycin as therapyfor Legionella pneumonia have been presented in four studies [Parolaet al. (lo)] in a total of 79 patients (Table 1). Overall cure rates were excellent (approaching loo%), indicative
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W
5hl2
U
3 m
; m
2
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Legionella Table 2 Clinical Studies with Azithromycin
Author
criteria Entry
Myburgh et al. Kuzman et al.
CAP Retrospective
Dorrell et al.
CAP -failure Erythromycin/ rifampin CAP
Rizzato et al.
Dose Outcome study mg days mg days (5) mg 5d mg (iv) mg5d 500 mg
3d
Total no. in
66
-
818
4/4
of the fact that probably only mild cases of Legionnaires’ disease were treated. However,given the fact that in one of these studies, (U), 15 patients had failed previous therapy, this suggeststhat clarithromycin can be safely prescribed in patients with Legionnaires’ disease. The recommended therapy is250-500 mg bid for at least days. Moreover, Abbott has data on file on an additional 50 patients (84% previously treated with other antibiotics) in whom a 100% clinical cure rate was seen. (J.C. Craft, personal communication). An even smaller numberof patients have beentreated with azithromycin (12) with similarly good results. The duration of therapy ranged from to 5 days (12-15). It is known that this drug has an extremely long half-life and accumulates intracellularly in the phagocyte, where L . pneumophila is located (16). In acute exacerbations of chronic bronchitis and community-acquired pneumonia, days of therapy with this azalide has given excellent results, comparableto p-lactam antibiotics (16). Currently, neither azithromycin nor clarithromycin is available for intravenous administration. Based on intracellular susceptibility testing, animal models, and clinical studies, we believe that either of these drugs can safely replace erythromycin orally, which must be administered four times daily and isnot well tolerated. Clarithromycin shouldbe given for at least days, 250-500 mg twice daily (the highest dose we personally favor). Azithromycin, when prescribed at 500 mg daily for 10 days, is certainly as effective as clarithromycin, and therapy forshorter a period is likelyto be effective. Azithromycin showsno capacity to induce or inhibit cytochrome P-450 enzymes. This may be an advantage in immunocompromised patients treated in cyclosporineor methylprednisolone and in patients also receiving theophylline and digoxin. A compassionate use program for the IV formulation existsfor both clarithromycin and azithromycin.
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At ICMAS 111, six posters were presented on Legionella spp. Segreti et al. (abstract 1.01) showed that in their fibroblast MBC model clarithromycin was more effective than azithromycin or erythromycin. Martin et al. (abstract 1.02) showed in their extracellular MIC model that 14-hydroxy clarithromycin was additive to its mother compound; however, we have already discussedthe value of extracellular susceptibility testing. Pendland et al. (abstract 1.03) showedthat in the vitro activity of the streptogramin RPR 106972 againstLegionella spp. is similar to that of erythromycin and ciprofloxacin, and the same authors (abstract 1.04) showed in their extracellularmodel that RPR 106973ismorerapidlybactericidal than erythromycin at 2 m MIC. The study of Kuzman (abstract 1.05) was recently published (14). Additionaldata on the efficacy of clarithromycin in Legionnaires’ disease came from Parola et a1 (abstract 1.06), who treated 20 patients with proven Legionnaires’ disease(out of 200 with CAP) with this drug 500 mg bid for IV days and continuedtreatment orally for 10-15 days; all patients were cured (12). Every investigator who treats patients with community-acquired pneumonia with azithromycin and finds casesof Legionnaires’ disease is urged to report these in the literature, especially if failures occur, because this can guide expertson the dose and durationof optimal therapy.In severe cases, combination with rifampin remains advisable. Fluoroquinolones may become the drugs of choice for these severe cases, as it has been shown that they are very active in animal models and in patients, some of whom were . severely ill or immunocompromised. Exceptionally high cure rates with this class of drugs have been reported.
DISEASES CAUSED BY SPIROCHETES Erythema Chronicum Migrans Erythema chronicum migrans (ECM)the is most important skin manifestation ofLyme disease. This tick-borne disease (the most important one, accounting for over 90% of such diseasesin the United States) is caused by the spirochete Borrelia burgdorferi. Lyme disease is a systemic infection which particularly targets the skin,. joints, heart, eyes, and the nervous system. In 1994, over 13,000 Lyme cases werereported to theCenters for Disease Control, although the diagnosis may bequestioned in someproportion of these. As with syphilis, Lyme borreliosis presents in stages, all requiring different treatment regimens. The primary lesion occurs at the site of the tick bite, 3-14 days after the feed. It begins as a macule or papule that expands to become an annular lesion witha raised, red border and central
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hetes Legionella and
ug
clearing called ECM. It slowly expands, the center may become necrotic and new adjacent rings may form. Inthe untreated patient, the skin lesion usually disappears over a period of weeks. Thereafter, a period of spirochetemia associated with constitutional symptoms occurs before the viable spirochetes settle invariousorgansystems,leading to immunologically mediated damage. The second stage consists of neurologic and cardiac abnormalities, andarthritis marks the third stage. Treatment of ECM is aimed at abolishing early disease and preventing late-stage disease. Therefore, therapeutic studies should always use the development of late-stage disease as part of the evaluation criteria.There is no current consensus on the optimal antibiotic or duration of treatment, and theissue of whetherB. burgdorferi causes intracellular infection in vivo is unresolved. In general, oral doxycycline or amoxicillin have been used for 2-3 weeks to treat ECM (Table 3), whereas intravenous ceftriaxone is recommended for neurological,arthritic, and cardiac disease. The in vitro activityof the new macrolides, azithromycin, clarithromycin, and roxithromycin was compared in different studies. Tests using at least 10 clinical isolates yield M I G s of 0.06 pg/ml for erythromycin,0.015 pg/ml for azithromycin and clarithromycin and0.03 pg/ml for roxithromycin (17), comparing favorably with MIC90s for ceftriaxone 0.06 pg/ml, tetracycline 0.5 pg/ml, and ampicillin 0.25 pg/ml(l8). Although azithromycin wasmore potent than other macrolides in some experimental infections (19), other studies in mice, using an arthritis model, show that p-lactam antibiotics effectively eliminate the disease, whereas macrolides do not (19). Studies in humans show conflicting results. Disappointing results have been seenwith roxithromycin (20) and azithromycin (21) in comparison to penicillin. However,Strle et al. (22) treated ECM with either azithromycin (3 gin 5 days) or doxycycline 100 mg bid for 14 days, and a study by Weber et al. comparing with penicillin V (23) yielded favorable results with azithromycin. A comparative studyby the group of Steere also found comparaTable 3 Therapy of Lyme Borreliosis ~~
Clinical (stage) Early (stage ECM
1)
Tetracycline
10-21 qid PO mg 250 10-21 bid PO mg 100 500 10-21 tid PO mg Amoxicillin plus Probenicid 10-21 tid PO mg mg 250 PO10-21 qid Erythromycin Cefuroxime 21 bid PO 500 mg
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ble results among azithromycin (1.5 g), amoxicillidprobenicid, and doxycycline for the treatment of early Lyme disease (24). However, patients with Lyme arthritis treated with oral antibiotics (doxycyclineor amoxicillin plus probenicid)may still develop neuroborreliosis (25). At the third ICMAS, five posters were presented on Lyme disease. One poster (abstract 1.07) from Bryskieret al. showed that roxithromycin and cyclines (e.g., tetracycline) displayed synergistic activity. Another study from Gasser (abstract 1.08) showed that MICs for Borrelia spp. increase when the temperature declines from38°C to 30°C, a finding which may be important in ECM. Strle et al. (abstract 1.09) presented follow-up data from their previousstudycomparingazithromycin (AZI) 3gin 5dayswith doxycycline (DOX) 100 mg bidfor 14 days (22). During the follow-up of 12 months, one patient in each group developed majorlate manifestations of Lymeborreliosis. In 10patients(16.4%) treated with AZI and in 11 (23.4%) receiving DOX, minor manifestations were observed. GoriSeket al. (abstract 1.11) treated 30 adults with ECM withAZI 1g on day 1and mg on days 2-5. Of 25patients studied,only two were documented late complications. deMarco (abstract 1.12) studied a cohort of 89patients in a retrospective fashion to determine the effectiveness of clarithromycin, in late Lyme disease.lbenty-eight were given CL 250 mg bid for 8 weeks, 37 were given CL 500 mg for 8 weeks, and 24 were given CL 250 mg or qd500 mg in combination with a cephalosporin. In the lower-dose group 71% (20/28) improved, comparedto 78% inthe high-dose group (29/37), whereas the combination group displayed slightly less improvement. However, these alldata should be considered premature, because in our opinion more long-term follow-up data are needed to judge efficacy of a regimen in Lyme (ECM).
Syphilis It isespeciallydifficult to interpret the efficacy of therapy for syphilis because of the interaction between host defenses andthe infecting organism, on the one hand, and the natural history that includes late recurrence of tertiary disease, on the other (26). In the preantibiotic era, in the absence of therapy, host immunity brought active syphilis under control, but tertiary disease still followed in about one-thirdof untreated cases. Treatment with arsphenamine eventually succeeded in producing a clinical cure of the early lesions of syphilis in all cases, but a late relapse to tertiary disease occurred in5-770ofcases. The later in the natural course of disease that therapy was given,the lower wasthe likelihood of late disease, further illustrating the lifetime importanceof host immunityafter the initial beneficial effect of therapy.
chetes Legionella and
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Penicillin represented a spectacular advance in therapy. Active disease was eliminated with vastly shorter courses of treatment. Standard regimens were developed during careful prospective study of thousands of patients under a wide variety of dosage schedules. Initially it could not have been known, butnow it appears clear that accepted courses of penicillin also prevent late syphilis. Some authorities point out that careful prospective studies have not been completed, but, as reviewed elsewhere (26), virtually all anecdotal retrospectivedata suggest that penicillin is effective in producing a lifetime clinical cure ofsyphilisin the absence of HIV infection. Several linesof evidence suggest that Treponema pallidum may persist after therapy, indicating that ongoing host immunity plays some role in preventing a recrudescenceof infection (26). Alternative drugs such as erythromycin have been used to treat relatively tiny numbers of cases. Courses of erythromycin therapy totaling20 g were associated with > 20% early failure rate and, although one study showed uniform cureof patients using g of erythromycin, others have reported failures in 5-10%ofcases at that dosage (27,28). It is simply unknown whether erythromycin abolishes the risk of tertiary syphilis, although it greatly reduces it. Similar problems exist with availabledata on tetracycline or, more recently, ceftriaxone (29), although it seems reasonable to believe that ceftriaxone will provide cure rates identicalto those of penicillin. Evaluation of any newantibiotic regimen must be carried out with the understanding that penicillin remains a clear regimen of choice because, by now, the high rate of clinical cure throughout life is certain. Roxithromycin, azithromycin, and/or clarithromycin have been shown to be effective in vitro against Treponema pallidum and in animals experimentally infected with this organism A prospective study of 16 patients who had early syphilis using500 mg azithromycin dailyfor days appeared to yield good results, although not entirely free of relapseheinfection The work of Professor Mashkilleyson, Dr. Gomberg, and their colleagues in Moscow, as reportedat this conference, isof great importance This group has treated 100 patients who have primary or secondary syphilis using azithromycin 0.5g daily for 10 days. At the third ICMAS, they report a uniform clinical cure during observation for up to years. Serologic response was seen in 90% of cases. In the context of the very small numberof patients previouslyreported after treatment with erythromycin, this experience with azithromycin is large and important. Based on available in vitro and in vivo (animal) data, as well as available clinical observations, azithromycin might be preferredto erythromycin. Compliance will almost certainly better, be and the Russian experience
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suggests an excellent short-term clinical response. However, very strong preference should continue to be given to penicillin, for which adequate data are available on long-term outcome. Alternative therapy should be given only to those patients who have a convincing history of penicillin allergy.Such patients should be followed closely for short-term clinical response and be advised that a long-term cure is not assured. Failure to prevent congenital syphilis by treating pregnantwomen is welldocumented not onlyfor macrolides (36) but also for penicillin Finally, the efficacy of macrolides in treating syphilis inthe presence of HIV infection has not been reported.
REFERENCES 1. Niederman MS, Bass Campbell GD, Fein AM, Grossman R, Mandell LA, Marrie TJ, SarosiGA, Torres A, Yu VL. Guidelines forthe initial management of adults with community-acquired pneumonia. Am Rev Respir Dis 1993; 148~1418-1426. 2. Mane TJ. Coxiella burnem’ (Q fever) pneumonia. Clin Infect Dis 1995; 21 (SUppl3):253-264. 3. Edelstein PH. Legionnaires’ disease. Clin Infect Dis 1993; 16741-749. 4. Edelstein PH. Antimicrobial chemotherapy for Legionnaires’ disease: a review. Clin Infect Dis 1995;5 (suppl3):265-276. 5. Hubbard RB, Mathur RM, MacFarlane JT. Severe community-acquired legionellapneumonia: treatment, complicationandoutcome. Quart JMed 1993; 86:327-332. 6. Havlichek D, Saravolatz L, Pohlod D. Effect of quinolones and other antimicrobialagents on cell-associated Legionella pneumophila. Antimicrob Agents Chemother 1987;31:1529-1534. 7. Edelstein PH, Edelstein MAC. In vitro activity of azithromycin against clinical isolates of Legionella species. Antimicrob Agents Chemother 1991; 35: 180-181. H, Tomonaga A, 8. Saito A, Sawatari K, Fukuda Y, NagasawaM,Koga Nakazato H, Fujita K, Shigeno Y, Ama YS, Yamaguchi K, Izumikawa K, Hara K. Susceptibility of Legionella pneumophila to ofloxacin in vitro and in experimental Legionella pneumonia in Guinea pigs. Antimicrob Agents Chemother 1985; 28:15-20. 9. Fitzgeorge RB, Lever S , Baskerville A. A comparison of the efficacyof azithromycin and clarithromycin in oral therapy of experimental airborne legionnaires’ disease. J Antimicrob Chemother 1993; 3l(suppl E):171-176. 10. Parola D, De Maio F, Minniti R, Tronci M. Clarithromycin in Legionnaires’ disease. ICMAS 3,1996; abstr 1.06. 11. Hamedani P, Ali J, HafeezS , Bachand R Jr, Dawood G, Quereshi S, Raza R, Yab S. The safety and efficacy of clarithromycin in patients with Legionella pneumonia. Chest 1991; 100:1503-1506.
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Rizzato G, MontemurroL,Fraioli P, Montanan G, Fanti D, Pozzoli R, Magliano E. Efficacy of a three day course of azithromycin in moderately severe community-acquired pneumonia.Eur Respir J Myburgh J, Nagel GJ, Petschel E. The efficacy and tolerance of a three-day course of azithromycin in the treatment of community-acquired pneumonia. J Antimicrob Chemother (suppl Kuzman I, Soldo I, Schonwald S, Culig J. Azithromycin for treatment of community acquired pneumonia caused by Legionella pneumophila-a retrospective study. Scand J Infect Dis Dorrell L, Fulton B, Ong ELC. Intravenous azithromycin as salvage therapy in patient with Legionnaires' disease. Thorax HoepelmanIM,Schneider "E. Azithromycin: the first of the tissueselective azalides. Int J Antimicrob Agents Preac-Mursic V, WilskeB,Schierz, SUB E, GroBB.Comparative antimicrobial activity of the new macrolides against Borrelia burgdorferi. Eur J Clin Microbiol Infect Dis E. In vitro and 18. Preac-Mursic V, Wilske B, Schierz G, Holnburger M, in vivo susceptibility of Borrelia burgdorferi. Eur J Clin Microbiol Moody K D , Adams RL, Barthold SW. Effectiveness of antimicrobial treatmentagainst Borrelia burgdorferi infectioninmice.AntmicrobAgents Chemother Hansen K, HovmarkA, Lebech A-M, LebechK,Olsson I, Halkier-S0rensen L, Olsson E, Asbrink E. Roxithromycin in lyme borreliosis: discrepant results of an in vitro and in vivo animal susceptibility study and a clinical trial in patients with erythema migrans. Acta DermVenereoll992; Luft BJ, Dattwyler R, et al. Azithromycin compared with amoxicillin in the treatment of erythema migrans. A double-blind randomized, controlled trial. Ann Intern Med, Strle F,Preac-Mursic V, CimpermanJ,Ruzic E, Maraspin V, Jereb M. Azithromycin versus doxycycline for treatmentof erythema migrans: clinical and microbiological findings. Infection Weber K,Wilske B, Preac-Mursic V et al. Azithromycin versus penicillin V for the treatment of early lyme borrellosis. Infection Massarotti EM, Luger SW, Rahn DW, Messner RP, Wong JB, Johnson RC, Steere AC. Treatment of early Lyme disease. AM J Med Steere AC, Levin RE, Molloy PJ, Kalish RA, Abraham JH, Liu N Y , Schmid CH. Treatment of Lyme arthritis. Arthritis Rheumatism Musher DM, Baughn RE, Hamill RJ. Effect of human immunodeficiency (HIV) infection on the course of syphilis and on the response to treatment. Ann Intern Med Willcox RR. Treatment of early venereal syphilis with antibiotics. Br J Vener Dis Lucas JB, Price EV. Co-operative evaluation of treatment for early syphilis. Br J Vener Dis
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29. Dowel1 ME, Ross PG, Musher DM, Cate TR, Baughn RE. Response of latent syphilis or neurosyphilis to ceftriaxone therapyin persons infected with human immunodeficiency virus. Am J Med1991; 93:481-488. 30. Stamm LV, PamshEA. In-vitroactivity of azithromycinand CP-63,956 against Treponema pallidum. J Antimicrob Chemother1990; 25:ll-14. 31. Lukehart SA,Baker-Zander SA. Roxithromycin(RU 965): effective therapy for experimental syphilis infection in rabbits. Antimicrob AgentsChemother 1987; 31:187-190. 32. Lukehart SA,Fohn MJ, Baker-Zander SA. Efficacy of azithromycin for therapy of active syphilis in the rabbit model. J Antimicrob Chemother 1990; 25~91-99. 33. Alder J, Jarvis K, Mitten M, Shipkowitz NL, Gupta P, Clement J. Clarithromycin therapy of experimental Treponema pallidum infections in hamsters. Antimicrob Agents Chemother1993; 37:864-867. 34. Verdon MS, Handsfield HH, Johnson FU3.Pilot studyof azithromycin for treatment of primary and secondary syphilis. Clin Infect Dis 1994; 19:486-488. 35. Mashkilleyson AL, Gomberg MA, Mashkilleyson N, Kutin SA. Treatment of syphilis with azithromycin.Int J STD AIDS 1996; 7:13-15. 36. El Tabbakh GH, Elejalde Br, Broekhuizen FE. Primary syphilis and nonimmune fetal hydrops in a penicillin-allergic woman. A case report. J Reproduct Med 1994; 39:412-414. 37. McFarlin BL, Bottoms SF,Dock BS, Isada NB. Epidemic syphilis: maternal factorsassociatedwithcongenitalinfection. AM J Obstet Gynecol 1994; 170535-540.
16 Mycoplasma and Chlamydia J. Thomas Grayston University of Washington Seattle, Washington
Pentti Huovinen National Public Health Institute Thrku, Finland
Data on the incidence of total pneumonia and pneumonia due to Mycoplasmapneumoniae and Chlamydiapneumoniaewas presented. Pneumonia occurs most often in the youngest and oldest persons the in population. In a 12-year study in a SeattleHMO, the annual incidenceof pneumonia averaged 1in 80. Similar rates have beenreported from other studies, including a nationwide survey of practitioners in the United States. Similar data also have been reported from Europe. These data suggesting that, on average, everyone has one episode of pneumonia during his lifetime is undoubtedly an underestimationdue to the failure of diagnosis in milder cases. In clinical and microbiologically controlled studies, it has been shown that clinical findings do not correlatewell withthe organism specific diagnosis of pneumonia. Although this sessionwas primarily concerned with atypical pneumonia, it was pointed out that the classification of pneumonias bythe typical and atypical criteria is difficult, because of the overlapping of symptoms and the rarity of typical lobar pneumococcal pneumonia. Both M. pneumoniae and C. pneumoniae are periodic. Efforts to define a cycle of a regular period of years for both these organisms have 219
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failed. Whereas countrywide epidemics have seen beenin the Nordic countries of Europe, fairly consistent endemic rates of 1620% for the two organisms have been seen Europe in and the United States in recent years. Although C. pneumoniae is far more apt to be associated with hospitalized pneumonia than M.pneumoniae, it is unclear how often the more severe cases of hospitalized pneumonia associated with TWAR infection are due to that organism alone. Dual infections with bacteria and viruses are found frequently. There was general agreementthat the macrolides werethe antibiotics of choice for treatment of atypical pneumonia. Data were presented from recent studies showingthat azithromycin was highly effective against pneumonia caused by M. pneumoniae and by C. pneumoniae. Some of these studies were compared with the effect of erythromycin which was also highly effective.A discussant stated that roxithromycin had been shownto be highly effective in atypical pneumonia. Another discussant told of the study showingthat clarithromycin.was equally effective as erythromycin in treatment of pneumonia due to M. pneumoniae and C. pneumoniae. It appears that the newer macrolides or azalides are at least as effective as erythromycin intreatment of C. pneumoniae and M.pneumoniae and that they have fewer side effects. A question was raised about whether any treatment was necessaryfor M.pneumoniae infection and perhaps evenC. pneumoniae infection, even when a clinical syndrome of pneumonia was present. The suggestion was that these patients gotwell spontaneously andthe cure rate with antibiotics was falsely inflated. There was little support for this position, although it was acknowledgedthat asymptomatic and mildly symptomatic pneumonitis due to bothorganismswaswelldocumented. The fact that prolonged moderately debilitating cough illnesses often followed untreated or inadequately treated C. pneumoniae lower respiratorytract infection argued for appropriate treatment of this infection. In a discussion of antibiotic resistance, no one in the audience had an isolate of either M.pneumoniae or any Chlamydia species that was resistant to macrolides or tetracycline.Duringtherapy, the MICs(minimal inhibitory concentrations) of thesemicroorganismsusuallystays at the same level or changes onlyone step up or down. There have been reports of cases where the MIC of C. pneumoniae jumped by dilutions after receivingazithromycin. There havebeenclinicalcaseswhereseveral courses of macrolides were neededfor clinical cure, although the MIC of C. pneumoniae isolated remained unchanged. Studies of isolates of C. trachomatis from patientswith clinical failure have been tested and always found to be susceptibleto macrolides and tetracyclines.It is probable that a 1 or 2 dilution difference in MIC susceptibility is an insignificant variation
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because of the cell culture technique used for measurement. The only known antimicrobial agent to which resistance has developed in these organisms is rifampin. There is a report that the MIC of chlamydia can be pushed up to the MBC (minimal bactericidal concentration) level by several passages. However, when concentrations of antibiotic at the MBC level have been reached, the organisms always die. Someof the problems of measuring the MIC of these organisms was discussed. For example, control of the pH of the media is important when testingM. pneumoniae. wide varietyof different cell typesare now usedfor measuring antibiotic susceptibility. There is a need for standardizationof the susceptibility testing methodsfor mycoplasmas and chlamydias. In the past decade, abouthalf of M. hominis isolates have provento be resistant to tetracycline, carrying tetM-resistantdeterminant. The same determinant can be seen in Ureaplasma urealyticum. In the preceding decade, isolates of these organisms were susceptible to tetracycline. This phenomenon is similar to that that has been seenfor Nekseria gonorrhoea. Several attempts in the laboratory to put the tetM-resistant determinant into M . pneumoniae have failed. Among the mycoplasma, there is concern about M. hominis, which can cause chronic systemic infectionsof the joints. The best antimicrobial has been the tetracycline group, but there is now resistance to these antimicrobials. It remains to be seen what effect some of the newer quinolines, such as spadoxacin will have on this organism, as it is known that temafloxacin and ciprofloxacinare not effective inM. hominis infections. There was a discussion on whatthe future holds in the way of antibiotic resistance if the newer macrolides are increasingly used for treatment of mycoplasmas and chlamydias.The concern expressed wasnot that resistance would develop in these organisms against these antibiotics, butthat the wideruseofmacrolidescould further promote resistance in other organisms such asStreptococcus pneumoniae. There was considerable discussion about the use of macrolides as a first-line drug inthe treatment of outpatient pneumonia. One of the issues was that because classical pneumococcal pneumonia is rarely seen, pneumococcal pneumonia often cannot be differentiated from pneumonia due to M . pneumoniae and C. pneumoniae. It was stated that persons who die of pneumococcal pneumonia duringthe first 24 h will die despite therapy and that the figures on such deaths have not changed in 70 years. It was suggested that if this type of infection is to be successfully treated, the target may be the cytokine response. The best predictorof mortality and serious complicationsthe is severity of the pneumonia illness at presentation. The nature of the patient has to be taken into account when planning antimicrobial treatment. When the
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patient has risk factors (e.g., age, smoking habits, diabetes), it is necessary to plan treatment according to these risk factors.The macrolides in uncomplicated outpatient pneumonia should be safe and effective. Future improvement in fatal and severe disease with pneumonia will probably be best effected by efforts to develop active vaccination programs andto develop new vaccinesfor young children. The next major topicfor discussion was specific diagnosis of clinical cases of atypical pneumonia. It is recognized that even specialists do not carry out laboratory tests for specific diagnosisof atypical pneumonia unless theyare involved in a clinical trial. This behavior is strongly influenced by the availability of effective rapid laboratory diagnostic methods. For M. pneumoniae, there are many antigen-detection tests and DNA/RNA probes available. However,the specificity of these tests islow. A PCR (polymerase chain reaction) test now is available, but how effective it will be inthe clinical setting is not yet known. Diagnosis of Chlamydia pneumoniae infection outside the research laboratory is difficult. Cell culture techniques for isolation of the organisms have yielded widely variable results. For the most part, isolation of the organism has been difficult. This is duepart in to the fact that the infection isin the lung and the specimens are taken from the throat. Obtaining specimens from a lung is rarely justified. There is general but not unanimous agreement that the MIF (microimmunofluorescent) serologic test is the most sensitive for diagnosingC. pneumoniae infections. However, because of the requirement for a convalescent serum specimen, this method is usually not practical for immediate clinical decisionsabout therapy, but it may be useful in cases with prolonged illnessesthat are not responding to treatment. Several PCR testshavebeendevelopedandmaymeet the criteria for more rapid diagnosisin the future. Even with the development of more rapid and sensitive tests to identify the organisms, there will remain decisions to be made clinically about the significance of the identified organism. Some asymptomatic camage of both organisms has been demonstrated. There was discussion of a need to diagnose M. pneumoniae or C. pneumoniae infection when pneumonia was not present. In the case of bronchitis caused by these organisms, there is general agreementthat specific therapy is useful. Although it is true that children and some adults may have mild infections with these organisms and recover fairly rapidly without treatment, some patients are debilitated for months and benefit from treatment. The question of treating asymptomatic infection was also discussed. Whereas asymptomatic female patients with genital C. trachomatis are always treated, there is no information to show whether patients with asymptomatic respiratory infections needto be treated.
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Finally, there was a discussion of the use of the new macrolides to treat M. hominis genital tract infection. M . hominis in the lower genital tract is usually a commensal. There are reports that M. hominis can cause pelvic inflammatory disease, but these appear to be rare cases. Infectionsin newborns and immunocompromised patients are different and treatment should be considered.
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Staphylococci, Streptococci, and Pneumococci Jacques F. Acar Fondation HSpital Saint-Joseph Paris, France
J& Melo-Crlstino University of Lisbon Lisbon, Portugal
The authors of abstracts and discussants developed an overview of S. aurew, S. pneumoniae, and S. pyogenes reporting on susceptibility and susceptibility testing; epidemiology of resistant strains and insights on therapy of infections. The susceptibility and susceptibility testing of S. uurew to the macrolides and macrolide-related antibiotics should be considered regarding the different classesof compounds: C,, macrolides, azalides,C,, macrolides, lincosamides, and streptogramins and B. Table 1summarizes the morefrequent resistance characters and the corresponding resistance phenotypes. Inactivation mechanisms with different enzymes are seldom reported. The E m genes . . . the major resistance determinants found in clinical isolate. Because the new macrolides are in the C,, group, this gene mediates the resistance to the whole group of compounds (including the azalides) inthe inducible state as well as in the constitutive state. In the consti-
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tutive form, E m mediates resistance to C,, macrolides, lincosamides, and streptograminB. The prevalence of macrolide resistance (usually in the constitutive form) in methicillin-resistant Staphylococcus aureus (MRSA) ranges from 40% to 95% of isolates. On the contrary, methicillin-susceptibleStaphylococcus aureus (MSSA) often remain susceptible with a prevalence of resistance only from 5% to 15%. Coagulase-negative staphylococci share the same resistance phenotype with Staphylococcus aureus. However, efflux mechanisms were recognized in coagulase-negative staphylococci. Pneumococcal resistanceto the macrolides tendsto increase inEuropean countries.The penicillin resistanceof pneumococci has beenreported more frequently associatedwith resistance to other antibiotics, particularly the macrolides. However, the prevalence of pneumococci resistant to the macrolides varies widely from country to country: 10-20% reported from France,whereas the prevalencein the Netherlands is only 1% and in Germany 1.5%. The occurrence of penicillin resistance in S. pneumoniae strains was variable, asreported at this meeting.A Spanish studyby Garcia de Lomas et al. with 296isolates demonstratedthat the resistance to penicillin (intermediate and high levels) in healthy carriers was higher in strains recovered from children than adults (70% in children and 42.5% in adults). The authors found the coexistence of susceptible and resistant strains in 17 specimens from children and 8 from adults. Resistance rates to macrolides (erythromycin and clarithromycin) were 5.6% among children and 6.3% among adults. Paris et al. studied the incidence of disease and antibiotic susceptibility of S. pneumoniae from a population-based study in Dallas County in 19921993; 732 S. pneumoniae isolates were identified. Penicillin-intermediate strains accounted for 14% and resistant strains 3%. Theoverall susceptibility to the macrolides, clarithromycin and azithromycin (MIC d 1pg/ml) was estimated at 93%. Visalli et al. determined the activity of erythromycin, azithromycin, and clarithromycin against 120 pneumococci isolated from the United States. Results ofMICandtime-killstudiesshowed that clarithromycin was the most active macrolide. The authors suggested that clarithromycin has a potential for use against infectionsbycaused penicillinsusceptible and -intermediate and -resistant pneumococci in the United States, provided that the strains are susceptible to macrolides. From Germany, Knothe et al. studied 710 S. pneumoniue isolates from non-hospitalized children. Only 21 strains showed decreased susceptibility to penicillin (18 intermediate and 3 resistant). Roxithromycin was tested as representative of the macrolides and only11(1.5%) of the strains were resistant (MIC 4 pg/ml). These results are in agreement with the favourable situationin Germany regarding antibiotic susceptibility of pneu-
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mococci. S. pneumoniae strains isolated from acute otitis media in children were less resistant to the macrolides, azalides, and streptogramins (MAS) than to other antibiotics commonly used to treat otitis media (p-lactams andco-trimoxazole)in the reports ofMcLinnwithazithromycinand clarithromycin, and Blocket al. with clarithromycin and clindamycin. The effect of CO, incubation in antimicrobial testing of pneumococci was discussed in two studies. Moore et al. and Fasola et al. found differences in two incubation atmospheres (CO2 and air). The latter authors made the point that the dissociationinvitro(diskmethod)between macrolide and lincosamide susceptibility inS. pneumoniae is artificial due to the better expression of resistance to the macrolides. They made recommendations for macrolide and lincosamide testing of pneumococci, which include the following:minimuminhibitoryconcentration(MIC)breakpoints for erythromycin 0.5 pg/ml,susceptible; lpg/ml, resistant; clindamycin 0.25pg/ml, susceptible; OSpg/ml resistant; no intermediate category should be used. Testing of these agents can be reliably performed by agar dilution, E-test and disk diffusion with incubation in 510% CO,. Testing of these agents by microdilution should be performed under 5-10% CO, or aerobic incubation prolongedto 48 h. P. Appelbaum stressed the cross- resistance of pneumococci to macrolides, azalides, and lincosamides and the susceptibility of such strains to the ketolides and the streptogramins. Streptococcus pyogenes and pharyngitis/tonsilitis werethe subject of a few posters. The efficacyofazithromycinwas stressed by several authors. O’Dohertyet al., from Ireland, demonstrated in a doubleblind prospective studyof 489 patients, that azithromycin at a doseof 20 or mg/kg/day for 3 days was as safe and effective as penicillin V in the treatment of pediatric patientswith acute pharyngitis/tonsillitis. The MAS resistance in S. pyogenes was presented by Katala et al. from Finland, a country where an increasein the erythromycin resistance was found in the beginning of this decade. A novel resistance phenotypein which clindamycin resistance could not be induced by erythromycin has increased and accounted for70% of the resistant strains isolated in1994. Ninety-five percent of these strains belonged to the T4 serotype (DNA analysis showed genetic heterogenicity among T4 serotypes), whereas 91% of the isolates showingthe inducible typeof resistance belongedto theT28 serotype. The authors concludedthat erythromycin resistance in Finland is due to the spread of a few serotypes of S. pyogenes. In Italy,anationalmulticentersurveyconductedduring1995included 2739 strains and revealed that resistance in S. pyogenes was 8.2% for clindamycin, 14.7% for tetracycline, and 24.7% for erythromycin. Disk diffusion was the prevailing technique adopted. The authors stressed that these findings raise serious concerns given the fact that severe S. pyogenes-
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associated diseasesare increasing worldwide and that in patients allergic to p-lactams, resistance to alternative agents may cause serious therapeutic problems. In contrast to the two previous studies, Van Asselt et al. from the Netherlands, presented a study of 180 Xpyogenes strains, none of which was resistant to macrolides, which confirms the low rate of erythromycin resistance in this country. Three methods (E-test, microbroth MIC, and diskdiffusion)wereused to study the susceptibility of S.pyogenes to azithromycin, clarithromycin, erythromycin, and roxithromycin. Clarithromycin and azithromycin were compared with erythromycin, vancomycin, and amoxicillin for prophylaxis of viridans streptococcal experimental endocarditisby Rouse et al. from United States. Azithromycin and clarithromycin were as effective as amoxicillin, and erythromycin was less effective (p < than amoxicillin. A report from Shibl et al., from Saudi Arabia evaluated roxithromycin postantibiotic effect (PAE)on several Streptococcus species. The data indicate that PAE could not only be considered as prolonged suppression of bacterial growth but also could include an inducedstate of decreased microbial virulence through impaired adherence, diminished tissue invasion, and modulation of bacterial susceptibility to phagocytosis. Three new compounds were evaluated in several studies and discussions: RP 59500 (Quinupristiddalfopristin),Synercida, a new injectable streptogramin developedby RhBne-Poulenc-Rorer, was testedby Pankuch et al. using time-killing curves of penicillin-susceptible, -intermediate, and resistant pneumococci. They found that MICs, for all groups were 1pg/ml. Only RP 59500 killed pneumococci on initial exposure and within 1 h of exposure and it also was the most active at 2 h.RP 59500 yieldedthe most rapid killingof all drugs tested and was equally active against erythromycinsusceptible and -resistant strains. Reinert et al. from Germany also found a goodactivityagainstallpneumococcitested(penicillin-resistantstrains were not tested), including erythromycin-resistant isolates, and suggested that RP 59500 might be useful for the treatment of pneumococcal infections. Tarasiet al. fromthe United States tested 217 pneumococcal isolates that included 200 penicillin-resistant strains intermediate (89 and 111highly resistant) and4 isolates with specific resistanceto third-generation cephalosporins. The isolates included representatives of five major multiresistant pneumococcal clones as defined with pulsed-field gel electrophoretic patterns. More thanhalf of the highly penicillin-resistant isolates were representatives of the 23F capsular type. RP 59500 was active against all these isolates, with MIC, = 0.25 andMIC, = 0.5 pg/ml, includingthe 49 strains that were resistantto erythromycin. Using a rabbit modelof experimental meningitis, Denver et al. of the United States suggested that there is suffi-
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cient penetration through the inflamed meninges of RP 59500 to cause extensivecidaleffectinpneumococcalmeningitis. Further studies are needed to confirm this finding. Berthaud et al. from France studied in vitro bactericidal activity of the compound against S.aurew and compared it with vancomycin. Both drugs, at concentrations achieved in blood, demonstrated potent a bactericidal activity, but the bactericidal effect of RP 59500 was more rapid than that of vancomycin. Tenoveret al. fromthe United States developed provisional disk diffusion breakpointsfor testing RP 59500. Using a susceptibility breakpoint of < 2 pg/ml, the best correlation zone sizes and MICs was achieved with 5/10(quinupristiddalfopristin) ratio disk and a zone diameter of > 18 mm. Clinical correlation data are needed to choose the optimal disk concentration for testing. Studies with RPR 106972, a new oral streptogramin developed by RhBne-Poulenc Rorer, was presented. Berthaud et al. from France verified that in vitro, the drug showed promising antibacterial activity against gram-positive cocci (staphylococci, streptococci, and enterococci) and certaingram-negativebacteriaresponsibleforrespiratory tract infections (Neisseria, Moraxella, H . influenzae) and mycoplasmas. In vivo, in experimentalgram-positiveinfectionsinmice,theydemonstrated that RPR 106972 was active in the treatment of infections caused by sensitive and macrolide-lincosamide-streptogramin B (MLS,)-resistant S. aurew and erythromycin-sensitiveand-resistant S. pneumoniae. The antipneumococcal activity of RPR 106972 was also studied by Spangler et al. from the UnitedStates.These authors compared its activitywith9 oral lactams against 75 penicillin-susceptible, 55 penicillin-intermediate,and 73 penicillin-resistant pneumococci and verified that RPR 106972 hadthe lowest MICs (< 0.5 pg/ml) of the compounds tested. Macrolide resistance did not interfere with the results because MICs distributions were similar for the different subgroups of susceptible and resistant strains. RU 004 is representative of a new class of 1Cmembered macrolide antibacterials, the ketolides, generatedby Hoechst Marion Roussel. Ketolides are characterized by a keto function in position of the macrolactone ring, which replaces the cladinose moiety. Agouridas et al. from France evaluated in vitro and in vivo, the antibacterial activity of the compound against respiratory pathogens in experimental murine septicemia and pne monia models. They showed that this compound demonstrates outstanding activity against macrolide-susceptible, macrolide-inducibleMU,-resistant staphylococci, constitutively MLS,-resistant cocci(e.g., pneumococci), except staphylococci.The compound showed activity similar to azithromycin against H.influenzae and also was very active against bacteria such as Legionella, Mycoplasma, and Chlamydia spp. The in vivo studies confirmed
Staphylococci, Pneumococci Streptococci, and
231
the potential usefulness of RU 004 in respiratory infections causedby H. influenzae and pneumococci resistant to macrolides, p-lactams, and other drugs. Edine et al. fromthe United States studied the activity of RU 004and compared it with15 other agents against228 erythromycin-susceptible and -resistant pneumococci and found that the compound had excellent activity against penicillin- and erythromycin-sensitive and -resistant strains,showing, in all cases, MICs, values much lower than those of erythromycin, azithromycin, and clindamycin.
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l8 Pharmacokinetics and Pharmacodynamics Charles H.Nightingale Hartford Hospital Hartford, Connecticut
Fritz Sorgel Institute for Biomedical and Pharmaceutical Research Nurnberg-Heroldsberg, Germany
This workshop discussed several important pharmacodynamic and pharmacokinetic issues.The most important issue involved understanding how the current pharmacodynamic model appliesto the macrolides. Currently, it is widely believedthat for concentration-dependent killing antibiotics (aminoglycosides, quinolones), the pharmacodynamic parameter that correlates with bacterial eradication is the area under the plasma concentration versus time curve/minimal inhibitory concentration(AUCMIC) ratio and, under certain conditions, the C#IC ratio. For concentration-independent killing drugs such as p-lactams, the important pharmacodynamicparameter is the time that the serum concentrations remain abovethe MIC (t > MIC). In both cases, the serum concentrationis indexed to the organism’s MIC, and for antibiotics to eradicate organisms, the serum or, by extension, the interstitial fluid concentrations needto exceed the MIC in someway. For macrolides, it can be demonstrated that these models hold for most organisms (e.g., S. pneumonaie), but for other organisms such asH. influenme, these models predict drug failure. Clinical studies indicated that macrolides are quite effective in the treatment of diseases where H . influ-
234
Nightingale and Sorgel
mzae is a major pathogen. This disconnect between the model and reality is an indication that the existing model cannot completely describe the pharmacodynamics of the macrolides. Upon further discussion, it was felt that the current model assumesthat the liquid surroundingthe microorganism contains a homogenous concentration of drug. This is probably not correct for drugs such as macrolides that are capable of accumulating in mammalian tissue. As drug leaves these tissue repositories, a concentration gradient is established with the highest drug concentrations just on the outer membranes of the mammalian cell and decreasing the further one travels away fromthat site. Measurable interstitial fluid levels represent an average drug concentration as do measurable serum or blood concentrations. For drugswith little tissue uptake (e.g., p-lactams), serum and interstitial fluid levelsare sufficiently high, even on average,to eradicate target pathogens; the current model predicts their activity very well. For drugs like macrolides, the same situation occurs (i.e., for very sensitive organisms, the average blood and interstitial fluid levels are sufficientlyhigh to eradicate the organism), however, when the organism’,is moderately susceptible, (i.e., has a higher MIC),the current modelis incapableof predicting organism eradication. important issue isto determine wherethe organism resides inthe body in relationship to where the drug is at high concentration. It is believed that pathogens reside attached to surfaces (i.e., the outside membranes of cells). It is further believed that due to the concentration gradient of macrolides, the bacteria reside in just the place where interstitial fluid concentrations are highest. Additionally, macrolides accumulate in macrophages. Macrophages migrate to infection sites to engulf bacteria. Upon doing they can release drug, thus increasing drug concentrations in bacteria andmay also changethe permeability of drug intothe bacteria, in effect reducing the in vivo MIC.It was generally feltthat the in vitroMIC is higher than the in vivo MIC and this may explain, in part, the lack of concordance between clinical studies and pharmacodynamic predictions for macrolides. It is believed that such a modified model is capable of describing the clinical observations associated with macrolides. Regarding pharmacodynamic parameters in general, it is felt there,is on a sigmoidal dose-response curve. For concena gradient of effects based tration-dependent killing drugs, the dose-response curve is verysteep and the drug is usually cidal using therapeutic doses. For p-lactams, the curve is less steep; at lower concentrations, the drugs’ actions may be bacteriostatic, and at higher concentrations, bactericidal. Macrolides may have a dose-response curvethat is less steep than that of p-lactams. Regarding the postantibiotic effect, it could not be decided whether this was generally goodor problematic. It was thought to be good because
acodynamics and Pharmacokinetics
235
it allows infrequent dosing; however, there was some indication that prolonged exposureto ineffective concentrationsmay result in mutations and selection of resistant organisms. To clarify this requires differentiation between postantibiotic effect and subtherapeutic concentrations. The workshop also focused on tissue distribution issues, noting that lactams are distributed extracellularly, aminoglycosides are similar, whereas quinolones have some intracellular distribution and macrolides have the most intracellular distribution. Not all macrolides are alike, however, because roxithromycin shows a distribution pattern similar to p-lactams. It was also pointedout that erythromycin pro-drugs may cause erythromycin to behave more like azithromycin than clarithromycin. It was postulated that the pro-drug can penetrate rapidly into tissue, be hydrolyzed, and egress out of the tissue, resulting in prolonged but low serum erythromycin concentrations. There was some discussion about azithromycin absorption probably due to loss by first-pass effects in the liver. Also, bioavailability of the suspensiondosageformdidnotchangewithage of the patients. Clarithromycin suspension dosage forms was stated to have AUC/24 hthat were similar to the adult dosage form, including similar Cm,’s and Cmin’s. The reporting of tissue concentration data was discussed. The point was made that tissue/serum ratios provide little useful information. It is possible to have a high ratio if the numerator is high, and it is possible to have a high ratio if the denominator islow. Antibiotics kill bacteria based on drug concentrationsat theplace wherethe bacteria reside. Actual tissue concentrations should be compared, not the penetration ratios. Penetration ratios have marketing value but little scientific merit and its use should be discouraged. Finally, the difficulties of conducting tissue uptake studies were discussed. It is realized that there is a wide variety oftechniques usedfor this purpose withlittle standardization. It was suggestedthat standardized methodologies be established as a guide in conducting these studies that the results canbe more easily compared from different study centers.
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19 Campylobacter and Helicobmter pylori Jean-Paul Butzler University Hospitals, St. Pierre, Brugmann, and Queen Fabiola Bmsels, Belgium
Francis M6graud Hopital Pellegrin et Universitkde Bordeaux 2 Bordeaux, France
MACROLIDES IN CAMPYLOBACTER ENTERITIS
In the last 10 years,Cumpylobucterjejuni has emergedas the most common cause of bacterial gastroenteritis in humans. The symptoms C. jejuni infection are usually mild but systemic and postinfectious manifestations such as the Guillain-Barr6 syndrome may occur (1,2). In general, Campylobacter enteritis is a self-limiting disease and the isolation of campylobacters from stools does not warrant chemotherapy. In the absence of chemotherapy, the feces of patients remain positive for about 2-7 weeks after the illness. Antimicrobialtreatment, when started within 4 days of onset, has a clinical benefit and shortensthe fecal excretionof C. jejuni. Salazar-Lindo et al. evaluated the efficiencyof erythromycin, starting immediatelyon presentation. They found that early administration of erythromycin significantly reduced the duration of both diarrhea and fecal excretion the organism in infants and children with acute C. jejuni dysentery. In children, roxithromycin treatment initiated within 4 days the onset of C. jejuni diarrhea appeared to reduce the camage of the
237
238 Mkgraud
and
Butzler
pathogen (4). It was also effective in reducing boththe severity and duration of the diarrhea and seemed to accelerate the disappearance of abdominal pain. Antimicrobial treatment is indicated in prolonged disease with severe symptoms, in high fevers or with bloody stools, in relapses, in pregnancy,andinimmunocompromisedpersons. The clinicalcourseof C. jejuni diarrhea in AIDS patients very often requires long-term treatment. Campylobacterjejuni shows rather unusual antimicrobial sensitivities. The macrolides, furazolidone andthe aminoglycosides are the most active compounds; all strains are resistant to benzylpenicillin, cephalothin, colistin, vancomycin, rifampicin, and trimethoprim. Up to 20% of strains are resistant to tetracycline. Susceptibility to ampicillin, metronidazole, and trimethoprim sulphamethoxazole is variable.C. jejuni is susceptible to the newer macrolides such as roxithromycin, clarithromycin, and azithromycin (Table 1). In industrialized countries, macrolides continue to be the drugs of choice for the treatment of Campylobacter infections because the preva(2-5%) and stable lence of erythromycin-resistantstrainsremainslow Several studies showthat macrolides like clarithromycin and azithromycin have invitro activities similar to that of erythromycin and superiorto that of roxithromycin (6). The situation for quinolones is totally different. Several authors have showna remarkable increasein the level of resistance since the introduction of norfloxacin and ciprofloxacin andthe inclusion of enrofloxacin in veterinary practices (5,7,8). Sanchez et al. ( 5 ) studied the evolution of antimicrobial susceptibilities of 275 clinical Campylobacter strains isolated in their institution duringa 5-year period and Tabk l
Susceptibility of Campylobacter jejuni to Various Antimicrobia'l Agents MIC(mgn) Range
Erythromycin Clarithromycin Roxithromycin Erythromycylamine Dirithromycin Flurithromycin Azithromycin Josamycin
90%
4.0
0.50
0.50
Spiramycin
Miokamycin Rokitamycin
50%
0.5->4
4.0
Campylobacter andpylori Helicobacter found a stable macrolide activity with a rapid development of quinolone resistance. The evolution of resistance (percent resistance in 1988 versus percent resistance in 1992) was as follows: erythromycin, 2.6 versus 3.1; clarithromycin, 2.6 versus 3.1; azithromycin, 2.6 versus 3.1; ciprofloxacin, versus 49.5; norfloxacin, 2.6 versus 55.5; ofloxacin, 0 versus 45.6. A number of studies (5-7) have confirmed that the species C. coli is much more resistant to erythromycin and the other macrolides than C. jejuni. Several investigators demonstratedthat the strains of C. coli isolated in pigs were those withthe highest resistance rate, close to 70% (9). This phenomenon may be dueto the indiscriminate useof the macrolide antibiotic tylosin (analogousto erythromycin) for fattening these animals, which should leadto the appearance of strains with cross-resistanceto erythromycin, which would subsequently be ingested by humans (7,9). Endtz et (8) determinedtheminimalinhibitoryconcentrations (MICs) of erythromycin and three new macrolide antibiotics for 36 quinolonesusceptible and 106 quinolone-resistantC. jejuni strains. The MIC, values of azithromycin,clarithromycin,roxithromycin,anderythromycinwere 0.5, 4, 16, and 4 m&,respectively. difference was found between macrolide activity against the quinolone-susceptible and the quinoloneresistant strains. Taylor and Chang (6) studied the MICs of azithromycin and erythromycin for 20 Campylobacter coli and 20 Campylobacter jejuni strains. The results demonstratedthat for Campylobacter species, all highlevel erythromycin-resistant strains were also resistant to azithromycin and that-azithromycin did not exhibit increased potency in comparison with that of erythromycin. Yan and Taylor (10) investigatedthe mechanism of resistance to erythromycin. Erythromycin resistance (MICs, greater than 1024 pglml) in three clinical isolates of Campylobacter jejuni and one C. coli isolate was determined to be constitutive and chromosomally mediated. If patients are caught in the early stages of the disease or if they are distressed, appropriate chemotherapy is justifiable. Erythromycin has been advocated as the agent of choice for the treatment of Campylobacter enteritis. It hasexcellentinvitroactivity,lowtoxicity,afairlynarrow antibacterial spectrum, and relatively low cost. Resistant strains are, on the whole, rare and almost confinedto C. coli. It probably matters little what preparation of erythromycinisgiven (other thanenteric-coatedpills). There are theoretical reasons for favoringthe stearate, at least for adults. Apart from being acid resistant andstable, it is incompletelyabsorbed, there is a chance of a contact action in the bowel lumen as well as a systemic action in the blood. A dosage of 500 mg twice daily for 5 days has proved satisfactory in practice; higher doses of the stearate are liable to cause acute abdominal pain. Erythromycin ethylsuccinate at 40 mg/kg/day in divided doses is recommended for children.
240
Butzler and Mkgruud
Although the newer macrolides (roxithromycin, clarithromycin, and azithromycin) may have a future because of their pharmacologic advantages, there is not sufficient evidence to prove that they are superior to erythromycin, but theymay be better tolerated. Funke et al. (11) reported the development of resistance to clarithromycin in vivo during treatment of an invasive Cumpylobucterjejuni infection in anAIDS patient. Because of the chemical relatednessof clarithromycin and erythromycin, resistanceto clarithromycin may be due to the same mechanism, suggested by the fact that the isolatesbecameresistanttobothdrugs at the sametime. As previouslymentioned,TaylorandChang (6) found that erythromycinresistant C. jejuni and C. coli strains always showed cross-resistance to azithromycin. The clinical course of C. jejuni diarrhea in AIDS patients very often requires long-term treatment that may not be successful. with other long-term antimicrobial therapies, clinicians should be aware of in vivo development of macrolide resistance in immunocompromized as well as in immunocompetent patientstreated for C. jejuni infection. In the industrialized countries, dehydration caused by C. jejuni is infrequent, but fluid and electrolyte replacementare sometimes necessary in infected infants.The best treatment forC. jejuni infections in developing countries could well prove to be different. Factors such as low socioeconomic status and malnutrition may determine the severity of a C. jejuni infection and its great prevalence in very young children. Vomiting and watery diarrhea are frequent, and sometimes, oral rehydration is required in children. Antibiotics should be reserved for very severecases. It is certain that education andbetter hygiene havefar greater roles in reducing infections than do antibiotics. In conclusion, the current consensus isthat antibiotic therapy is indicated in patients with Campylobacter infection who are acutely ill with enteritis, have persistent fever, bloody diarrhea, more than eight bowel movements per dayor significant volume loss,or more than a 7-day history of diarrhea. HIV-infected individualsor immunocompromised persons and pregnant women should receive antibiotic treatment. When antimicrobial therapy is indicated, erythromycin is the drug of choice, given its efficacy, low toxicity, and low cost. There is not sufficient evidence to prove that the newer macrolides, roxithromycin, clarithromycin, and azithromycin, are superior to erythromycin, but theymay be better tolerated. HELICOBACTER PYLORIAND MACROLIDES The discovery of Helicobacterpylorihas beenone of the most important in the field of medical bacteriology.The diseases of the stomach, in particular peptic ulcer for which numerous causes had been advocated in the past
Campylobacterandpylori Helicobacter
241
including diet, stress, and forth, are now recognized as infectious diseases (12). Furthermore, for the first time, a bacterial infection is considered to be at the origin of a carcinogenic process as wasthe case for viral infections (e.g., hepatitis B) and parasitic infections. for anyother bacterial infection, H.pylori infection must betreated with antibiotics. WhenMICs were determined on this bacterium, most antibiotics were found to be effective, but clinical experience selected only a few compounds or groups of compounds: a p-lactam: amoxicillin, nitroimidazoles, and macrolides.
MICs of Macrolides on H.pylon When MICs were determined on strains of H.pylori, a small group of resistant strains was identified (e.g., 10% in France) (14). The MICs of susceptible strainsto clarithromycin at neutral pH range from to 0.06 m&. This compound isthe most effective macrolide with aMIC, of m& (i.e., almostaslowasamoxicillin). Other macrolideswithgood activity are azithromycin and roxithromycin When MICs are determined at a lowerpH, their values increase,but, again, clarithromycin isthe least affected compound (Table2). A question which was debated during ICMASI11 was the optimal way of testing strain susceptibilityto antibiotics and especially to macrolides. It was agreed that for macrolides, the method was not critical because all the methods can discriminate between susceptible and resistant strains (i.e., E-test, and even disk diffusion can be used); the agar diffusion method is not mandatory. However, it was stressed that a heavy inoculum should be plated.The reason is that a mixed population of susceptible and resistant strains may be involved and it may be missed if one uses a light inoculum or a single colony. Table 2 Susceptibility of Helicobacterpylon Strains to Various Antimicrobial Agents at Three Different pHs
pH 7.5 Erythromycin Roxithromycin Dirithromycin Clarithromycin Azithromycin Spiramycin Mdecamycin
0.2 0.25
pH 6.5 2
pH 5.5 16 4
1
1 4
0.03 0.12
0.06 1
1 1
8
32
2
4
16 0.25 8
242
and
Butzler
Mbgraud
However, there are asmallnumber of strains with MICs slightly higher than normal (i.e., 0.5-1 m&), the question remains: Where should the breakpoint be set? Based onU.S.and European studies (16,17), a breakpoint of 0.5 m& was proposed but was not accepted by the U.S. Food and Drug Administration. In fact, noone knows what to do with the isolates which have an MIC of 0.5-1 m& and which are seldom found. It would be interesting to know if they possess the same genetic mutationas the high-level resistant strains. More data on clinico-bacteriological correlations are needed before concluding onthe breakpoint values.
Pharmacokinetics of Macrolides in the Stomach It is well known that macrolides have the ability to concentrate in tissue. This phenomenon also seemsto apply in the gastric mucosa. A study was performed with azithromycin given to patients before stomach resection. Azithromycin concentration was determined at different time intervals. Concentrations of azithromycin of 2.27 mg/g were still present in gastric tissue h or more after oral administrationof 500 mg of azithromycin. The concentration in gastricmucus was much lower mg/g, standard deviation: 0-1.6) but still of potential interest (18). More recently, clarithromycin concentrations have been determined in gastric.mucosaof patients receiving this antibiotic alone or in association with omeprazole. Whereas in two instances clarithromycin concentration did not differ significantly the at antrum and fundus level, its concentration in the mucus increased by a factor of 10 when omeprazole was given also. Information is lackingfor the other macrolides.
H.pylori Resistance to Macrolides Recent studiesreported during this meeting by Versalovicet al. showedthe 2058 molecular basisof this resistance which is a point mutation in position or 2059 on the 23s ribosomal RNA genes. It has been shown for Mycobacteria, a genus with a singly copy of ribosomal RNA genes, that a point mutation is responsible for resistance. The best evidence now is that H. pylori has copies of ribosomal RNA genes and, despite this, resistance can occur. One mutation seems sufficientto cause macrolide resistance in H. pylori; this is contradictoryto the dogma that when multiple copies of ribosomal RNA genes are present, point mutations do not lead to resistance. Versalovicet al. reportedthe case ofa heterozygotestrain with a one point mutationon one gene andthe wild type onthe other gene which was resistant. Furthermore, the mutation seemsto be present whenthe MIC of the strain is 2 mgL(l9).
Campylobacter andpylori Helicobacter
243
0.5% 5 - 10%
10
4 I
-
Resistance of H. pylori to clarithromycinin European countries in1994.
Although the mechanism is most likelyto be the same for all strains, only 10 strains fromthe United States have been tested, it is important to study this problem further. The frequency of this mutation has been studied in the past and is foundto be in the range of lo-’ to which is lowerthan for nitroimidazoles (20). Based on this mechanism, it is easy to understand that macrolide resistance is definitive and not reversible, aswas wrongly proposed inthe past. There are different situations with regard to the level of this resistance in different parts of the world. The 1994 data presented during the meetings in Edinburgh and Berlin in 1995 are summarized in Fig.1. Three countrieshavealevel of resistance of approximately 10%: Belgium, France, and Spain. A level of 7% was reported in Hungary, but in other countries likethe United Kingdom,the resistance level is almost nil. These data most likely reflectthe common use of macrolides for respiratory infections in these different countries inthe past. It was noted in Belgium, for example, that the resistance rate was 1.7% before 1992 and it has risento 10.5%sincethen(21). A dramaticincreasewasobservedinSpainby Lopez-Brea (22). Baquero also reported at this meeting an increase from to 12% between 1987 and 1995 in Madrid. We have evaluated the resistance of all our strains isolated in Bordeaux since 1985 and were surprised to note that, during thisperiod, resis-
Butzler and Mdgraud
244
tancehasbeen in the range 8-11%; thisresistance rate waspossibly reached between 1982, the date of the recommendation to use macrolides for respiratory infections, and 1985 (14). The audience concluded that the resistance problem should be kept in mindbut that it does not seem to be a menacing problemat this stage.
Antibiotic Regimens to Eradicate H. pylori
The history of H.pylori treatment is short but already rich. In 1990, the firsteffective triple therapyincludingbismuthcompoundswasrecommended Because of side effects and poor compliance, alternatives were then proposed, especially bitherapies associating a proton pump inhibitor with an antibiotic. The best result was obtained with clarithromycin but the eradication rate did not surpass 75% and, therefore, was considered insufficient. Accordingly, triple therapiesare now favored, especially combinations ofproton pump inhibitorswith clarithromycin and amoxicillin or clarithromycin and metronidazole. These regimens allow a cure of the infection in more than90% of patients (Fig.2). However, a question debated during the session wasthe comparative advantages of bitherapy versus tritherapy. Bitherapies using clarithromycin (1.5 g/day) with aproton pump inhibitor during2 weeks leadto an eradica100
80
70 60 50 40
30 20 10 0
0-A 0 ”- C - A
Figure 2 Meta-analysis of the results obtained with different eradication therapies (based on data from patients). (FromRef. 24.)
Cumpylobacter and Helicobacter pylori
245
% eradication
"l
Figure 3 Eradication rates obtained in studies using clarithromycin as the only antibiotic during 14 days in ulcer patients. American trial (Ref. 25):(1) Clari = clarithromycin 500 mg tidyOme = omeprazole 40 mg;(2) Clari = clarithromycin 500 mg tid;(3) OME = omeprazole 40 mg. European trial(Ref. 26):(4) Clan = clarithromycine 500 mg tid, Ome = omeprazole 40 mg; (5) Ome = omeprazole 40 mg.
tion rate of 80% at best (Fig. whereas clarithromycin at a lower dose administered with amoxicillin and a proton pump inhibitor during only 1 week leads consistentlyto eradication rates higher than 90%. There was a consensus that the best way to prevent the development of resistance isto have the highest possible eradicationrate. With regardto bitherapies, different populations seem to have different response rates. For example, northernEuropeans tend to have higher eradication rates than southern Europeans. Because clarithromycin can inhibit omeprazole metabolism andtherefore improve the treatment efficacy, one hypothesis is that there is some variability in this interaction between populations. An interesting report is that clarithromycin given alone, despite an unsatisfactory eradication rate, was able to heal ulcers (27) and even perform better than antisecretory drugswith regard to pain relief. Obviously, suchdata need to be confirmed, butif they are, it would be additional proof of the infective nature of peptic ulcer disease.
Butzler and Mbgraud
246
At this stageit is stillthe rule to use an antisecretory drug, butwhich one is the best? Omeprazole and lansoprazole seem to be equally effective. The new compound, ranitidine bismuth citrate, which brings forth ranitidine activity has been tested with clarithromycin, but there has not yet been a head-to-head comparison between omeprazole and this compound. It is also difficultto compare the results of these studiesto others because only the rates of eradication in healed ulcers were considered. With .regard to in vitro data, some results concerning the association between roxithromycinandlansoprazolewerepresentedandshowed that there was an additive effect (28). Most of the invivo data have been obtained with clarithromycin; however, some studies have been performed with other macrolides. There were two multicenter studies using roxithromycin aspart of a triple therapy regime. Dammann et al. in Germany associated roxithromycin (300 mg bid) with metronidazole andthe H, antagonist roxatidine during 7-10 days and obtained eradication rates ranging from 78% to 90% (31). The same dose was used by Lamouliatte et al. in association with amoxicillin and lansoprazole during 2 weeks the andresult was a promising 85% eradication rate (32). Azithromycinwasusedin two studies in Eastern Europe. In the Czech Republic, Shonovaet al. used 250 mg/day of azithromycin during a week with omeprazole (85% eradication rate)(23), and in Croatia 1g/day during 6 days with ranitidine (68% eradication rate) (34). There was also one report on the use of spiramycin in association with metronidazole and lansoprazole during 2 weeks in children with a 72% successrate (35). The cost-effectiveness of this new triple-therapy regimen must be considered. All the studies performed have shown that eradication therapy is considerably cheaper than the traditional antisecretory drug therapy. Short-term triple therapies which were not considered the in study by Vakil et al. (36) are also cost-effective comparedto dual therapies andstandard triple therapiesor quadruple therapiesif the rate of eradication observedis as high asthe 90% reported. In conclusion, macrolidesand, in particular, clarithromycin have confirmed their important role in therapeutic regimens designed to eradicate H . pylori. At this stage, tritherapies seem the most efficient. They canbe used on a short-term basis, but there are still questions remaining, especially with regard to the possibility of expansion of antimicrobial resistance.
REFERENCES 1. Butzler JP, Glupczynski Y, Goossens H. Campylobacter and helicobacter infections. Curr Opin InfectDis 1992; 5:80-87.
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2. Mishu Allos B, Blaser M. Campylobacterjejuni and the expanding spectrum of related infections. Clin Infect Dis1995; 20:1092-1101. 3. Salazar-Lindo E, Sack RB, Chea-Woo E, Kay BA, Piscoya Early treatment with erythromycin ofCampylobacterjejuni-associated dysentery in children. J Pediatr 1986; 109:355-360. 4. Butzler JP, Levy J, Goossens H, Glupnynski Y ,De Mol P. Macrolides in campylobacter enteritis. In Macrolides, Chemistry, Pharmacology, and Clinical Uses. Bryskier A, Butzler J-P, Neu HC, lklkensPM, eds. Arnette-Blackwell, Paris:1993. 5. Sanchez R, Fernandez-Baca V, Diaz MD, Munoz P, Rodriguez-Creixems M, Bouza E. Evolution of susceptibilities of Campylobacter spp. to quinolones and macrolides. Antimicrob Agents Chemother 1994; 38(9):1879-1882. 6. Taylor DE, Chang N. In vitro susceptibilities of Campylobacter jejuni and Campylobacter coli to azithromycin and erythromycin. Antimicrob Agents Chemother 1991; 35(9):1917-1918. 7. Reina J, RosMJ, Serra A. Susceptibilitiesto 10 antimicrobialagents of 1,220 Campylobacter strains isolated from 1987 to 1993 from feces of pediatric patients. Antimicrob Agents Chemother1994; 38(12):2917-2920. 8. Endtz H P , Broeren M, Mouton RP. In vitro susceptibility of quinoloneresistant Campylobacterjejuni to new macrolide antibiotics.Eur J Microbiol Infect Dis 1993; 12(1):48-50. 9. Wang WL, Reller LB, Blaser UT. Comparison of antimicrobial susceptibility patterns of Campylobacterjejuni and Campylobacter coli.Antimicrob Agents Chemother 1984; 26(3):351-353. 10. Yan W, Taylor DE. Characterization of erythromycin resistance inCampylobacter jejuni and Campylobacter coli. Antimicrob Agents Chemother 1991; 35(10):1989-1996. 11. Funke G, Baumann R, Penner JL, Altwegg M. Eur J Clin Microbiol Infect Dis 1994; 13(7):612-615. 12. Mtgraud F,Lamouliatte H. Helicobacterpylori and doudenal ulcer. Evidence suggesting causation. Dig Dis Sci 1992; 37:769-772. 13. Schistosomes, Liver Flukes and Helicobacterpylori;IARC, Lyon, 1994. 14. Camou C, Ancelle J, Lamouliatte H, Mtgraud F. Evolution of the resistance of Helicobacter pylon to macrolides, The Third International Conference on the Macrolides. Azalides and Streptogramins, Lisbon, 1996;abstr 5-12. 15. Darmaillac V, Bouchard S, Lamouliatte H, MBgraud F. Effects of pH on the susceptibility of Helicobacter pylori to macrolides. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon 19%; abstr 5-13. 16. Ghoneim AT, Langdale P, Edmonds A, Green R, Reitmayer R, Flamm RK, Tanaka SK. Interpretive criteria for in vitro susceptibility testingof clarithromycin against Helicobacter pylori from a multicenter European clinical trial. The Third International Conference on the Macrolides, Azalidesand Streptogramins, Lisbon, 1996; abstr 5-05. 17. Graham DY, VersalovicJ, Flamm RK, Clamdge JE, Evans DG, Edmonds A, Cox S, Tanaka SK. Interpretive criteria for in vitro susceptibility testing of
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27.
29.
Butzler and Mdgraud clarithromycin againstHelicobacterpylori from a multicenterUS clinical trial. The Third International Conference on the Macrolides, Azalidesand Streptogramins, Lisbon, 1996; abstr 5-03. Harrison JD, Jones JA, Morris DL. Azithromycin levels in plasma and gastric tissue, juice and mucus, Eur J Clin Microbiol Infect Dis 1991; 10:862-864. Versalovic J, ShortridgeD, Kibler K, GriffyW ,Beyer J, FlammRK, Tanaka SK, Graham DY, Go ME Mutations in 23s rRNA are associated with clarithromycin resistance in Helicobacter pylori. Antimicrob Agents Chemother 1996; 40. Haas CE, Nix DE, and SchentagJJ. In vitro selection of resistant Helicobacter pylori, Antimicrob AgentsChemother 1996; 34:1637-1641. Glupczynski Y, Goutier S, Van den Borre C, Butzler JP, Burette A. Surveillance of Helicobacterpylori resistance to antimicrobial agents in Belgium from 1989 to 1994, Gut 1995; 37(suppll):A56. Lopez-BreaM,Martinez MJ, Doming0 D, SanchezTomero I, Sanz JC, Alarcon T. Evolution of the resistance to several antibiotics in H.pylori over a 4 year period. Gut 1995; 37 (suppl 1):A97. Helicobacter pylori: causal agent in peptic ulcer disease? A working team report. Gastroenterol Hepatoll991; 6:103-140. Chiba N, Wilkinson JM, Hunt RH. Clarithromycin (C) or amoxicillin (A) dual and triple therapies in H. pylori (Hp) eradication: a meta-analysis.The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 5-26. Hunt R, Schwartz H, Fitch D, Fedorak R, AI Kawas F, Vakil N. Dual therapy of clarithromycin (CL) and omeprazole (OM) for treatment of patients with duodenal ulcers (DU) associated with H. pyloninfection. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 5-18. O'Morain C, Logan RPH. the Clarithromycin European H. pylon Study Group. Clarithromycin (CL)in combination with omeprazole (OM) for healing of duodenal ulcers (DU), prevention of DU recurrence, and eradication of H.pylon (HP) in European studies. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 5-25. Cave D, Vakil N, Hunt R, Graham DY, the clarithromycin H. pylori Study Group, Therole of clarithromycin without anti-secretorytherapy in the treatment of H. pylon and prevention of duodenal ulcer recurrence. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 5-29. MBgraud F, Briigmann D, Darmaillac V. Roxithromycin-"ICs and bactericidal effecton Helicobacterpylori. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 5-01. Bardhan KD, Dallaire C, Eisold H, Duggan AE. The treatment of duodenal ulcer with GR122311X (ranitidine bismuth citrate) and clarithromycin. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 5-33.
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Peterson WL, Sontag SJ, Ciociola AA, Sykes CL, McSorley DJ, Webb DD. Ranitidine bismuth citrate (RBC) plus clarithromycin (CLAR) is effective in the eradication of Helicobacter pylori and prevention of duodenal ulcer relapse. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, abstr Dammann HG, Walter TA. Roxithromycine, metronidazole and roxatidine acetate triple therapy andH. pylori eradication rates.The ThirdInternational Conference on the Macrolides, halides and Streptogramins, Lisbon, abstr Lamouliatte H, Coumer A,Mion F, MBgraud F, Rio Y,Reverdy ME, FlBjou JF, Topeza M. Roxithromycin mg bid in association with amoxicillin and lansoprazole on eradication of Helicobacter pylori: results of an open noncomparative multicentre study. The Third International Conference on the Macrolides, halides and Streptogramins, Lisbon, abstr Shonova Petr P, Hausner 0. hithromycin as a promising part of helicocidal regimens. The Third International Conference on the Macrolides, halides and Streptogramins, Lisbon, abstr Desnica B, Burek V, Makek N, Azithromycidranitidine combined treatment of H. pylori in patients with duodenal ulcer and chronic gastritis-a pilot study. The Third International Conference on the Macrolides, halides and Streptogramins, Lisbon, abstr Raymond J, Ralach N, Bergeret M, Benhamou P, Barbet P, Briet F, Flourie R, Senouci I, Gendrel D, Dupont C. A controlled studyof efficacy of lansoprazole in combinationwithtwodifferentdualantibioticassociationsduring Helicobacterpylori gastric infection in children.The Third International Conference on the Macrolides, halides and Streptogramins, Lisbon, abstr Vakil N, Fennerty B, Wi U. Economic modeling of medical therapy for H. pylori related peptic ulcer disease. The Third International Conference on the Macrolides, halides and Streptogramins, Lisbon, abstr
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20 Haemophilus influenme,Enterococci, and Anaerobes Vincent T. Andriole Yale University Schoolof Medicine New Haven, Connecticut
Ian Phillips United Medical and Dental Schools of Guy’s and St. Thomas’s Hospitals London, United Kingdom
The difficulties of in vitro susceptibility testing in relation to the MAS group of antibiotics in general, and this otherwise heterogeneous group of microorganisms in particular, were repeatedly stressed throughout the session. Wide discrepancies in results obtained byanyof the conventional susceptibility-testing methods may be related to differences in the media used, including subtle changes in the composition of what purports to be the same medium as well more obvious differences between media such as Haemophilus Test Medium, Mueller-Hinton medium with additives such as L-cysteine, hemin, horse or sheep blood, and on. Differences in pH, even within the NCCLS-recommended range, usually associated with incubation in atmospheres containing carbon dioxide which may be necessary for the adequate growth some these bacteria can have profound effects, particularly on macrolides and azalides. Finally, the sizeof the bacterial inoculum may affect results, evenfor otherwise robusttests such as the E-test (which is also affected by carbon dioxide). 251
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.Phillips
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HAEMOPHILUS INFLUENZAE
After considerable discussion of the technical problems associated with susceptibility testingof this organism, includingthe additional factor ofthe osmolarity of the medium for an organismthat readily produces L-forms, it wasgenerallyagreed that forerythromycin and, to alesser extent, clarithromycin (even allowing for potentiation by the 14-OH metabolite) minimal inhibitory concentrations (MICs) cluster around the generally recognized breakpoints. The same may be true for dalfopristidquinupristin when breakpoints are established. On the other hand, azithromycin and, on the basis of the preliminary results, the ketolide RU 64004 appear to have rather greater anti-gram-negative activity, and thusdo not pose quite the samedifficulty. It isindicative of the problems that Barry,having proposed higher breakpoints for clarithromycin, still felt that infections caused by strains identified as having intermediate sensitivity might be well treatable as easily as those caused by fully sensitive strains. It was generally agreed that high-level MAS resistance has not yet been convincingly demonstrated for H. influenzae, but there was concern that somestrainsmightbegenuinelylesssensitive to macrolidesand azalides,some of thembeingampicillin-resistantP-lactamase-negative strains, which are rare but do occasionally cause infection. It also appears that these strainsof border-line resistancemay not share withStrep. pneumoniae and Strep. pyogenes, the property of susceptibility to streptogramins. Several contributors feltthat although properly controlled susceptibility testing is essential for epidemiological purposes, it contributes little at present to the management of the individual patient. However, when MAS treatment of haemophilus infection fails, it is advisable to carry out full invitro susceptibility teststo identify resistance whenit occurs. For this purpose, it was suggested that the E-test might be useful. Results of the use of MAS inexperimental septicemia and pneumonia added the important dimension of differential pharmacokinetics to the discussion, and the importance of area under the plasma concentration versus time curve (AUC)/MIC as opposedto @la/MIC for some of these antibiotics was mentioned. Considerable amountsof clinical data were availablefor both clarithromycin and azithromycin, demonstrating their reliable microbiological and clinical efficacy in the treatment of patients with acute otitis media which (in antibiotic concentrations in middle-ear fluid do appear to be adequate), acute sinusitis, bronchitis, and lower respiratory tract infection in children. Clearly, H. influenzae is notthe only or even the most common pathogenin these conditions, but the overall good results imply that these macrolides are generally effective for this organism as well asthe others. However, we
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Enterococci, and Anaerobes
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were reminded that at least a proportionof the patients with eachof these infections recovers without chemotherapy, although this may not be a practicable optionin the face of patients’ and parents’ expectations.
ENTEROCOCCI This session was introduced with an historical perspective of the emergence of multidrug-resistant enterococci. The following reflections were emphasized. Although the field of antimicrobial therapy has experienced many changes since the discovery of the sulfonamides more than 60 years ago, advances inour knowledge of molecular bacteriology have led to the development ofnewer and better antibacterial agents. New agents were designed to inhibit and kill bacterial pathogens by interfering with factors essential for bacterial survival and multiplication. Despite this progress in antimicrobialtherapy,bacterialpathogenshavebeenable to develop mechanismsof resistance,even to the newestclassesof antibacterial agents. One of our major problems today is the increasing incidence of serious infections caused by gram-positive bacteria, especially those that are now resistant to previously effective antibiotics. These gram-positive bacteria include strains of Staphylococcus aureus and coagulase-negative staphylococci resistant to methicillin, Streptococcus pneumoniae resistant to penicillin and macrolides, and enterococci, particularly, Enterococcus fuecium, resistant to vancomycin, gentamicin, and other antibiotics. Thus, there is an urgent need to develop new antimicrobial agents which will be effective inthe treatment of these life-threatening infections. The development of the streptogramins represents a new and unique class antibiotics, which inhibit protein synthesis at the ribosomal level and which are active against many gram-positive and gram-negative bacteria. Quinupristiddalfopristin (Q/D) currentlyis the mostdeveloped streptogramin. There wasconsiderablemeaningfuldiscussion about recent data, both in vitro and in vivo, which evaluated the potential efficacy of Q/D against enterococci, particularly against vancomycin-resistant Enterococcus faecium. Three issues were emphasized. The firstissueinvolved the role of erythromycinresistance of vancomycin-resistant Enterococcus faecium (VREF) as a predictorof Q/D susceptibility of these VREF strains. Although some data suggested that erythromycin-resistant strains of VREF were not killed in vitro by Q/D, other data suggested that erythromycin resistance had no impact on susceptibility of VREF strains to Q/D. This dichotomy was resolved favorably because two different definitions of erythromycin resistance were used in
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these studies. Specifically, high MICs to erythromycin correlated with loss of bactericidalactivity to Q/DagainstVREF,whereaslowerMICs to erythromycin (even though these MICs were relatively high) had no predi tive value of VREF resistance to Q/D. The second issue involved the clinical efficacy of Q/D onthe outcome of VREF bacteremia in surgical patients with comorbidities ofshock, dialysis, mechanical ventilation, liver failure, and immunocompromised state. Mortality was high in boththe Q/D treated patients andthe control group. However, the QD-treated cohort was less likely to die as a direct consequence of VREF infection, which suggested a beneficial effect of Q/D. However, larger clinical trialsare needed to determine the true efficacy of Q/D treatment. In this context, much discussion centered on the proper identification of VREF bacteremia. Specifically, blood cultures obtained from contaminated intravascular lines and defined as serious VREF bacteremia was considered an inappropriate definition for true VREFbacteremia because it would interfere with proper efficacy studies, not only of Q/D treatment but also for any new antimicrobial agent. We all agreed that proper technique was to obtain blood cultures from a site peripheral to an intravascular line. The third issue involved considerable discussion about the potential value of combination antibiotic therapy in the treatment of VREF infections because,in vitro, the combination of a new oral streptogramin, RPR with vancomycin or ampicillin enhanced the in vitro activity compared to either agent used alone. Additional benefit was also seen when RPR wascombinedin triplecombinations ofvancomycinplus gentamicin, or ampicillinplusgentamicin.Clearly,clinicalstudies are needed to assess the in vivo efficacy of combination therapy. All agreed that we need innovative approaches to hopefully treat serious VREF infections successfully.
ANAEROBES Again there was considerable discussion of the technical problems of susceptibility testing of anaerobes for these antibiotics.It was suggested that the rather high MICsobtained in a multicenterEuropean study might have been the result of the inactivating effect of Gcysteine, whereas those of other studies might have been adversely affected by carbon dioxide andthe consequent loweringof pH. It was agreed that MAS antibiotics are generally more active against gram-positive than gram-negative anaerobic species, although azithromycin is less imbalanced than the macrolides in this respect. Givenappropriate in vitro conditions,the ketolide RU and streptogramin combina-
Haemophilis influenzae,
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tions such asdalfopristidquinupristinhave potentially usefulantianaerobe activity, as indeed do erythromycin and clarithromycin. Even the gramnegative anaerobes such asBacteroides fragilis are probably within therapeutic range. The intrinsically macrolide-resistant fusobacteria such as F. varium are resistant to RU but strainswith acquired resistancemay not be More information is needed on cross-resistance of anaerobic bacteria among MAS antibiotics. The report of bactericidal activity of the streptogramin antibioticRU for the single strainof B. fragilis tested as well as the considerable postantibiotic effect were noted. Perhaps paradoxically in view of in vitro results, azithromycin appears to be the only one of the new MAS agents to have been tested against experimental E. colilB. fragilis infections. Treated animals fared significantly better in terms of early mortality andlate abscess formationthan did untreated controls. We were reminded that erythromycin in combination with an aminoglycoside has a long and apparently successful history as a bowel “sterilizer’’ before major largebowel surgery forthe prevention of postoperative infection. Although this does not confirm erythromycin asantianaerobe an drug, as mostof these infections involve anaerobes as well aerobes, as it is an interesting pieceof collateral evidence.The best evidence proferred of the clinical efficacy of MAS antibiotics against anaerobic infections again paradoxically relatedto azithromycin. In a placebo-controlled trial, azithromycin was shownto be useful adjunctto standard dental maneuvers inthe treatment of periodontal disease, both in terms of the depth of pockets and in the reduction of counts of Porphyromonas gingivalis and spirochetes. Clearly,beforenew MAS antibiotics are accepted as good agents for the treatment of anaerobicinfections,moreexperimentalworkis required-and justified.
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21 The Newer Macrolides,halides, and Streptogramins for the Management of HIV-Associated Opportunistic Infections Kenneth H.Mayer Memorial Hospital of Rhode Island Pawtucket, RhodeIsland
J. Allen McCutchan University of California Schoolof Medicine San Diego, California
The discussion of the uses of macrolides and azalides (MAS antibiotics) to HIV-associated infections during the workshop was dividedinto three parts: . the first two focused on the treatment and prophylaxis of Mycobacterium avium complex (MAC) infections. The third area of discussion focused on MAS antibiotics for prophylaxis and treatmentof other opportunistic infections:toxoplasmosis, Pneumocystitiscarinii, bartonellosis(rochalimeae) and cryptosporidium.
TREATMENT OF MAC The MAC treatment discussionwas introduced bydefining therapeutic goals in microbiologic and clinical terms. Microbiologically,the goal is the elimination of both MAC bacteremia and tissue reservoirs, which have 257
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recently been shown to be both anatomically and quantitatively highly variable among patients at autopsy Clinically, the goal is reliefof the symptoms of fever, weight loss, diarrhea, and fatigue. Although multidrug regimens had reduced levels of circulating mycobacteria and symptomsof MAC (3,4), the recent introductionof clarithromycin dramatically improved and simplified the therapy of MAC (5), and azithromycin has also shown promise (6). The major limitation of monotherapy with these drugs is recurrence after a few months of MAC bacteremia with organisms resistant to both macrolides. An expert panel recommended that macrolides be used in combination ethambutol, with rifabutin, clofazimine, or other drugs with demonstrated activity against MAC (7). The effectiveness of this approachin prolongingthe control of MAC by clarithromycin had not been demonstrated, however, at the time of those recommendations. This question has been subsequently addressed in a seriesof comparative studies utilizing clarithromycin in combination with two or three additional drugs to suppress resistance. As presented at this meeting (8), the California Collaborative TreatmentGroup (CCTG) demonstrated that the addition of ethambutol delayed emergence of resistance, but a significant proportion of patients (about half of the responders by around nine months) still broke through with MAC bacteremia. In a complementary study, clofazimine had no effect when added to ethambutol and clarithromycin (5). About two-thirds of patients responded initially, regardless of the number of typeof drugs addedto clarithromycin, suggestingthat clarithromycin drives the dramatic clearance of MAC bacteremia. Thus, ethambutol appears to delay emergenceof clarithromycin-resistantMAC, but clofazimine does not. The high rate of recurrent bacteremias with clarithromycin-resistant MAC documented by the CCTG study necessitates acontinuedsearch for amore potent seconddrug to augment clarithromycin intreatment of MAC bacteremia. During the discussion, Dube addressed several unsolved. technical problem in MAC treatment studies: the definitions of both response and relapse bymicrobiologicandclinical criteria. He showed that twomicrobiologic definitions of relapse utilized in the CCTG trial led to substantiallydifferingestimates of the rates of relapse by Kaplan-Meier survivalanalysis. The first(protocol-defined)methodwhich required sustained recurrence of bacteremia appeared to seridemonstration ously underestimate the rate of relapse (only 5% relapsed on clarithromycinplus ethambutol and clofazimine). A second(posthoc)method (i.e., any positive blood culture for MAC after any negative culture in patients who responded to treatment) appeared to be moreclinically relevant (about half of patients relapsed) but might not be capable of
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distinguishing relapses due to poor compliance from those due to failure of treatment. A third method based on the isolation of clarithromycin-resistant MAC from blood may be preferable, as such breakthroughs presumably reflect failure of sustained treatment, rather than noncompliance. In other words, only patients who fail during a sustained exposure to clarithromycin should relapse withclarithromycin-resistant MAC. Noncompliant patients would be more likelyto relapse withclarithromycin-sensitiveMAC. In fact, the third method agreed closely in the CCTG study with the second microbiologic definitionof relapse: a positive blood culture following a negative culture. Thus, recurrent macrolide-resistant MAC bacteremia may be the best measureof true treatment failure, and recurrent macrolide-sensitive MAC may measure noncompliance withtreatment. Another methodological problem has been to identify clearlythe clinical manifestationsof relapse in the face of multiple other potential causesof fever, wasting, and diarrhea in advanced AIDS patients. Future studies will also need to evaluate the role of other new drugs to add either to clarithromycin or azithromycin for improving the duration of treatment responses centered on the aminoglycosides (e.g., amikacin), quinolones (e.g., ciprofloxacin and sparfloxacin), newer rifamycins (e.g., rifabutin and KR”1648), and immune modulators (such as gamma interferon andGM-CSF). ACTGstudy is currently examining rifabutin, ethambutol, and both drugs added to clarithromycin for MAC treatment.
PROPHYLAXIS OF MAC Issues of prophylaxis of MAC were introduced by a comparison of the regimens for which substantial evidence of efficacy has been developed. The first, rifabutin mg, was the original drug approvedfor this indication (9). The three other regimens were discussed in abstracts of this conference (8, 10-18). Clarithromycin is licensed inthe United States for treatment for MAC and is widely used for prophylaxis (500 mg) based on a large,placebo-controlledstudy (13). Inaddition,twotrialscomparing azithromycin 1.2 g weekly either to placebo (19) or to rifabutin alone and to the combination of rifabutin and azithromycin(16) have establishedthe value of weekly azithromycin for MAC prophylaxis. Daily clarithromycin and weekly azithromycin appearto have similar efficacy(about 70%) and are superior to daily rifabutin (50% efficacy). In order to compare the four regimens, the annual costs of prophylaxis for 100 patients was divided by the estimated number of cases pre-
Mayer and McCutchan
260 Table I
Agent
Candidate Regimens for MAC Prophylaxis in AIDS Other Costlcase Annual Cases mstb prevented8 Efficacy
benefits prevented ~
Rifabutin 300 mg qd Clarithromycin 500 2O1,94gc21 Azithromycin 70% 1200 Azithromycin 85 PCP 1200 qw, and rifabutin 300 mg
50%
%
15
297,367
19,842
21
355,741
16,940 respir. PCP infect. ? Giardia 9,617 Respir. infect.,
25.5
Respir. 19,580 499,315
?
infect.,
.Annually per 100 patients assuming per annum incidence in target population (< 100 CD4 cells). bEstimated pharmacy dispensingcost (“Red Book” wholesale cost of drug + 30% + $8.50 dispensing fee every 2 months) for 100 patients. CBased on price of 2 600-mg tablets approved for prophylaxis.
vented in 100 patients to generate the cost per case prevented (Table 1). The number of cases prevented per 100 patients is calculated by multiplying the annual incidence of MAC in patients with CD4 < 100 (approximately 30 cases/100 patientdyear) times the prophylacticefficacy(e.g., 50% for rifabutin, 70% for either azithromycin or clarithromycin, and 85% for azithromycin plus rifabutin). The costs for each drug were calculated using wholesale acquisition costs fromthe ‘Red Book‘ to which pharmacy dispensing costsare added as outlinedin footnote b of Table 1. The cost of azithromycin was based on the 2 600 mg, lactose-free capsules used in the Pfizer/CCTG study. Costsper case of MACprevented for daily clarithromycin($16,940) and for daily rifabutin ($19,842) are higher than for weekly azithromycin ($9,617). The most effective regimen, the combination of azithromycin weekly and rifabutin, is more costly ($19,580 per case prevented) and requires more than four times as many capsules per week as azithromycin given weekly for monoprophylaxis. In addition to the issues of cost, both rifabutin and clarithromycin have potential drug interactions with multiple drugs frequently taken by advanced AIDS patients. For example, fluconazole increases rifabutin conce trations which can result in anterior uveitis (20). Clarithromycin increases levels of theophylline, carbamazepine, and terfenadine. Thus, the use of macrolides for prophylaxisof MAC inAIDS appears well established, and initial estimatesof costs and benefits favors weekly azithromycin over other currently licensed drugs. More sophisticated pharmacoeconomic analysis
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taking into account benefitsof these drugsfor prophylaxis of other opportunistic infections andthe costs of drug interactions shouldbe undertaken to better estimate their total impact.
OTHER OPPORTUNISTIC INFECTIONS Although the combination of the sulfa drug plus a folic acid antagonist has been shown to be effective inthe treatment of HIV-infected patients who develop cerebral toxoplasmosis, relapses are not uncommon and intolerance to a one-drug regimen maybe particularly frequent in HIV-infected persons The dose-limitingtoxicity of pyrimethamine maybe leukopenia, whichmay or may not respond to treatment with colonystimulating factors, and many HIV-infected patients become sulfonamide intolerant over time-however, many will respondto desensitization protocols. The second-line treatment agents,suchasclindamycin,mayalso result in unacceptable toxicities, such as refractory diarrhea. Newer regimens may include drugs like atoviquone. However, the experience of using spiramycin in the treatment of nonimmunocompromised patients with a variety of toxoplasmosis syndromes has raised the question of whether the macrolides alone or in combination withother drugs may constitute effective therapy for HIV-infected patients with cerebral toxoplasmosis. Initial reports indicated that failures were seen in HIV-infected patients who received monotherapy witheither spiramycin or roxithromycin (24). Several patients have been reported to have developed toxoplasmosis whilereceivingclarithromycin Subsequent studies have suggested that pyrimethamine plus clarithromycinwas comparable to pyrimethamine plus clindamycin However, complete responses were noted in only about 50% of patients in both treatment groups, and patients who failed this regimenreported anemia, nausea, and increased liver function tests, as well as hypoacusis. At the current conference, Brun-Pascaud presented a poster which indicated that in a rat model, roxithromycin plus dapsone or in combination with a sulfonamide was active against both Pneumocystis carinii pneumonia(PCP)andtoxoplasmosis.Thisfindingraised the hope that the combination of a macrolide plus a sulfa, sulfone,or pyrimethamine could result in prophylasis against three very common opportunistic infections (i.e., PCP,toxoplasmosis,and M. avium-intracellulare infection). Craft, Crampton, and Henry presented several posters at the conference suggesting that among participantsin a placebo-controlled studyof clarithromycin 500 mg twiceper day to perventM. avium infection, there was significantly lessPCP versus 10% in the placebogroup,p-value = .021). In
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addition, there was less community-acquired pneumonia (7% versus 13%, p = .Ol), as well as less giardiasis(0.9% versus p = .048). However, the prevalenceoftoxoplasmosiswasnotshown to be prophylaxis of M. aviumsignificantlyreducedin the Craftstudy intracellulare infection with clarithromycin; but it is importantto note that the study was done in North American where the prevalence of toxoplasmosis antibodies is significantly lower than in Europe. It is possible that future prophylaxis studies conducted overseas might be ableto show some added benefit from the use of a macrolide for the prophylaxis or other opportunistic infections. However, atthe present time, the data are not sufficiently strong to suggest the use of a macrolide as first-line treatment for toxoplasmic encephalitis, or for the prophylaxis of patients who have positive toxoplasmosis antibody titers andlow CD4 counts. However,inthosepatientswho are notable to tolerate sulfonamides or pyrimethamine or who progress despite appropriate first-line therapy, the next choice wouldbe clindamycin. However, there is a substantial number people livingwith HIV whomay not be able to tolerate long-term clindamycin therapy because of diarrhea, and for those individuals, the useof amacrolideincombinationwith other activeantitoxoplasmosis agentsmaybeuseful. Another alternative wouldbe the use of atovaquone, and further studies have to be done on alternative salvage therapies for patients who do not respond to pyrimethamine/sulfadiazine. The macrolides have also been shown to have some efficacy against cryptosporidiosis in animal models (27). However, studies by Soave and colleagues have shown partial responses with monotherapy using azithromycin at high daily doses (up to 1.8 g per day), with frequent relapses (28). The useof intravenousmacrolidetherapy for thisopportunistic cause of diarrhea is also undergoing study. Another study by Blanshard and colleagues revealedthat azithromycin given 1 g as a loading dose and then 500 mg per day did not decrease the number of stools per day or oocyst burden (29). However, at the conference, roxithromycin given 300 mg twice per day for 4 weeks resulted in complete responses in two different studies from Brazil (Sprinzet al. and Uip et al.) and partial responses inabout an additional quarter of individuals. The authors indicated that in patients for whom there was not anappropriate clinical response,or when the response was onlypartial, they triedother macrolides such as azithromycin, occasionally with success, and alsoadded other active agents, such as paromomycin The consensus at the meeting was that more efficacious therapy of cryptosporidial diarrhea could possibly be achieved by the addition of a macrolideplusparomomycinand that thiscombinationwarrants more careful critical study.The discussion also notedthe high degree of variabil-
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ity with regardto the syndrome associated with cryptosporidial enteritis, in that in patients with higher CD4 counts who developed cryptosporidiosis, the disease may be self-limited or be more likely to respond to a short course of an oral macrolide; in other individuals with extremely lowCD4 counts, the likelihood that the macrolide could be adequately absorbed and efficacious would be less likely. Thus, the interpretation of nonrandomked, carefully controlled clinical trials is difficult, although the data presented the hope that the newer macrolides could play a role in the management of cryptosporidial diarrhea inAIDS patients. The workshop concluded with an increased awareness that the macrolides are highly efficacious for many diverse HIV-associated opportunistic infections. Thus, verysoon, virtually all patients with advanced HIV infection will likely receivea macrolide aspart of their prophylaxisor treatment regimen (31). This will raise many confusing management questions in the future if clinical illnesses such as M.avium-intracellulare, toxoplasmosis, or cryptosporidiosis occur while they are receiving an MAS antibiotic. By ICMAS IV, hopefully there will be new reports as to how macrolides may be used in future prophylaxis and therapeutic regimens. In addition, at this conference other novel uses of the newer macrolides were discussed, such as the treatment of bacillary angiomatosis (Paucaret al.) andother atypical non-MAC mycobacterial infections. Thus, the use of the newer macrolides can be expected to continue to increase in HIV-infected populations over the next few years, with precise indications being further refined as clinical trials are completed and clinical experience matures.
REFERENCES 1. Tbrriani FJ, McCutchan JA, Bozzette SA, Grafe M R , Havlir DV. Autopsy findings in AIDS patients with mycobacterium avium complex bacteremia. J InfectDis 1994; 170:1601-1605. 2. Tomani FJ, Behling CA, McCutchan JA, Haubrich RH, Havlir DV. Disseminated mycobacterium avium complex: correlation between blood and tissue burden. J Infect Dis1996. 3. Chiu J, Nussbaum J, Bozzette S, Tiles JG, Young LS, Leedom J, Heseltine NR, McCutchan JA, the California Collaborative Treatment Group. Treatment of disseminated mycobacterium aviumcomplex infectionin AIDS with amikacin, ethambutol, rifampin, and ciprofloxacin. Ann Intern Med 1990; 113~358-361. Bartok AE, Leedom 4. Kemper CA, Meng TC, Nussbaum J, Chiu J, Feigal DF, JM, Tilles JG, Deresinski SC, McCutchan JA, the California Collaborative Treatment Group. Treatment of mycobacterium avium complexbacteremia in AIDS with a four-drug oral regimen. Ann Intern Med 1992; 116:466-472.
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5. Chaisson RE, Benson CA, Michael DP, Leonid BH, Korvick JA, Elkin S, Smith T, CraftC, Sattler FR. Clarithromycin therapyfor bacteremic mycobacterium avium complex disease: a random, double-blind, dose-ranging study in patients with AIDS. AnnIntern Med 1994; 121:905-911. 6. Young LS, Wiviott L,Wu M, KolonoskiP, Bolan R, Inderlied CB. Azithromycin for treatment of mycobacterium avium-intracellulare complex infection in patients with AIDS. Lancet 1991;338:1107-1109. 7. Mazur H, etal. SpecialReport: recommendations on prophylaxis andtherapy for disseminated mycobacterium avium complex disease in patients infected with the human immunodeficiency virus.N Engl J Med 1993;898-904. 8. Dube M, Sattler F, Torriani F, See D, Havlir D, Kemper C, Dezfuli M, Bozzette S, Bartok A, Leedom J, Tiles J. McCutchan J. Clarithromycin plus clofazimine, withor without ethambutol for the prevention of relapse of MAC bacteremia in AIDS. In: Program and Abstracts of the Third International Conference on Macrolides, Azalides and Streptogramins, Lisbon, 1996;abstr 7.07. 9. Nightingale SD, Cameron W, Gordin FM, Sullam PM, Cohn DL, Chaisson RE, et al. lbo controlled trials of rifabutin prophylaxis against mycobacterium avium complex infection in AIDS. N Engl J Med 1993; 329:828-833. 10. Miller DA, Clarithromycin:safeandeffectiveprophylaxisagentagainst MAC. In: Program and Abstracts of the Third International Conference on Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 7.01. 11. Sinnott JT, HoltDA, Houston H, Bergen G, Sakalosky P, Larkin J. Clarithromycin 500 mg BID as prophylaxis for MAC disease: a follow-up review. In: Program and Abstractsof the Third International Conference on Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 7.02. 12. Grieger-Zanlungo P, Zev C. Clarithromycin 500 mg BID for dMAC prophylaxis. In: Program and Abstracts of the Third International Conference on Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 7.03. 13. Pierce M, Crampton S, Henry D, Craft C, NotarioG. Theeffect of MAC and its prevention on survival in patients with advanced HIV infection. In: Program and Abstracts of the Third International Conference on Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 7.04. WH, IndorfAS. 14. File TM, Klippel DC, LongstrethSJ,SignsDJ,Ruby Clarithromycin prophylaxis for disseminated mycobacterium avium (dMAC). In: Program and Abstracts of the Third International Conference on Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 7.05. 15. Craft J, Henry D, Olson CA, Notario G, Hom R, Crampton S, Pierce M. Prevention of resistance to clarithromycin during prophylaxis for disseminated mycobacterium avium complex (MAC). In: Program and Abstracts of the Third Internationql Conferenceon Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 7.06. 16. Havlir DV, Dube M P , Sattler FR, et al. Prophylaxis against disseminated Mycobacterium avium complex with weekly azithromycin, daily rifabutin or both. N Engl J Med 1996;335:392-398.
Newer Macrolides, Azalides, and Streptogramins and H N infections
265
17. Crampton S, Craft JC, Notario G, Henry D. Prevention of pneumocystis carinii pneumonia in AIDS patientsby clarithromycin prophylaxis for mycobacterium avium complex (MAC). In: Program and Abstracts of the Third International Conference on Macrolides, Azalides and Streptogramins, Lisbon, 1996;abstr 7.09. 18. Crampton S, Craft JC, Henry D. Clarithromycin prophylaxis for MAC in reduces the incidence of AIDS patients with CD4 counts < 100 ~elldmrn-~ community-acquiredpneumonia.In:ProgramandAbstracts of the Third International Conference on Macrolides, Azalides and Streptogramins, Lisbon, 1996;abstr 7.10. 19. Oldfield et al. Third National Conference on Retroviruses, Washington,DC, 1996. 20. Havlir D, Torriani F, Dube M. Uveitis associated with rifabutin prophylaxis. Ann Intern med 1994; 121510-512. 21. Richards FO, Kovacs JA, Luft BJ. Preventing toxoplasmic encephalitis in persons infected with human immunodeficiency virus. Clin Infect Dis 1995; 21(~~ppl):S49-S56. the acquired immuno22. Luft BJ,et al. Toxoplasmic encephalitis in patients with deficiency syndrome. N Engl J Med 1993; 329:995-10oO. 23. Porter SB, Sande MA. Toxoplasmosis of the central nervous system in the acquired immunodeficiency syndrome. N Engl J Med 1992; 327:1643-1648. 24. Saba J,et al. Pyrimethamine plus azithromycin treatment for of acute toxoplasmic encephalitis in patients with AIDS. Eur J Clin Microbiol Infect Dis 1993; 12~853-856. 25. Ruf B, Schurmann D, Pohle HD. Failure of clarithromycin in preventing toxoplasmic encephalitis in AIDS patients (letter). J Acquired Immune Defic Syndr 1992; 5530-531. 26. Leport C, et al.Combination of pyrimethamine-clarithromycin for acute therapy of toxoplasmic encephalitis. A pilot study in 13 AIDS patients. 30th ICAAC, 1990;abstr 1158. 27. Blanshard C, et al. Cryptosporidiosis in HIV-seropositive patients. Quart J Med 1992; 85307-308. 28. Soave R, et al. Oral diclazuril therapy for cryptosporidiosis. Abstract of the VI International Conference on AIDS, San Francisco, 1990. 29. Blanshard D, et al. Azithromycin, paromomycin and letrazuril in the treatment of cryptosporidiosis. Third European Conference on Clinical Aspects and Treatmentof HIV Infection, Pans, 1992;abstr P28. 30. White AC, et al. Paromomycin for cryptosporidiosis in AIDS: a prespective double-blind trial. J Infect Dis1994; 170:419-429. 31. Centers for Disease Control:USPHSLIDSA Guidelines for the prevention of opportunistic infections in persons infected with Human Immunodeficiency Virus. MMWR 1995; 44:l-24.
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22 Special Pathogens John E. McGowan, Jr. Emory University Atlanta, Georgia
Joel Ruskin Kaker Permanente Medical Center andUCLA School Los Angeles, California
Medicine
The development of new macrolides, azalides, and streptogramins has led to unique and unusual applications of these antimicrobial agents. There was extensive discussion in the seminar regarding how best to utilize the drugs withinthe clinical context ofcurrent therapeutic options, what advantages new regimens would offer in terms of increased efficacy, safety and tolerance, and whether the new data presented at the meeting might, in fact, alter the treatment of a varietyof infectious diseases.
HUMANAND ANIMAL BITE WOUND INFECTIONS Human and animal bites may cause a wide spectrum tissue damage, ranging from superficial breaks in the integument to cellulitis, septic arthritis, and osteomyelitis. In an emergency-room setting, the bite victim is often treated empirically with an oral antibiotic. The drug selected should possess activity against the polymicrobial flora most likely to cause such an infection. The culpable bacterial species include streptococci, staphylococci 267
268
McGowan and Ruskin
(usually S. aureus), Pasteurella multocida (especially important in cat bites), oral anaerobes (Prevotellu and Porphyromonas spp. are common, whereas Fusobacterium nucleatumis not), and Eikenellu corrodens. Amoxicillidclavulanate is generally consideredthe best choice to cover infection with these organisms. However, penicillin-allergic patients need alternative treatment. Goldstein and Citron (Santa Monica, CA) evaluated the in vitro efficacy of the newer macrolides azithromycin, clarithromycin, and roxithromycin against isolates commonly cultured from bite wounds. Azithromycin was foundto be the most active against many of the aerobes, including R multocida [minimalinhibitoryconcentration for 90% of isolates (MI(&,) < 2pg/ml] and E. corrodens, and was 2 to 4 dilutions more active thanerythromycinversusanaerobes.Overall,clarithromycinwasless activethanazithromycin,androxithromycinwasevenlessactive than erythromycin. Neither Goldstein nor others in the workshop had substantive clinical experience with the newer macrolides in bite wound management. Yet, in light of the above in vitro data, there was consensusthat at least azithromycin merited clinical evaluation in the penicillin-allergic patient. However, two caveats were stressed. First, in the emergent care of bite wounds, particularly clenched-fist injuries, copious irrigation followedby elevation of the affected extremity is essential for cure; no antimicrobial is likely to be of benefit without such intervention. Second,if the bite wound extends to bone and osteomyelitis ensues, the consequences may be devastating. In this scenario (which is not uncommon when the infection is due to P. multocida), it is probably bestto avoid therapy with any ofthe macrolides.
LISTERIA AND CORYNEBACTERIA
A concern of the 1990s is the growing microbialthreat posed byresistant croorganisms. Bacteria resistant to p-lactam drugsare now seen to be resistant as well to glycopeptides. Among these are the Corynebacteria, which have begunto play an increasingly important role as nosocomial pathogens. Corynebacterium jeikeiumis the most common organism of thisgroup and is often associated with intravascular infection. In studies of this bacterium, Holloway and associates (Wilmington, DE) determined its in vitro susceptibility to the streptogramin quinupristiddalfopristin (RP59500).Susceptibility to quinupristiddalfopristin could addto our therapeutic armamentarium an alternative drug for strains resistant to the current antibiotics of choice, vancomycin andthe quinolones. The early stage of these investigations demonstrated differencesin in vitro susceptibilityof the organism to quinupristiddalfopriistin when testing was performed in glass tubes asop-
Special
269
posed to plastic containers. Ultimately, the laboratory test system to be used must be defined by a clinically relevant gold standard. However, none is yet available, as few patients have beentreated with this drug. Listeriu monocytogenes, another organism of nosocomial importance, wasconsideredinpresentationsfrom the United States andGermany. Variable in vitro susceptibility to quinupristiddalfopristin was noted in different test media. The study from Hof and colleagues (Mannheim,Germany) suggested that this drug, as well as clindamycin and other macrolides, are much less active against Listeria when present inside cells with an efflux pump. The clinical impact of these fascinating observations remains to be determined.
BRUCELLOSIS Brucellosis, although uncommon in the United States, still exists worldwide, especially in the Middle East, the Indian subcontinent, and in parts of Mexico and Central and South America. Rubinstein and colleagues (Tel Aviv, Israel) reported findingsfromamurinemodelofinfectionwith Brucella melitensis. In Rubinstein’s view, the model mimics the subacute, disseminated granulomatous infectionin man, where brucellae localizein organs (viz. liver and spleen) rich in elements of the reticuloendothelial system. In the murine model and in man, combination antibiotic regimens have been shownto be the optimal therapyof Brucella infection. Excellent results have been achievedwith tetracycline plus streptomycin,or tetracycline plus rifampin. Single-drug treatment, notably with cotrimoxazole or the quinolones, has been unsuccessful. It was proposed that single-drugtherapywith one of the newer macrolides might yieldbetter results. To test this hypothesis in their murine model of the infection, Rubinstein and his colleagues administered azithromycin or clarithromycin for 7-14 days, beginning 7 days after intraperitoneal inoculation of B. melitensis. Azithromycingiven for either 1 or 2 weekswasnearly100%efficacious.Moreover,azithromycinprevented relapse for a 4-week follow-up period. other antimicrobial previously tested in this model produced such dramatic results. Rubinstein believes these data warrant a clinicaltrial of azithromycin therapy of human brucellosis. Infected infants and pregnant women, in whom the use of tetracyclines is precluded, would be desirable subjects. On the other hand, he was less sanguine about the use of azithromycin or macrolides at this time for the treatment of the osteoarticular complications of brucellosis. Unfortunately,the latter have been notoriously refractory to protracted courses of standard drug combinations, including those that incorporate parenteral streptomycin.
McGowan and Ruskin
270
PERTUSSIS Whooping cough continuesto be an important causeof infectious morbidity in New Zealand. Brett and co-workers at the Communicable Disease Center (Porirua, NZ) wereinterested, therefore, in assessingthe activity of roxithromycin andother drugs against recent isolates there. Roxithromycin was found to bebactericidal at easilyachievableconcentrations.They noted inthe discussion that clinical trialsof this drug, a more easily administered alternative to erythromycin, are planned. InZagreb, Croatia, Bace et al. used azithromycin in a clinical trial in children with pertussis. Bacteriologic eradication byday 7 wasseeninall15children treated, and no adverse effects were seen. They suggested to the group that the advantage of the drug in preference to erythromycin (shorter treatment course and thus better patient compliance) madefurther evaluation of azithromycin in whooping cough reasonable.
SALMONELLOSIS Multidrug-resistant salmonellae, includingS. typhi, are being isolated with increasing frequency throughout the world. Treatment regimens with chloramphenicol, ampicillin,or cotrimoxazole are no longer reliable. The quinolones (notably ciprofloxacin) have been utilized with some success, but concerns remain about their use in children and pregnancy. Azithromycin is known to achieve huge intracellular concentrations in intestinal lymphoid tissue, where salmonellae reside and multiply. Kelneric and co-workers from a veterinary institute (Zagreb, Croatia) studied the activity of azithromycin and erythromycin against animal isolates of S. enteriditis and S. typhimurium, as well as against isolates of S. virchow and S. typhi recovered from humans. More than 90% of the 67 strains tested were inhibited by azithromycin at concentrations between 2 and 4 &ml, whereas high-level resistance to erythromycin was demonstrated. Rubinstein and Ruskin noted that there is already-published salutary human experience withazithromycinin treatment of salmonellagastroenteritis as well as typhoid fever. In viewof these supportive in vitrodata from Croatia, azithromycin and possiblyother new macrolides merit testing in large-scaletreatment trials of clinical salmonellosis.
MALARIA Malaria remains a leading cause of morbidity and mortality in the developingworld; 100-300millioninfectionsand1-1.5million deaths occur yearly. The highly developed world is by no means immuneto this “tropi-
271
Special
cal” disease; in 1994, 1229 cases werereported in the United States. New and safe prophylactic agents effective against R falciparum are urgently needed. Currently, mefloquine is recommended for some settings and is about 90% effective. However, mefloquine-resistant strains already exist and are likely to spread withincreasinguseof the drug.Moreover, mefloquine has disadvantages: It is expensive and is not recommended in pregnancy, in infants weighing4 mg/L for inducibly and constitutively MLS,-resistant strains, respectively, as shown by induction experiments carriedout in parallel.
Susceptibility Testing (1) In vitro susceptibility tests were performed by using a twofold agar dilution method. Mueller-Hinton agar medium (pH 7.4; Diagnostic Pasteur, France) was used throughout the study. The medium was appropriately supplemented to support the growth of some fastidious microorganisms
In Vitro Antibacterial Activity
R U 004
397
(4% globular extractfor Haemophilus injluenzae;7% horse bloodfor streptococci and pneumococci). Reference organisms were included for quality control: S. aureus ATCC 29213 and E. faecalis ATCC 29212. A standard inoculum of 104CFW (colony-forming units)/ spot was used throughout. All plates were incubated at 36°C for 20 h and the MIC was defined as the lowest concentration at whichnovisiblegrowthwas detected on agar plates.
Determination of Minimal Bactericidal Concentrations First, a broth microdilution test was used to determine MICs. Subcultures on Mueller-Hinton agar or other appropriate medium were made from those dilutionsin microdilution plates which failed to show visible growth. The agar plates were read after 24 h incubationat 36°C. The lowest dilution of the antibiotic yielding no bacterial growth on the agar subculture was taken as the minimal bactericidal concentration (MBC).
In Vitro Resistance Studies The development of resistance to RU was studied with several bacterial strains using a macrodilution method. An M C . of RU 004 was first determined in Mueller-Hinton broth against a test strain. Bacteria from the highest concentration of RU 004 showing growth were subculturedto tubes containing doubling concentrations of RU 004. Again, an MIC was determined andthe highest concentrationof RU 004 was subcultured once more in doubling concentrations the antibiotic. This subculture process was repeated several timesto study possible buildupof resistance.
RESULTS Results are shown in Table1 and Figs. 1and 2. Tuble I
In Vitro Antibacterial Activity of RU
(GM,m a ) ~~
Strains (No.)
Phen.
Staphylococci (65i)b Enterococci (13i+19c) Streptococci (27) Pneumococci (38i+34c)
ET-R Ery-R ET-R
H. influenza (80)
Ery-R -
RU 0.027 0.20
0.027 0.039 0.23
ERYa
CLA
AZI
PRI
>40 22.7 7.4 29.1 1.59
23 17.7 3.7 23.6 1.77
>40 33.6 15.1 >40 0.16
0.12 1.14 0.057 0.22 0.37
'ERY: erythromycin M I : azithromycin; C L A : clarithromycin; PRIS: pristinamycin. c: inducible, constitutiveM L S , resistance.
Agouridas et al.
398
Figure I
In Vitro activity of RU
against ERY-R gram-positive
DISCUSSION
In Vitro Activity of RU
Against Relevant Nonatypical Respiratory Pathogens Gram-PositiveBacteria
I
Against strains susceptible to macrolides, the activity of RU 004 is one (staphylococci, P-hemolytic streptococci, pneumococci) or two orders of magnitude (enterococci) higher than that of other available drugs [e.g., clarithromycin and erythromycin A (data not shown)]. Because RU 004 does not induce M L S , resistance, all clinical isolates inducibly resistantto macrolides are highly susceptibleto the ketolide at a level identicalor very close to that found with susceptible bacteria (staphylococci, enterococci, pneumococci). It isconsiderablymoreactivethanjosamycin,a 16membered-ring macrolide considered the as reference against strains exhibiting inducible macrolide resistance. With the exception of staphylococci, RU 004exhibits high activity against all other constitutively resistantMU, gram-positive cocci. In this respect, RU 004 displays potent activity against pneumococci resistant to macrolides andor penicillin G andor several
In Vitro Antibacterial Activity of RU 004
399
Figure 2 In VitroActivity of RU againstatypicalbacteria. (A) Antichlamydial activity (MLC) (after Haider F, et al. 35th Intersci Conf Antimicrob Agents Chemother, 1995; abstr. F-165).(B) Antibacterial activity against45 strains of Legionella (afterBorstein N, et al., 35thIntersciConfAntimicrob Agents Chemother, 1995; abstr. F-166).
Agouridas et al.
400
Gram-Negative Bacteria The activity of RU 004against H. influenzae is high, closeto that found for azithromycin. In addition, the new compound does not show dissociated activity against isolates susceptible or resistant to ampicillin. RU 004shares the same antibacterial potency the as 14-membered-ring macrolides against Moraxella catarrhalis. RU 004 is slightlymore activethan available macrolides against several species of the family Enterobacteriaceae, with the exception of Salmonella sp. against which its efficacy is slightly less than that of azithromycin (data not shown). Other Properties of RV 004 RU provides MBC values equivalent to the MICs for pneumococci and H. influenzae. The Potential selectionof mutants by RU 004 wasstudied by serial passaging in broth. In contrast to josamycin, the increase in MICs was very slow (data not shown). Thus, the emergence of resistance to the ketolide is expected to be slow for pneumococci and enterococci.
In Vitro Activity of RU Against Atypical Bacteria (Fig. 2) Mycophma The new ketolide RU was very active in vitro against all mycoplasma species tested, M. pneumoniae, M. genitalium, M. penetrans, U. urealyticum, and even M. hominis and M.fermentam. Its activity was better than the other available drugs (macrolides or azalides). Furthermore, this antibiotic was bactericidal against most mycoplasma species tested, except M. homink (data not shown). Chlamydia RU displays very high activity against the three species of Chlamydia tested far, a resultwhich confirmsits well-balanced spectrum.There is a clearbactericidaleffect of the drugwhichmay eradicate intracellular chlamydia. Legionelha RU 004 displays excellent activity against clinical and reference Legionella strains (MI% 0.03 and 0.03 m& compared with 0.5 and 0.12 m& for erythromycin). In comparison with macrolides,RU 004is the only drug whose MICs are never above 0.06 m&. Its activity against L. pneumophila is identical to that of the most active macrolide, clarithromycin.It is superior to all macrolides againstother species of Legionella.
In Vitro Antibacterial Activity of R U 004
401
Considering these results, as well asthe high intracellular accumulation inside phagocytes,RU 004 appears to be a promising new agent for the therapy of infections due to intracellular pathogens. In vivo experiments are now needed to assess the potential clinical value of the new ketolide.
CONCLUSIONS RU 004 isactiveagainstmultiresistantpneumococciincluding penicillin-resistant and macrolide-resistant strains. It possesseswell-balancedantibacterialactivityagainstrelevant pathogensinvolvedinRTI(gram-positivecocci, H . influenme, and M. catarrhah, regardless of resistance antibiophenotypes, as well as atypical bacteria). Unlike macrolides, it does not induce the so-called MU, resistance (staphylococci or pneumococci), thereby exhibiting potent activity against all macrolide inducibly-resistant strains and opening a fundamentaldebate on the role of cladinose inthe induction of resistance. These findings strongly suggest a mechanism action dissociated fromthat of macrolides. Its physicochemicalpropertiesand structure give RU 004 high stability in acidic medium, little influence of pH on antibacterial activity, and high intracellular accumulation, accompanied by adequate efflux kinetics whichensure total elimination of the drug. Conforming to its in vitro profile,RU 004 displays potent therapeutic efficacy in animals infected by all relevant strains of bacteria. RU 004thus appears as avery promising agent inthe treatment of infections causedby difficult-to-treat respiratory pathogens.
REFERENCE 1. Lorian V. Antibiotics in Laboratory Medicine, Baltimore,MD: Williams and Wilkins, 1993.
Antistaphylococcal Activity of Quinupristidlalfopristin, a Well-Defmed Mixture of Chemically Modified Streptogramins Christof von Eiff and Georg Peters Westjalische Wilhelms- Universitat Munster Munster, Germany
INTRODUCTION Pristinamycin is a mixtureof naturally occurring streptogramin antibiotics isolated fromStreptomyces pristinaespiralis. It consists of a complex oftwo main componentsof water-insoluble compounds, pristinamycin I, (a group B streptogramin) and pristinamycin 11, (a group A streptogramin), which have synergistic antibacterial activities. Pristinamycin I, and 11, have been chemically modified to provide a well-defined mixture : 70) of watersoluble semisynthetic derivates suitable for parenteral administration (1). In our study, the in vitro activity of this mixture, quinupristiddalfopristin (RP59500) was compared with thoseof other antimicrobial agents against methicillin-susceptible and -resistant S. aureus and speciated coagulasenegative staphylococci.
402
Antistaphylococcal Activity of QuinupristiniDalfopristin
MATERIAL AND METHODS A total of 247 staphylococcal strains freshly isolated from clinical material was tested. Only one isolate per patientwas included. In addition, because methicillin-resistant Staphylococcus aureus strains can cause epidemics, we used several criteriato avoid multiple copiesof the same strain: (i) strains were from different geographic locations (both within hospitals and from different hospitals), which would reduce the chance of obtaining a single clone, (ii) collecting strains over a period of years would also make a single clone unlikely, and (iii) when strains with similar antibiograms and phenotypes were obtained, we performed pulsed-field gel electrophoresis and selectedonly one clone. The S. aureus strainsincluded 29 penicillinsusceptible S. aureus (PSSA), 40 methicillin- susceptibleS.aurews (MSSA), and61methicillin-resistant S. aureus (MRSA). The coagulase-negative staphylococci (CONS) comprised24 methicillin-susceptible S. epidermidis (MSSE), 28 methicillin-resistant S. epidermidis (MRSE), 20 methicillinsusceptible S. haemolyticus (MSSH), 23 methicillin-resistant S. haemolyticus (MRSH), and 22 other coagulase-negative staphylococci belongingto novobiocin-susceptiblecoagulase-negativestaphylococci [S. hominis (7), S. warneri S. simulans (3), S. capitis (2), S. lugdunensis (l)] and to novobiocin-resistant coagulase-negative staphylococci[S. saprophyticus ( 9 , S. cohnii (l)]. The minimalinhibitoryconcentrations(MICs)were determined on Mueller-Hinton agar (Difco, Augsburg, Germany) using the agar dilution technique. A dilution of a fresh overnight culture was applied to agar platesby using a multipoint inoculator (MastLaboratories Ltd., Bootle,England),yieldingafinalinoculum of105 CFU (colonyforming units). Isolates were confirmed to be methicillin resistant by supplementation of the agar with 2% NaCl (48 h, The antibiotics were tested in 13 different concentrations ranging from 0.031 m& to 128 m&. The following substances were used and obtained from their respective manufacturers: RP 59500, vancomycin, and ciprofloxacin. The results were read after 18 h incubation at 36°C. Additionally, the following reference strains were included:S. aureus: ATCC 25923, ATCC 29213;Enterococcus faecalis: ATCC 29212; Escherichia coli: ATCC 25922, ATCC 35218;Pseudomonas aeruginosa: ATCC 27853.
RESULTS The ranges of MICs, MIC,$, and MIC,s for the 130 strains of S. aureus are showninTable 1 and the correspondingvalues for the 117 strains of coagulase-negative staphylococci tested are giveninTable2. The MIC
von Eiff and Peters
404 Table I
In vitro activity of RP 59500,Vancomycin, and Ciprofloxacin Against
130S.aureus Strains Range
S. awe@
nwa
mc50 ~~
RP 59500 Vancomycin Ciprofloxacin
PSSA MSSA
RP 59500
MRSA
Vancomycin Ciprofloxacin RP Vancomycin Ciprofloxacin
0.5 1-2 0.5-2 0.25-1 1-2 0.25-32 0.5-1 1-2 0.5->l28 64
0.5 1 0.5 0.5 1 0.5 0.5 1 16
0.5 2 1 0.5 1 1 1
2
‘Number of strains tested: PSSA, penicillin-susceptibleS. aureus (29) MSSA, methicillin-susceptibleS. aureus (40) MRSA, methicillin-resistantS. aureus (61)
Table 2 In Vitro Activity of RP 59500,Vancomycin, and Ciprofloxacin Against 117Strains of Coagulase-Negative Staphylococci ~~~~
~
~~~
Range
CONS= MSSE
0.25-0RP .5 59500 Vancomycin Ciprofloxacin
MRSE
1-2 0.25-64
0.25-1 RP 59500 Vancomycin Ciprofloxacin
RP
MSSH
Vancomycin Ciprofloxacin RP 59500 Vancomycin Ciprofloxacin RP Vancomycin Ciprofloxacin
MRSH CoNS (others)
MIGO
MI%
0.5 2
0.5 2 0.5 0.5 2 64
0.25
0.5
2 0.25-64 0.5-10.5 1-2 0.25->l28 0.5 1-4 64 0.125-64 0.25-2 0.5-4 0.063-32
~~
‘Number of strains tested: MSSE, methicillin-susceptibleS. epidemidis (24) MRSE, methicillin-resistantS. epidemidis (28) MSSH, methicillin-susceptibleS. huemolyticus MRSH, methicillin-resistantS. huemolyticus (23) CoNS, other coagulase-negative staphylococci(22)
2
1 0.5 2
2
0.25
2 0.5 2
0.5 2 16 0.5 2 0.25
2 2 1
Antistaphylococcal Activity of &uinupr&inlDalfoprktin
405
Table 3 In Vitro Activity of RP 59500, Vancomycin, and Ciprofloxacin Against
Reference Strains Reference strain
Vancomycin RP Ciprofloxacin 59500
S. aurew
25923 ATCC 29213 ATCC
2
1
1
1 0.5
E. faecah
29212 ATCC
4
8
E. coli
0.031
8 25922 ATCC 8 35218ATCC
0.031
P. aeruginosa
27853 ATCC
>128
>l28
values for the control reference strains were within the expected range throughout the testing andare given in Table
DISCUSSION Depending on local epidemiological conditions, a significant number of staphylococci are resistant to penicillin, methicillin, clindamycin, erythromycin,aminoglycosides,and/orquinolones(2).Additionally,emerging glycopeptide resistance in some staphylococcal species is a newpotential threat particularly in nosocomial infections. Occasionally, clinical CONS with elevated MICs for vancomycin have been isolated and transformagene has been tion of S. aurew strainswithavancomycin-resistant achieved under laboratory conditions(4). In this context,the ability of RP 59500 to inhibit staphylococcal strains resistantto methicillin is potentially of major clinical importance.In ourstudy, all staphylococci were inhibited by 2pg of RP 59500 per milliliter. The new streptogramin was equally active against both methicillin-susceptible and methicillin-resistant strains of S. aureus, the MI&, values being0.5 pg/ml and 1pg/ml, respectively.A similar pattern was seen for all other Staphylococcus spp. studied. The results concerningthe antimicrobial susceptibilities of our strains were generally in agreement with those reported in previous studies showing that RP 59500was as activeas or moreactive than vancomycinagainst methicillin-susceptible and -resistant staphylococci (1,5-8), even though we tested a larger number of methicillin-resistant strains and excluded multiple copies of the same strain. Additionally, we tested widely used reference strains and a major number of different species of coagulase-
von Eiff and Peters
406
negative staphylococci, separating the methicillin-susceptible and -resistant strains. In summary, our data indicate that RP 59500 exhibits wide-spectrum antistaphylococcal activity against both methicillin-susceptibleand -resistant strains, stimulating further in vitroand, particularly, in vivo evaluation of this promising new antimicrobial agentfor therapy of infections due to multiple-resistant staphylococcal microorganisms.
REFERENCES 1. Bamere JC, Bouanchaud DH, Paris JM, Rolin 0, Hams SmithC. Antimicrobial activity againstStaphylococcus aureus of semisynthetic injectable streptogramins: RP 59500 and related compounds. J Antimicrob Chemother 1992; 30 (supplA):l. 2. Voss A, Milatovic D, WallrauchSchwarz C,Rosdahl W, Braveny I. Methicillin-resistant Staphylococcus aureus in Europe. Eur J Clin Microbiol Infect Dis 1994; 1350. 3. Sanyal D, Johnson AP, George RC, Cookson BD, Williams AJ. Peritonitis due tovancomycin-resistant Staphylococcus epidermidis. Lancet 1991;33754. 4. Noble WC, Virani Z, Cree RG. Co-transfer of vancomycin and other resistancegenesfrom Enterococcusfaecalis NCTC12201 to Staphylococcus aureus. FEMS Microbiol Lett 1992; 72:195. 5. Brumfitt W, Hamilton Miller JM, ShahS. In-vitro activityof RP 59500, a new semisynthetic streptogramin antibiotic, against gram-positive bacteria. J Antimicrob Chemother 1992; 30 (suppl A):29. 6. vonEiff C, Peters G . In vitro activity of RP 59500, a new semisynthetic injectable pristinamycin against staphylococci. Zbl Bakt. 1966; 283:497. 7. Fass RJ. In vitro activity of RP 59500, a semisynthetic injectable pristinamycin, against staphylococci, streptococci, and enterococci. Antimicrob Agents Chemother 1991; 35553. 8. Leclercq R, Nantas L, Soussy Duval J. Activity of RP '59500, a new parenteral semisynthetic streptogramin, against staphylococci with various mechanisms of resistance to macrolide-lincosamide-streptograminantibiotics. J Antimicrob Chemother1992; 30 (suppl A):67.
E-Test for Susceptibility Testing. of Streptococcus pyogenes to Azithromycin, Clarithromycin, Erythromycin, and Roxithromycin Gerard J. van Asselt University Hospital Leiden, The Netherlands WesteindeHospital Den Haag, The Netherlands
Jacobus H. S l m University Hospital Leiden, The Netherlands
INTRODUCTION The macrolides are alternatives for treatment of group streptococcal infections after penicillin failure and are often used as empirical therapy for infections of the respiratory tract. Compared with erythromycin, the new macrolides azithromycin, clarithromycin, and roxithromycin have an expanded spectrum, reduced adverse effects such as dose-related epigastric distress, improved oral bioavailability, increased tissue penetration, and longer clearance times. Reports on the activity of the newmacrolides against Streptococcus pyogenes are scarce. 407
van Asselt and Sloos
408
STUDY OBJECTIVE The purpose of the study was to determine the in vitro activity of the macrolides azithromycin, clarithromycin, erythromycin, and roxithromycin against group A streptococci and to compare the results of the E-test method, the broth microdilution method, and disk-diffusion assay.
MATERIALS AND METHODS Group A streptococcal strains included 180 clinical isolates originating from 6 regions in The Netherlands. W Olow-level erythromycin-resistant group A streptococci from Leiden, a high-level erythromycin-resistant group A streptococcus from Finland, and the Enterococcus faeculis ATCC29212 strain were included as controls.
E-Test Method The inoculum was 0.5 McFarland at 5% sheep blood agar plates; the time of incubation was 18-24 h.
Broth Microdilution Method The inoculum was 106 CFU (colony-forming units)/ml in Mueller-Hinton broth; the time of incubation was 18-20 h. The concentration of macrolide ranged from 0.0039 to 4.0 pg/ml (extended to 128 pg/ml for resistant strains).
Minimal Bactericidal Concentration The inoculum was 1 p1 of the minimal inhibitory concentration (MC) dilutions, appliedto sheep blood agar platesby a multipoint inoculator;the time of incubation was 24 h.
Disk-Diffusion Assay The inoculum was a tenfold dilution of a 0.5 McFarland standard at 5% sheep blood agar plates;the time of incubation was 18-24 h. The disk load was 15 pg. All tests were repeatedon a separate occasion.
RESULTS AND DISCUSSION The results are summarized in Tables 1 and 2. The MICs and minimal bactericidal concentrations (MBCs) of erythromycin and clarithromycin
nge
E-Test for Susceptibility Testing Table I
MIC for
of S. pyogenes
409
Erythromycin-Susceptible Group A Streptococci
MIC(Pg/ml)MIC microbroth method by byE-test
(Pg/ml)
Macrolide
MC,
Azithromycin Clarithromycin Erythromycin Roxithromycin
0.094
Range
MIC,
.
were lower than those of azithromycin and roxithromycin. The MICs obtained by microbroth and E-test methods were reproducible: For 2 95% of strains, the outcome of the repeated experiments was within two dilution steps. No major discrepancies were found among the E-test method, the microbroth MIC method,and the disk-diffusionassay. The somewhat lower MIC values obtained with the E-test method compared to the microbroth method could be explainedby use of different media with different pH conditions (sheep blood agar versus Mueller-Hinton broth). The reproducibility of the MBCs was low: For 27%of strains, the values of the repeated experiments differed more than two dilution steps (possibly because of the inhomogeneous suspensionsof some groupA streptococci and the small volume of inoculum).
SUMMARY The E-test method was comparedto a microbrothM C method and a diskdiffusion assay for susceptibility testing of 180 clinical isolates of group A streptococci to the macrolide antibiotics azithromycin, clarithromycin, Table 2 Zone Diameters and MBC for Streptococci
diameters Zone (mm)
an Range Macrolide Azithromycin Clarithromycin Erythromycin Roxithromycin
Erythromycin-Susceptible GroupA (Pg/W MBc microbroth by
method MBGO
MJ3c,
410
Sloos
van Asselt and
erythromycin,androxithromycin. MBCs alsoweredetermined. The MIGs of azithromycin, clarithromycin, erythromycin, and roxithromycin for group A streptococci were &m1 (MI% obtained by the E-test was 0.250, 0.047, 0.094, and 0.250 pg/ml, respectively). The bactericidal pg/ml) were activities of clarithromycin and erythromycin (MBC,, reached at lower concentrations than those of azithromycin and roxithromycin (MBC,, pg/ml). Both the MIC and MBC data proved that clarithromycin and erythromycin have higher antistreptococcal activities than azithromycin and roxithromycin. MICs obtained with the E-test were one or two steps lower than those found with the microbroth method. Only minor discrepancies were observed among the three methods. The Etest method is applicable in a diagnostic laboratory for susceptibility screening of group A streptococci forthe macrolides tested in this study. None of the 180 strains was resistant to erythromycin and the other macrolides, confirming the low rate of erythromycin resistance (0.5%) in The Netherlands, as foundin our previous study.
REFERENCE 1. van Asselt GJ, Sloos Mouton RP, Van Boven CPA,van de Klundert JAM. Susceptibility of Streptococcus pyogenes to azithromycin,clarithromycin, erythromycin and roxithromycin in vitro. J Med Microbiol 1995; 43:386-391.
Efficacy of Clarithromycin Against Experimental Pulmonary Infection Caused by Streptococcus pneumoniaeStrains with a Novel Macrolide Resistance Mechanism J. A. Meulbroek, M. J. Mitten, A. Oleksiew, V. D. Shortridge,
S. K. Tanaka, and J. D. Alder Abbott Laboratories Abbott Park, Illinok
INTRODUCTION Macrolide resistance has been increasingly frequent in Streptococcus pneumoniae. Although onlyl-2% of all S. pneumoniae are resistant to erythromycin, >50% of penicillin-resistant S. pneumoniae are also erythromycin resistant (1). The most thoroughly described mechanism of macrolide resistance is a ribosomal methylase encoded by e m , a large family of related genes found in many different bacterial species. In Sfreptococci, methylase, production is encodedby e m A M (2). PCR analysis has recently shownthat the majority of S. pneumoniae strains with intermediate erythromycin resistance lacked DNA homologous to known e m sequences (data not shown). These strains have a novel macrolide resistance mechanism. In thisreport, the in vitro susceptibilities of S. pneumoniae strains bearing the novel resistance mechanism are de411
412
Meulbroek et al.
scribed.Additionally, the comparativeefficacies of clarithromycinand azithromycin against rat pulmonary infection caused by susceptible and novel resistant strainsof S. pneumoniae are reported.
MATERIALS AND METHODS All strains of S. pneumoniae were clinical isolates maintained the in clinical culture collection of Abbott Laboratories. Minimal inhibitory concentrations (MICs) were determinedby agar dilution testing using guidelinesset by the National Committee for Clinical Laboratory Standards. Twentyfour-hour cultures,grown in BHI broth with 5% sheep blood, were usedas inocula.Inoculawereprepared bymixing undilutedculturewith 2% molten agar in a1:3 ratio. Sprague-Dawley (male)rats, 7-10 weeks of age (Charles River Breeding Laboratories, Wilmington, MA), were inoculated intratracheally, per via a blunt-end feeding tube, delivering a volume of 0.2 m1 of the inoculum. Medications were given orally, beginning at 5 h postinoculation, then twice daily (7 A . M . /P.M.) ~ for 2 additional days. Untreated rats were determined not to be bacteremic at the termination of the efficacy trial. Lungs were harvested 12 h following the last medication. Homogenates wereplatedonColumbiaCNAAgar with 5% blood.Colonieswere counted 24 h later.
RESULTS The results are summarized in Tables1-3.
Tabh I
Comparison of MIC Values of Sensitive and Resistant S. pneumoniae MIc (CLg/ml)
Resistance
MIC
ery-Sensa
0.008 MIcso 0.015 MIC, 2 MIGO 4 MIC, 16 MGO MIC,>l28 >l28
ery-Novel ery-Em .
Clan = clarithromycin; = penicillin.
Claria
Azi
0.03 0.03 8 32
Clinda
0.06 0.12 0.12 16 >l28
Pen
0.03 0.06 1
4 2 4
= azithromycin; ery = erythromycin; Cliida = clindamycin; Pen
S. pneumoniae
MacrolideinResistance
413
Tabk 2 In Vitro Susceptibility of Streptococcus pneumoniae to Clarithromycin, Azithromycin, Clindamycin, and Penicillin
esistanceStrain
Claria
Pen
Clinda
Novel Novel Novel Susceptible Clan = clarithromycin;Azi = azithromycin; Clinda= clindamycin; Pen = penicillin.
DISCUSSION Streptococcus pneumoniae which bear the novel resistance mechanism to macrolides are a subset of penicillin-resistant pneumococci. These organisms are distinct in their resistancepattern from pneumococci bearinge m gene sequences: The novel resistant organisms have a moderate level of resistance to macrolides andare sensitive to clindamycin (Table 1). A rat pulmonary infection was utilizedto determine the comparative efficacies of clarithromycin and azithromycin against a number of strains Table Efficacies of Clarithromycin and Azithromycin Against Rat Lung Infection Caused by Novel Resistant S. pneumoniae
from lungs Log Cmr recovered Strains of S. pneumoniae
of
Untreated rats
Clarithromycintreatedb rats
Azithromycintreatedb rats
f
& f f f
f -t f f
2 f
f
% Reduction (compared to untreated rats) following Rx with
Clan
Azi
.With respect to macrolide susceptibility, strains 5649, 5654, and 5659 bear the novel resistance mechanism; strain 5739 is a susceptible strain (see Table Inoculum doses for the individual trials are strain 5649, log 6.15 CFU; strain 5654, log 6.69 C F U ; strain 5659, log 6.60 C F U ; strain 5739, log 6.78 CFU.( C F U = colony-forming units.) bFor individual involving strains 5649, 5654, and 5659, rats were treated with macrolide at 100 mglkg per day, twice per day, for days. For the trial involving strain 5739, rats were treated with macrolidesat 5 mg/kg per day, twice per day, for days.
Meulbroek et al.
414
bearing the novel resistance mechanism (Table 2). As indicated in Table clarithromycin therapyat 100 mgkg per day resulted in greater a reduction in the bacterial burden recovered fromthe lungs of infected rats than did azithromycin fortwo of the novel resistant strains tested (strains and Against a third novel resistant strain (strain 5654), the efficacies of clarithromycin and azithromycin were comparable. Both macrolides were highlyefficaciousagainstasensitive strain (strain These results indicate a potential role for the use of clarithromycin against a subset of penicillin-resistant pneumococci.The novel resistance mechanism isunder investigation inour laboratories.
CONCLUSIONS Clarithromycin has efficacy superior to azithromycin for the treatment of pulmonary infection caused by novel resistant Streptococcus pneumoniae. Novel resistant S. pneumoniae make up alargeportion of penicillinresistantpneumococci and are still potentially treatable withclarithromycin. The use of this macrolide as a treatment option for penicillinresistant pneumococci needsto be more thoroughly investigated.
REFERENCES 1. Lonks JR,Medeiros AA. The growing threat of antibiotic-resistant Streptococcus pneumoniae. Med Clin N 1995; 79523-535.
2. Leclercq R,Courvalin P.Bacterial resistance to macrolide, lincosamide, and streptogramin antibioticsby target modification. Antimicrob Agents Chemother 1991; 35:1267-1272.
Macro- and Microautoradiographic Studies on Penetration of Azithromycin in Bacterially Infected Mice Issei Nakayama and Emiko Yamaji Nihon University Schoolof Medicine Tokyo, Japan
Kaoru Shimada Tokyo Senbai Hospital Tokyo, Japan
Shuichi Yokoyama, Kazumi Miura, Hideya Muto, Kazunori Enogaki, Masatoshi Ogawa, andKino Shimooka Pfizer Pharmaceuticals, Inc. Nagoya, Japan
INTRODUCTION Azithromycin is a new acid-stable 15-membered-ring macrolide developed at Pfizer Inc. inthe United States. Azithromycin well is absorbed following oral administration in mice, rats, dogs, and cynomolgus monkeys, exhibits a uniformly long elimination half-life, and is distributed exceptionally well into all tissues compared with erythromycin(1).In vivo efficacy has been observed in various infection models, which suggest that penetration into infectionsitesishigherthannoninfectedtissues (2-4). The ability of 415
Nakayama et al.
416
azithromycin to accumulate markedly in human phagocyte cells also has been demonstrated(5). According to studies on the intracellular accumulation of azithromycin, one potential for phagocyte transport to sites of infectionwassuggested.Inthisstudy,wedescribe the distribution of azithromycin in the infection site by autoradiography and the distribution of azithromycin in inflammatory cells by microautoradiography.
MATERIALS AND METHODS Radiolabeled Compounds The specific radioactivityof [W]azithromycin was 0.92 MBq/mg.(24.8pCi/ mg) and that of [3H]azithromycin was 13.2 MBq/mg (357 pCi/mg). The radiochemicalpurity of eachcompoundwas more than 98%, as determined by radio-thin-layer chromatography [Three types of solvents: chloroform/hexane/diethylamine/triethylamine(15 : 15 : 2 : 2), ethylacetate/hexane/diethylamine(15 : 15: 2), diethylether/methanoYammoniawater (85 : 15 : 2)]. 'Ib prepare the dosing solution, a radiolabeled compound was dissolved in0.05 M citrate buffer(pH 5.3) and unlabeled azithromycin was added to dilute the radioactivity.
Experimental Animals ICR mice (male, 4 weeks, SPF) were purchased from Nihon SLC Co. ('Ibkyo). Mice were given water and fed [CE-2, Nihon Clea (Tokyo)] ad libitum. Infection models were produced in mice as described byGirard et al. [4]. A localized infection was induced by implanting apaper disk (0.08 mm) impregnated with Staphylococcus aureus ATCC25923 (3 X CFU/ disk/mouse) on the mid-back of anesthetized mice.The incision was closed with wound stitchesto prevent removalof the suture.
Autoradiography of Infection Sitein Locally Infected Mice At 5 dayspostchallenge,micereceived 20 mgkg [Wlazithromycin by gavage. At 2,6,24, and 72 h after administration,mice were sacrificed by diethylether anaesthesia.The mice were rapidly frozen by immersion in an acetone-dry ice mixtureat -80°C, and the CMC block wasmounted to the sample holder of the cryotome. The frozen mice were sagittally sectioned (30 pm) and lyophilized. All cryosections were exposed for 3 days in an imaging plate(20x40 c m , FUJI film, Japan) before being scanned with the BioimagingAnalyserSystem(BAS2000 FUJI film, Japan). The autoradiographic optical densityof the film was measured and handled with 32 colors, with output expanding fourfold.
Autographic Studies
on Penetration of Azithromycin
41 7
Microautoradiography Mice were given 20mgkg [3H]azithromycin by mouthat 5 days after challenge. At 24 h after the dose, mice wereanesthetized with diethylether and the infection site wasexcisedandmountedusingO.C.T.compound (TISSUE-TEK, Miles Inc.). The tissue was sectioned into 4-pm sections withacryostat(CRYOCUT Leica) at -20°C.Each section was placed on slides precoated with emulsion for microautoradiography by the thaw mount method(6) and dried.The sections were exposedat -80°C for 2 weeks, developed with Dectol (Kodak), and fixed with Konik (Konica). Sections were then stained with hematoxylin-eosin for microscopic examination.Microautoradiogramsandphotomicrographswere prepared at x200 and ~ 1 0 0respectively. , In addition photomicrographs were madeat X400 to examine the cells.
RESULTS Infection Site Autoradiography Autoradiograms of the infection site following oral doses of [Wlazithromycin at 5 dayspostimplantation of a S. aureus-impregnateddisk are shown in Figs. 1 (whole-body autoradiogram) and 2 (autoradiograms of infected site). The distribution of radioactivity aroundthe implanted paper disk (infection site) was similar to that of noninfected sites 2 h after the dose. The level of radioactivityin the infectionsitewashigher than noninfected tissue siteat 6 h afterthe dose, and increased progressively by 24 h. In addition, the level in the infection site around the paper disk was
Z Whole-body autoradiogram of infected mouse showing the distribution of radioactivity after oral administration of [W]azithromycin (20 mglkg) 5 days postimplantation of a S. aureus-impregnated disk.
Nakayama et al.
2h
24h
72 h
Figure 2 Autoradiograms of infected sites showing the distribution of radioactivity after oral administration of [14C]azithromycin (20 mgkg) to mice, 5 days post implantation of a S. aureus-impregnated disk.
also higher than at noninfection site at 72 h and suggestedthat the tissue of infection site retained [14C]azithromycin.
Microautoradiography Microscopic examination of infection site showeda massive influx of polymorphonuclear leukocytes,a few macrophages and fibroblasts, and formation of large foci of S. aureus. Neutrophil accumulation and layered distribution were observed aroundthe implanted paper disk. The. silver grains indicating radiolabeled drug distributed around the paper disk and concentrated in the area of neutrophil accumulation. The distribution of the radiolabeled drug corresponded to the area of inflammatory cell aggregation.
Autographic Studies on Penetration
Azithromycin
419
DISCUSSION Autoradiograms of the infection site in mice following an oral dose of [Wlazithromycin showed that extremely high level of azithromycin accumulated at the local infection site where a large number of phagocytic cells infiltrated. In addition, microautoradiograms using [3H]azithromycin suggested that phagocytes retained high concentrations of azithromycin. Wildfeuer et al. reported that uptake of [Wlazithromycinby human neutrophil polymorphonuclear leukocytes and macrophages resulted in intracellular concentrations greater than the extracellular medium (5). Retsema et al. described the possibility of augmentation of azithromycin delivery to the infected sitewith the use of S. aureus thigh infection models in CD-l mice. Azithromycin concentrationsin the infected leg were 20-foldgreater than in saline-injected legsat 145 h (2). In other rodent infection models (e.g., S. aureus paper disk infection in rat and Salmonella models of acute systemic and tissue infection in rats), azithromycin was shownto be effective even though azithromycin plasma concentrations were belowthe minimal inhibitory concentration (MIC) at all times during and after the challenge In viewof the above two studiesand our results, highlevelsof azithromycin in the localized infection site may be a consequence of augmented drug distribution as a result of chemotaxis of azithromycin-loaded phagocyte cells and may help explain why azithromycin was effective even though plasma concentrations were below the MIC of key pathogens.
CONCLUSION Autoradiograms of the infectionsiteinmicefollowing oral doses of [“C]azithromycin showedthat an extremely high level of azithromycin accumulated in the localized infection site where large amounts of phagocytes infiltrated. Microautoradiograms of the infection site suggested that the phagocytes retained high concentrationsof azithromycin. High levels of azithromycin in the localized infection sites may be a consequence of augmented drug distribution as a result of chemotaxis of azithromycin-loaded phagocytic cells even though the azithromycin plasma concentrations were below the MIC of key pathogens.
REFERENCES 1. Girard AE, Girard D, English AR, Gootz TD, Cimochowski CR, Faiella JA, Haskell SL, Retsema JA. Pharmacokinetic andin vivo studies with azithromycin (CP-62,993), a new macrolide with an extended half-life and excellent tissue distribution. Antimicrob Agents Chemother 1987; 31:1948.
420 2.
3. 4. 5.
6.
Nakayama et al. Retsema JAYBergeron JM, Girard D, Milisen WB, Girard AE. Preferential concentration of azithromycininaninfectedmousethighmodel.JAntimicrob Chemother 1993; 31 (supplE):S. Girard AE, Girard D, Retsema JA. Correlation of the extravascular pharmacokinetics of azithromycin with in vivo efficacy in models of localized infection. J. Antimicrob Chemother 1990;25 (suppl A):61. Girard D, Bergeron JM, Milisen WB, Retsema JA. Comparison of azithromyroxithromycin, and cephalexin penetration kinetics in early and mature abscesses. J Antimicrob Chemother 1993;3l(suppl E):17. Wildfeuer A, Reisert I, Laufen H. Uptake and subcellular distribution of azithromycin in human phagocytic cells. Armeim.- Forsch/Drug Res 1993; 43(I), No. 4:484. Stumpf WE. Techniques for the autoradiography of diffusible compound. In: PrescottDM, ed. Methods in Cell Biology 13, New York: Academic Press, 1976:171.
In Vivo Antibacterial Activityof RU 004, a New Ketolide Active Against Respiratory Pathogens Constantin Agouridas, A. Bonnefoy, and Jean-Franqois Chantot Rowel-Uclaf Romainville, France
INTRODUCTION RU 004 is a new ketolide which displays excellent in vitro activity against all relevant pathogens involved in RTI , including erythromycin-resistant pneumococci, Huemophilus infruenzae (Hi) and atypical bacteria. We report here on the in vivo antibacterial activityof RU 004 in various murine experimental septicemia models and in two different murine models of pulmonary infections.
MATERIALS AND METHODS mouse septicemia modelwas usedto assess the protective efficacy of RU A (ERY), clarithromycin (CLA), azithromycin (AZI), josamycin (JOS), pristinamycin (PRI), and ampicillin inthe case of Hi. PD, (protective doses) were calculated according to the Reed and Munch method(1). In pneumonia models,the following bacterial strains were used:
004 in comparison with erythromycin
421
Agouridas et al.
422
Streptococcus pneumoniae (Sp)serotype01,originallyisolated fromabloodculture(strainSp6254) : erythromycin-resistant strain. Hi serotype b, originally isolated from cerebral spinal fluid (strain 87169); a P-lactamase-producing strain. Infections were induced in female Swiss (OF-l) mice, 6-7 weeks of age (Sp model) and in female C57BI/6 mice, 6 monthsof age (Hi model). Mice were infected intratracheally via the mouth with 105 CFU (colonyforming units)of Sp 6254; they developed bacteremic pneumonia and fatal diseasewithin 2-5 days.In the case of Hi, micewereinfected intratracheally via the mouth with 108 CFU; they developed inflammatory bronchopulmonary diseasethat was spontaneously cured. Therapeutic assays were conducted as follows;
-
-
Survival studies (Sp):Treatmentwas initiated 6 h (protective)or 18 h (curative) postinfection. Drugs were administered orally bidfor 3 days (n=10-15 mice per treatment group). Bacterialclearance(Hi):Kinetics of bacterialkillingwererecorded over a 24-h period following a singlePO administration of the drug 16 h postinfection(n=3 mice).
RESULTS Results are shown in Table1 and Figs. 1and 2. Table Z In Vivo Antibacterial Activityof RU 004 (PD,,, mgkg)
Strains S. pyogenes Ery-Sa 300 S. aureus Ery-S S. aureus Ery-Ri E.faecium Ery-R, Van-R S. pneumoniae Ery-S >50 S. pneumoniae Ery-Ri S. pneumoniae Ery-RC S. pneumoniae Ery-RC S. pneumoniae Ery-RC H. influenzae Amp-S H. influenzae Amp-R325
RU004 16 20 13 11 16 30 19 17 15 142 116
ERYa
>l00 >l00
>l00 >l00 >l00 >l00
CLA
AZI
PIU
AMP
-
16 13 >l00
16
-
>l00
-
>l00 >l00 >l00 >l00 346 110
18 >l00 >l00 >loo >l00 110 117
-
-
-
-
>l00
-
>IO0 >l00 >l00 67
-
-
-
5
aEry-Ri, Ery-RC: inducible, constitutive resistance to erythromycin A; ERY: erythromycin; C M : clarithromycin; M I : azithromycin;PRI:pristinamycin; A M P : ampicillin; VAN: vancomycin.
In Vivo Antibacterial Activity
423
RU 004
Experimentalmurinesepticemia.
Figure I
DISCUSSION In septicemia caused by gram-positive cocci susceptible to ERY, RU 004 shows in vivo activity similar to that of CLA but quite superior to that of ERY. In infectionscaused by ERY-R strains, RU displays a high antipneumoccocal efficacy, unlike available 14- or 16-membered-ring macrolides which are completely inactive. The protective doses fall withinthe range of values foundfor susceptible bacteria (15-42 mg/kg). PR1 showed measurable activity in only two out of five infections, with the PD,, at 67 and 76 mgkg. In Hi-induced septicemia, the ketolide was two to three
Q
1
2
3
4
6
e
7
e
o
l
Q
l
l
I
Z
Pmtecthre treatmant
Figure 2 Experimental murine pneumonia. (From Ref. 2.)
~
~
Curative treatment
~
B
~
Agouridas et al.
424
times more active than ERYor CLA. Conversely, PD,, were closeto those of M I . Moreover, RU 004 displays high efficacy inEnterococcus infections, whatever the phenotype of infecting strains (VAN-R and/or ERY-R) (Table l). In pneumonia induced by ERY-R Sp, RU 004 demonstrates the same level of efficacy as virginiamycin (Fig. 2). Despite a significant decrease inefficacy between protective and curativetreatments, the ketolide remains notably effective in severe conditionsof bacteremic experimental disease. In these experiments, no resistant mutants were isolated. In pneumonia induced by Hi, RU 004 exhibits an activity similarto that of AZI. The initial bacterial clearance was more efficient with the ketolide than with AZI.
CONCLUSIONS Unlike macrolides and streptogramins, RU 004 displays high therapeutic efficacy in mice infected by various common respiratory pathogens and gram-positive cocci, thus conforming to its well-balanced and potent in vitro activity. RU 004 appears to be very promising for the treatment of infections causedby difficult-to-treat respiratory pathogens.
REFERENCES 1. Lorian V. Antibiotics in Laboratory Medicine. Baltimore,MD: Williams and Wilkins, 1993. 2. Rajagopalan, Levasseur P, VallCe E, et al., 35th Intersci Conf Antimicrob Agents Chemother, 1993; abst F-173.
Open Noncomparative Studyof the Efficacy and Safetyof Azithromycin in the Treatment of Adult Tonsilitis (Epidemiological Studyof the Responsible Bacteria) A. Desaulty Centre Hospitalier, Rkgional Universitairede Lille Lille, France
INTRODUCTION Cases apparently commonplace bacterial tonsilitis occur frequently in patients of all ages. Such cases require treatment designed not only to relieve symptoms as rapidly as possible but also to avoid those complications which mightresult from infections due to group streptococci (rheumatic fever, glomerulonephritis, phlegmonous adenitis, peritonsillar abscess, etc.). Azithromcyin is a new azalide (nitrogen-containing macrolide) antibiotic that provides a useful alternative in the treatment such tonsilitis in view ofthe following: It has a suitable spectrum of activity; it shows excellent diffusioninto tonsillar tissue; and its pharmacokinetic parameters are such that it persists for a prolonged period in tonsillar tissue. The objective of the present trial was to evaluate the clinical efficacy and safety 425
Desaulty
426
of treatment with azithromycin (once-daily dose for 5 days) in adults with acute tonsilitis and also to define the bacterial epidemiologyof communityacquired acute tonsilitis.
METHODS Patients Patients included in the trial had to comply with the following criteria: age over 18; monitored as outpatients; presenting with acute erythematous or erythematous pustular tonsilitis accompanied bysignsof acute tonsilitis; treated with azithromycin when the reference therapy (penicillin)couldnot be used;havinggiveninformedconsentinwriting. A throat swabhad to be taken foreachpatientfrom the tonsillar area, purulent or cryptic areas, and/or the posterior wallof the pharynx for bacteriologic studies.
Treatment Patients received500 mg of azithromycin (2 X 250 mg capsules) on the first day and 250 mg of azithromycin dailyfor the following 4 days. Any other antibiotic therapy, digitalis glycosides, and ergot derivatives were prohibited throughout the trial.
FOllOW-up Clinical efficacy was assessed at day evaluation.
-t
2 during a complete physical
Assessment Criteria Success was defined as follows: Apyrexia (defined as a temperature equal to or below before the control evaluation at day 2 2 without the need for any other antibiotic; the disappearance of the following signs at day 10: pharyngeal pain (spontaneous or on swallowing), alteration in appearance of the tonsils and/or pharynx, and difficulty in swallowing. For the purposes of the analysis, all patients entered in the trial were included in the description of the population and evaluationof the safety of azithromycin. The efficacy of azithromycin wasevaluated in the population sample with streptococcal tonsilitis as documented by bacteriologic tests.
Azithromycin for Adult Tonsilitis
427
RESULTS Description of the Population Three hundred nine patients were included in the trial between December and July The population was balanced in terms of sex men, women) and had a large majorityof patients under years of age In compliance with protocol recommendations, all included patients exhibited analteration in the appearance of the tonsils and/or pharynx together with a bodytemperature equal to or greater than More than half the patients were suffering from severe pharyngeal pain and had great difficulty in swallowing or had already taken an antipyretic
Bacterial Epidemiology Bacteriologic tests in patients revealed bacterial tonsilitis in 55% of cases includingstreptococcaltonsilitisin 16% The other microorganisms most frequently detected were as follows: Staphylococcus aureus in cases; Haemophilus parainfluenzae in cases; Haemophilus influenzae in cases. Bacteriologic tests were negative in of the patients
Efficacy Evaluation The efficacy evaluation was carried out for a sample size of patients with streptococcal tonsilitis initially confirmed by bacteriologic tests. Treatment was successful in of cases Of the three cases regarded as failures, the test treatment had been discontinued and days after day 0 respectively as local and systemic signs either persisted or worsened, warranting the prescription of another antibiotic. No complications (particularly phlegmonous adenitisor peritonsillar abscess) werereported during the trial.
Safety Evaluation Ttventy-three out of patients reported at least 1 adverse event during the trial. Gastrointestinal symptoms were most frequently involved (six cases of diarrhea, four casesof abdominal pain, two casesof gastralgia, and two cases of nausea). patients had to discontinue treatment prematurely becauseof adverse reactions (diarrhea and exacerbation of hemorrhoids in one case, and nausea and erythemaof exposed parts in the other). None of the other adverse events mentioned was regarded as serious (i.e. leading to permanent sequelae, adversely affecting the patient’s prognosis for survival or to hospitalization).
Desaulty
428
DISCUSSION AND CONCLUSION The trial involved 309 adult patients suffering from acute erythematous or erythematopultaceous tonsilitis and provideddata concerning its bacterial epidemiology. The data may be compared with previously published relevant results (1): A high proportion of negative bacteriologic, results were obtained (45% of cases): the infection was most frequently diagnosed as streptococcal in origin during the trial (16% in the total sample size and 29% of the population with bacteriologically documented tonsilitis). However, this figure is lower that thanpresented in previously published results. The trial also confirmed the efficacy of once-daily azithromycinfor'5 days for the symptoms of acute streptococcal tonsilitis in adults.At day'l0, the success rate attained 91%; thisis similar to the results noted inother trials for the same indication (2,3). Safety analysis revealed that 7.4% of patients experienced at least one adverse event duringthe trial and confirmedthat none of the adverse events was serious.
REFERENCES 1. Gehanno P, Portier H, Longuet P. L e pointactuelsur1'6pid6miologie des angines aigueset dessyndromes post-streptococciques.La Revue du Praticien 1992; 42(3):284-287. 2. Hooton TM. A comparison of azithromycin and penicillinV for the treatment of streptococcal pharyngitis.Am J Med 1991; 9l(suppl3A):23S-26S. 3. Carbon C, etal. Economic analysis of 3 antibiotic regimens in acute pharyngia prospective naturalistic comparison of azithromycin versus roxithromycin. J Antimicrob Chemoth (in press).
Open Studyof Clarithromycin in the Treatment of Pneumonia Due to Streptococcus pneurnoniue Murat Hayran, MustafaErman, Deniz Gur, Murat Akova, and Serhat Una1 Hacettepe UniversitySchool of Medicine Ankara, %key
INTRODUCTION Clarithromycin is a novel macrolide antibiotic active against a variety of organisms responsible for community-acquired pneumonia. Streptococcus pneumoniue invariably has been the most commonly encountered pathogen and appears to be sensitive to clarithromycin in vitro. In this open, prospective, noncomparative study, 41 patients with community-acquired lobar pneumonia were evaluated to establish the efficacy and safety of clarithromycin.
PATIENTS AND METHODS Forty-one adult patients with community-acquired pneumococcal pneumonia were included.The diagnosis was established by the presence of symptoms which included cough, sputum production, dyspnea, pleuritic chest pain and rigors, x-ray findings, fever, and the demonstration of abundant 429
Hayran et al.
430
polymorphonuclear leukocytes( P m ) and gram-positive diplococci on microscopic examinationof sputum. Patientswith evidence oftuberculosis or malignancy, chronic liver or renal impairment, or receiving theophylline/ theophylline analogs, as wellas pregnant or lactating women were excluded. Patients with severe or complicated lower respiratory tract infections necessitating parenteral antibiotictreatment were not eligible. Complete physical examinationwas performed daily. Cough, sputum production, and dyspnea were recorded by using a grading scale as absent, mild, moderate, or severe. Pyrexia was considered as present when body temperature was greater than 37.8"C. Complete blood counts with differential, blood urea nitrogen, creatinine,total bilirubin, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were performed, and sputum samples for microscopic examination and culture were collected on the first day, during, andat the end of treatment, if available. Chest x-ray was obtained on the firstand tenth daysandrepeatedweeklyduring follow-up until pneumonia resolved. Blood cultures were taken before the initiation of treatment and daily as long as the patient remained febrile. Gram-stained sputum smears were examined and sputum cultures were performedon sheep blood and chocolate agar plates incubated at 37°C in an atmosphere containing 5-10% carbon dioxide daily as long as sputumsamplecould be obtained.Suspiciouscoloniesweretested for susceptibility to optochin. Susceptibility to penicillin and clarithromycin was determined by disk-diffusion method. n7vo tablets of clarithromycin (250 mg) were administered everyh for 10 days. Clinical curewas defined asthe resolution or improvement pretreatment symptoms and signs with defervescence of fever before study day
RESULTS Of the 41 patients, (54%) were males and 19 (46%) females.The mean age was 40.5 years (range:18-67). Four (9.8%) patients had underlying chronic obstructive pulmonary disease. Clinical characteristics of patients are summarized in Table1. Streptococcus pneurnoniae was isolated in the sputum cultures of 16 patients (39.0%). None of the patients was bacteremic. Susceptibility testing by the disk-diffusion method disclosed all isolates were sensitive to clarithromycin in vitro. Clinical cure with microbiological eradication was achievedin 37 patients(90.2%). Pretreatment signsandsymptoms resolved in days after initiation of clarithromycin treatment (median: days).In patients whodevelopedpleuraleffusion, treatment was switched to parenteral p-lactam antibiotics. Another two remained febrile
Clarithromycinfor S. pneumoniae Infection
431
Table Z Clinical Characteristicsof Patients Treated with Clarithromycin Characteristics Sex, FM (min, Mean Median (min, max) duration of infection (day) 2 (9.8) disease Underlying (COPD) (%) Abundant gram-positive diplococci in sputum smear (%) S. pneurnoniae culture sputum in (%) Cure (%) (90.2) eradication Microbiological (%) (95.1) Failure (%) (%) (7.3) Side Nausea (%) (2.4) transaminases Increase in (%) (2.4) discomfort Abdominal (%) (2.4)
19/22
40.5 (18-67) (1-5) 4/41 41 (100.0) 16 (39.0) 37/41 39/41 2/41 (4.9) 3/41 1/41 1/41 1/41
as long as the fifth day and were, therefore, assigned to have “indeterminate” response and the antibiotic regimen was modified. Clarithromycin was well tolerated by all but three patients who exhibited mild side effects of nausea, abdominal discomfort, and elevation of aspartate aminotransferase (ALT). However, none of these side effects required discontinuationof treatment.
CONCLUSION Clarithromycin is effective and welltolerated in the treatment of uncomplicated lower respiratory tract infections due to Streptococcus pneumoniae. The use of clarithromycin in severeor complicated pneumonia needsto be further evaluated.
REFERENCES 1. Fass RJ. Aetiologyand treatment of communityacquiredpneumoniain adults: an historic perspective. J Antimicrob Chemother 1993; 32 (suppl A): 17-27. 2. Uzun Hayran M, Akova M, Gur D, Akahn HE. Efficacy of a three day course of azithromycin inthe treatment of community acquired pneumococcal pneumonia: a preliminaryreport. J Chemother 1994; 653-57. 3. Chien SM, Pichotta P, Siepman N, ChanCK. Treatment of communityacquired pneumonia. A multicenter, double-blind, randomized study wmparing clarithromycinwith erythromycin. Chest1993; 103(3):697-701.
Clinical Studyof Rokitamycin on Pneumococcal Upper Respiratory Tract Infections in Pediatrics Yoshikiyo Toyonaga YamanashiRed Cross Hospital Yamanashi, Japan
INTRODUCTION Pediatricians most frequently encounter upper respiratory tract infections (URTI) in outpatient clinics. An especially high isolation rate of penicillininsusceptible (MIC 0.01-0.78 pg/ml)/resistant (MIC 2 1.56 pg/ml) Streppneumoniue (PISPPRSP) has become a recent problem (1) (MIC = minimal inhibitory concentration). To investigate the clinical efficacy of rokitamycin (RKM), a 16-membered ring macrolide antibiotic, on pediatric URTI caused by S. pneumoniue, we have reviewed the clinical performances of cases of pediatric URTI treated in the department and the results are reported below.
PATIENTS AND METHODS Rhinopharyngeal cultures were taken from pediatric who patients visitedthe department from February to October 1995. Patients with URTI purportedly due to S. pneumoniue were given RKM, cefditoren pivoxil (CDTR), or 432
Rokitamycin in Pneumococcal URTI
in Children
433
cefdinir (CFDN) at random, and the clinical efficacy and bacteriological effects were investigated. The dosages were mgkg, mgkg, and mgkg for RKM, CDTR and CFDN, respectively,three times daily.
RESULTS MIC Distribution Of the cases of URTI, significant organisms weredetected in cases. S. pneumoniae was isolated from52.2% of the cases cases), of which 80 cases were complicated infections with various other bacteria, such as Haemophilus influentae and Moraxella subgenus Branhamella catarrhalis. There were isolates of S. pneumoniae, of which isolates) were penicillin susceptible (MIC &ml) S. pneumoniae (PSSP) and isolates) were PISPPRSP. The MIC distributions of various antibacterial agentsfor isolated PSSP andPISPPRSP areshown in Figs. and 2. MIC distributionsof the penicillins (PCs) and the cephems (CEPs) for PISPPRSP were evidently inferior to those against PSSP. Among the macrolides (MLs), 14-membered-ring erythromycin (EM) and clarithromycin (CAM) showed marked increases inthe number of resistant strains
Figure l Antibacterial activity of various antibiotics against penicillin-susceptible S. pneumoniae.
Toyonaga
434
"0-
--
"e
--&
CFDN
-.d-.RKM
-e*---
Figure 2
Antibacterial activityof penicillin-insusceptiblelresistantS. pneumoniae.
for PISPPRSP; however, 16-membered-ring RKM showed similar MIC distributions forPSSP and PISPPRSP.
Clinical and Bacteriological Efficacy The clinical efficacyrates of RKM, CDTR, and CFDN were 89.5% (34/38), 87.1% (27/31), and 72.4% (21/29), respectively, for the PSSP infections, and 90.0% (18/20), 73.9%, (17123) and 71.4% (10/14), respectively,for the PISPPRSP infections, indicating no significant decrease in efficacy with these agents.On the other hand, the eradication rates of CDTR and CFDN for the PISPPRSP infections were evidently lowered compared with the PSSP infections, whereas a reduction in the eradication rate was not observed for RKM for the PISPPRSP infections (Table1).
DISCUSSION Streptococcus pneumoniae, in addition to H. influenzae and M. (B.) catarrhalis, is an important causal organism of pediatric upper respiratory tract infections. In recent years, PISPPRSP have been isolated at a high frequency, and insusceptibilityof the organism not only to PCs and CEPs but also to MIS (1) is a serious problem for pediatricians. Also in this study a marked insusceptibility of PISPPRSP toPCs and CEPs was found. This was reflected in bacteriological efficacy, and reduc-
Rokitamycin in Pneumococcal URTI
in Children
435
Table Z Bacteriological Effects Classified by Penicillin Sensitivity (Eradication Rate of S. pneumoniae)
Agent RKM
.
CDTR CFDN
'No. of strains eradicatemo. of strains isolated.
tions in eradicationrates of CDTR and CFDN were found inPISPPRSP infections, compared with PSSP infections. On the other hand, regarding MLs, PISPPRSP showed insusceptibilityto EM and CAM. However, the MIC distributionof RKM for PISPPRSP was similar to that for PSSP, and high eradication rates for bothPSSP and PISPPRSP were observed inthe clinical performance results. RKM is reported to have a MIC of 1.56-12.5 pglml for S. pneumoniae, for whichEM and other 16-membered-ring macrolideshaveMICs of notlessthan100 &m1 (2). The excellent antibacterial activityof RKM, even againstPISPPRSP, which are resistant to EM and CAM, suggests low cross-resistanceto other MLs. In addition, RKM enters polymorphonuclear leukocytes at higher concentrations than EM andCAM and may explainthe excellent eradicationrates achieved with RKM in this study.
CONCLUSIONS Rokitamycin showed similar MIC distributions for both PISPPRSP and PSSP and exhibited excellent clinical efficacy and bacteriological effects. With the recent problem of increasing PISPPRSP, RKM is considered useful for the treatment of URTI due to S. pneumoniae.
REFERENCES Working Group for Pc-resistant S. pneumoniae; organizer: Masatoshi K. An epidemiological study of penicillin-resistant Streptococcus pneumoniae in Japan. JJA Inf D Koichi D, et al. Study of susceptibilities of freshly isolated strains to macrolide antibiotics. Jpn J Antibiot Ishiguro M, Koga H, Kohno S, Hayashi T, Yamaguchi K, Hirota M. Penetration of macrolides into human polymorphonuclear leukocytes. J Antimicrob Chemother
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V PHARMACOKINETICS, PHARMACODYNAMICS, DRUG INTERACTIONS
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A Comparison of the Bronchopulmonary Pharmacokinetics of Clarithromycin and Azithromycin Kalpana B. Pate1 Arnold and Mane Schwartz College of Pharmacy, Long Island University Brooklyn, New York
Dawei Xuan, Charles H. Nightingale, Pamela R. Tessier, John H. Russomanno, and Richard Quintiliani Hartford Hospital Hartford, Connecticut
The purpose of this investigation wasto measure and comparethe concentrations of clarithromycin, 14-hydroxyclarithromycin, and azithromycin in plasma, epithelial lining fluid, and alveolar macrophage cells in healthy volunteers atthe end of a typical courseof therapy.
MATERIALS AND METHODS
Study Design This was a randomized, prospective, nonblinded trial in healthy adult volunteers. All potential subjects were required to be 18 years or older and within 10% of acceptable weight for their height according to the Metropolitan Life heightlweight tables(1). 439
Pate1 et al.
440
After giving informed consent, 42 volunteers underwent a complete medicalhistory,physicalexamination,andbaselinelaboratorytesting. lkenty-one (12 males, 9 females) were randomized to the clarithromycin group and 21 (11 males, 10 females) to the azithromycin group. The median age (range)of subjects in the clarithromycin and azithromycin were 28 (21-41) years and 29 (20-41) years, respectively. Clarithromycin, 500-mg tablets, was administered orally every 12 h for nine doses. Azithromycin, 500 mg on day 1followed by 250 250-mg capsules, was administered orally, mg daily for four doses. Prior to the first dose administration, a blood sample was collected.
Bronchoalveolar Lavage Standardized bronchoscopy and bronchoalveolar lavage (BAL) were performed at 4, 8, 12, and 24 h after the last dose of medication administration. Subjects were required to fast at least 6 h before the bronchoscopy. A total of four 50-m1 aliquots of normal saline were instilledinto the right middle lobe and each was immediately aspirated into a trap. The first aspirate was processed separately (first aspirate) fromthe second through the fourth aspirates, which were pooled (pooled aspirate).The volumes of the first and pooled aspirates were measured and recorded. Aliquots from the first and pooled aspirates were sent to clinical laboratoryfor cell count and differential. The measured volumes of the first and pooled aspirates werekeptoniceuntilcentrifugation at 400 g for 5 min. The supernatants were immediately removed fromthe cell pellet and then frozen at -70°C until assay. A small aliquot of the supernatant was frozen separately for urea assay.
Specimen Handling Blood samples were spunat lo00 g for min and the plasma separated and frozen until assay. An aliquot of plasma on the day of bronchoscopy was frozen separately for urea assay. The cells were resuspended with a potassiumphosphatebuffer,pH8.0, to a total volumeof 10% of the recoveredlavagefluidvolume. The cellsuspension was freeze-thawed three times and sonicated for 2 min before drug assay.
Drug Assay Clarithromycin (CLA), 14-hydroxyclarithromycin (14-OH CLA), and azithromycin (AZ) were assayed utilizing high-performance liquid chromatography (HPLC) techniques.
Bronchopulmonary Pharmacokinetics
of CLA and AZ
441
HPLC Instrumentation: Estimation of Epithelial Lining Fluid (ELF) Volumes and Determination of Drug Concentration in ELF and Alveolar Macrophage (AM) Cells The urea in plasma samples was measured by the clinical laboratory at Hartford Hospital. ELF volumewas determined by the urea dilution method (2). The volume of AM cells inthe cell pellet suspension was determined from the cell count and differential performed the on BAL fluid. Cell count was performed using a hemocytometer, andthe differential countwas performed after centrifugation of the specimen in a cytocentrifuge
STATISTICAL ANALYSIS Statistical evaluation of the effect of site sampled (AM cells, AM cells/ plasma ratio, and plasma) and time sampled (4, 8, 12, 24 h) on measured drug concentrations was performed using an analysis of variance method with a Sheffe’ F-test for multiple parameters. A P value
\A I 2
I
I
I
la
26
hours
I Bactericidal kinetics of Streptococcus pneumoniae exposed to variable concentrations following the serum pharmacokineticsof macrolides.
Pharmacodynamics of CLA and AZZ Table l
481
MICs of Macrolides
MIc ( m a ) Species (n) S. aureus S. pneumoniae
PEN-S S. pyogenes S. agafactiae
H.injhenrae AMP-S M.catarrhalis
Compound
MI%
Range
Azithromycin Clarithromycin Azithromycin Clarithromycin Azithromycin Clarithromycin Azithromycin Clarithromycin Azithromycin Clarithromycin Azithromycin Clarithromycin
0.25 0.06
0.06
0.5
man serum (C-) and its relation to the MIC of the respective pathogen (Table 1).Azithromycin kills a lower proportion of the initial population in comparison with clarithromycin (Table 2). Regrowth of the cultures after reduction of the initial inoculum[lo6CFU (colony-forming units) per milliliter] was observed during exposureto azithromycin (Table 2).
Reduction of Susceptibility to Macrolides An increase in MICs(8-16 times) was observed whenbroth cultures of all species included were exposed to a gradientof azithromycin concentrations following its serum pharmacokinetics (Table 2). Under identical conditions, no reduction in sensitivity occurred the in presence of clarithromycin. The selection of resistant mutantsby azithromycin was restricted to strains with MICs below 1m&. Strains with an MIC of 1mgL prior to exposure to azithromycin (e.g., S. aureus, H.infzuenzae) did not further increase their MIC inthe presence of azithromycin (Table 2).
DISCUSSION Serum concentrationsof antimicrobials have been regarded as a major and rational parameter for cutoff points and therapeutic perspectives. Evidence has accumulated for therapeutic efficacy of compounds found intracellulary not at all or only at very low concentrations(e.g.,P-lactams, aminoglycosides). Whether drugs with high intracellular but very low extracellular absolute concentrations (proportions may be misleading) are
et
482
Bauernfeind
al.
Table 2 Kinetics of Killing of Respiratory Tract Pathogens by Macrolides
Dosage
MIC (m@)
Minimum number achievable after time exposure of CmJ/ml
CFU/ml Organism h Compound End Initial (mg) hem. Streptococci hem. Streptococci hem. Streptococci S. pneumoniae S. pneumoniae S. aureus S. aureus S. aureus S. aureus H . influenzae H . infruenzae M.catarrhalis M.catarrhalis
26 h
CLA AZI AZI CLA AZI CLA CLA AZI AZI CLA AZI CLA AZI
2 1 .l 2X 1 1 1 1 1 1 1 1 1
250
500 250
500
500
0.03 0.06 0.13 0.06 0.13 0.25 1 0.25 1 1 1 0.13 0.03
0.03 1 2 0.06 1 0.25 1 2 1 1 1
110 2.0 lo4 16 1.0 I d 5.8 16 6.0 10 4.2 104 8.0 16 1.5 lo6 2.5 io4 1.3 lo6 510 4.7 Id
510 84.4 106 68.0 lo' 12 2.0 102, 64.6 lo6" 26. 6.0 26 4.2 104 62.7 lo8 2. 7.2 lo8 18 3.0 io4 28.0 lo8 24 110 108.4 104
'After 12 h.
safe in infections in which pathogens do not obligatorily reside only intracellularly remains a major issue. Although the macrolides show similar chemical structure (7), they are very dissimilar in their pharmacokinetic profiles. For some of them, the proportion between intracellular and extracellular concentration is much higher [e.g., azithromycin (2,8)] compared with others [e.g, roxithromycin (2)]. However, the therapeutic indication of none of the compounds is restricted to infections caused only by obligatory intracellular pathogens. In a pharmacodynamic model, both azithromycin and clarithromycin (excluding its 14-hydroxy metabolite) demonstrate bactericidal activity at concentrations reached in human blood at recommended dosages (azithromycin 500 mg qd, clarithromycin250 mgbid). For clarithromycin, boththe rate and the extent of killing are higher than for azithromycin. In both aspects, the clarithromycin 500-mgqddosingis superior to the clarithromycin 250-mg bid dosing. Mutants with between 8 and 16times reduced susceptibility in compari son withthe initial strain were selected during exposure to azithromycin(not to clarithromycin) with all strains with an initial MIC10.25 m a . No mu-
Pharmacodynamics of CLA and
483
tants with increased MICs were selected from strains with higher initial MICs (e.g., H . influenzae and S. aureus with MIC 1mg/L). These populations are able to proliferate unimpaired as their MICs are above the concentration of azithromycin achievable in the serum. Therefore, there is no selective advantage for mutants with increased MICs. Consequently, no selective enrichmentof mutants is observed.The selection of mutants with reduced susceptibilityto macrolides is not a phenomenon restricted to the in vitro model but has been observed during therapy of infections caused [e.g., by H . influenzue Helicobacter pylori (lo), Streptococcus pyogenes (11,12), Streptococcuspyogenes (11,12), and S. pneumoniue (13).
CONCLUSION The therapeutically predictive impact of these in vitro data is difficult to interpret; however, the findings might be relevant in cases where pathogens enter the bloodstream of patients with impaired defenses (neutropenia) in which the postulatedantibioticdelivery for internalizeddrugs via the phagocytic system becomes less effective.
REFlERENCES 1. Bauernfeind A. In vitro activity of dirithromycin in comparison with other new and established macrolides.J Antimicrob Chemother 1993; 31 (suppl C): 39-49. 2. Sorgel F, Kinzig M, Naber KG. Physiological disposition of macrolides. In: Bryskier A, Butzler JP, Neu HC, Thlkens PM, eds. Macrolides-Chemistry, pharmacology and Clinical Uses. Pans: 1993:421-435. 3. Kees F, Wellenhofer M, Grobecker H. Serum and cellular pharmacokinetics of Clarithromycin 500 mg q.d. and250 mg b.i.d. involunteers. Infection 1993; 23:168-172. 4. Mazzei T, Surrenti C, Novelli A, Crisp0 A, Fallani S, Carla V, Surrenti E, Petri P. Pharmacokinetics of azithromycin in patients with impaired hepatic function. J Antimicrob Chemother 1993; 31 (suppl. E):57-63. 5. Foulds G, Shepard RM, Johnson RB.The pharmacokineticsof azithromycinin human serum and tissues.JAntimicrob Chemother 1990; 25(suppl. A):73-82. Bauernfeind A, Jungwirth R,Petermuller C. Simultaneous simulationof the serum profilesof two antibiotics and analysis of the combined effect against a culture of Pseudomonas aeruginosa. Chemotherapy(Basel) 1982;28:334-340. 7. Kirst HA, Sides GD. New directions for macrolideantibiotics: structural modifications and in vitro activity. Antimicrob Agents Chemother 1989; 33: 1413-1418. Lode H, Boeckh M, SchabergT. Human pharmacokineticsof macrolide antibiotics.Bryskier A, Butzler JP, Neu HC, ThlkensPM, eds. Macrolides-
484
9. 10. 11. 12. 13.
Bauemfeind et al. Chemktry, Pharmacology and Clinical Uses.Paris: Arnette Blackwell, 1993: 409-420. Davies BI, Maesen FPV, Gubbelmans R. Azithromycin (CP-62,993) in acute exacerbations of chronic bronchitis: an open clinical,microbiological and pharmacokinetic study. J Antimicrob Chemother1989; 23:743-751. Glupczynski Y, Burette A. Failure of azithromycin to eradicate Campylobacter pylon from the stomach becauseof acquired resistance duringtreatment. A m J Gastroenteroll990; 8598-99. Seppala H , Nissinen A, Jarvinen H , et al. Resistance to erythromycin in group A streptococci.N Engl J Med 1992;326:292-297. Seppala H , Klaukka T,Lehtonen R, et al. Outpatient use of erythromycin: link to increased erythromycin resistance in group A streptococci. Clin Znfect D~.s1995; 21~1378-1385. Shah PM, Bryskier A. Epidemiology of tesistance to macrolide antibiotics. Bryskier A, Butzler JP, Neu HC, Tulkens PM, eds. Macrolides-Chemistry, Pharmacology and Clinical Uses.Paris: Arnette Blackwell, 1993:143-166.
VI HELZCOBACTER PYLON, CAMPYLOBACTER
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Comparison of Clarithromycin Efficacy in Ferrets and Humans for Treatment of Helicobacter-Induced Gastritis P. Ewing, J. D. Alder, M. J. Mitten, A. Conway, A. Oleksijew, K. Jarvis, L. Paige, and S. K. Tanaka Abbott Laboratories Abboft Park,Illinois
INTRODUCTION Helicobacter pylon infection of the gastric mucosa causes chronic gastritis and peptic ulcer diseasein humans (1).Eradication of H. pylori produces more rapid healing of ulcers and a decrease in ulcer recurrence. Clinical trials indicate that Helicobacter-associated ulcers can be successfully treated with combination therapy of antibacterial agents and proton pump inhibitors such as omeprazole. Animal modelsof Helicobacter-induced gastritisare useful for evaluating efficacy of both experimentaltherapeutic agents and modificationsof established therapeutic combinations. Ferrets have anatural infection with Helicobacter rnustelae, which results in gastric.pathology similar to that of humans infected with H. pylori (2). The purpose of this investigation was twofold: (1) to determine efficacy of clarithromycin andother antibacterial therapiesin a ferret model of ,
487
488
Ewing et al.
Helicobacter-induced gastritis and (2) to compare the efficacy to results achieved clinically in humans.
MATERIALS AND METHODS In Vitro Tests Minimal inhibitory concentration(MC) values were determinedfor clarithromycin, amoxicillin, omeprazole, and metronidazole against H.pylori and H. mustelae by agar dilution method (3).
In Vivo Evaluations Ferrets were inoculated on study day 0 with 107-CFUs(colony-forming units) of H.mustelae ATCC 43773 by oral gavage. Therapy was initiated 21 days after inoculation. Antibiotics were administered once daily for 14 days (days 21-34) and omeprazolewas administered once daily for 28 days (days 21-48). To determine gastric H.mustelae burden during the trials, gastric mucosal biopsy specimens weretaken from anesthetizedferrets on day 35 (end of antibacterial therapy) or day 49 (end of omeprazole therapy). On day ferrets were sacrificed and sections of pylorus and fundus were excised for determination of H. mustelae burden and histopathologic evaluation.Hematoxylinandeosin(H&E)-stainedsections of pylorus and fundus were graded 0-5 for acute inflammation (polymorphonuclear cell density) and chronic inflammation (mononuclear cell density) according to criteria defined by the modified Whitehead scale. Warthin Starry (WS)stained sectionsof stomach were graded0-5 for Helicobacter density using the modified Whitehead scale. The following trials were conducted: Comparison of monotherapy with 1. Double Versus Triple Therapy: omeprazole to double therapy with clarithromycidomeprazole (C/O; clarithromycin days 21-34, omeprazole days 21-48)or triple therapy with amoxicillidmetronidazole/omeprazole amoxicillidmetronidazoledays 21-34, omeprazole days21-48) 2. Length of Therapy Trial: Comparison of 1week clarithromycinl4 weeks omeprazole therapy to 2weeks clarithromycid4 weeks omeprazole therapy Triple Therapy Trial: Comparison of triple therapy with clarithromycin/metronidazole/omeprazole( W O ; clarithromycidmetronidazole days 21-34, omeprazole days 21-48) to monotherapy with omeprazole (days21-48)
of Gastritis
Clarithromycin Treatment Efficacy in
489
4. PharmacokineticTrial: Single-dose and multiple-dose pharmacokinetic analyses
RESULTS Minimum Inhibitory Concentration The MIC of clarithromycin againstH . mwtelae was 0.015 pg/ml, which was twofold to fourfold lowerthan the MIC values against H . pylori pg/ml). The MIC value of amoxicillin against H . mwtelae was 0.5 pg/ml, which was one to two logs higher than the MIC value range against H . pylori pg/ml). MIC valuesfor metronidazole and omeprazole against H . mustelae were 4 and pg/ml, respectively, compared to MIC values of 1-4 and pg/ml, respectively, againstH . pylori.
Double Versus Triple Therapy Data aresummarized in Table1. Table Z Helicobacter Burden and Histopathology Scoresof Gastric Samples from Ferrets Treated with AntibacteriaYOmeprazole Combinations
Mean log Helicobacter & SD (No. positive1total)’ Mean histopathology scores, Group
Day 35 biopsy
0.00 f 0.W Clari/omec Fundus (014) (014) h o d m e t / 0.49 2 0 . 6 9 omee v41 Omef 3.35 -C 0.47 (414) 5.49 Untreated 3.33 -C 0.73 Fundus (414) (414)
Day 63 pylorus 0.00
0.W
day 63b Region Pylorus
MNC
1.0
1.0 1.0 2.5
0
2.5 2.0
4.8 4.0
0.5
4.43 f 1.74e Pylorus Fundus 3.5 1.5 (414) 5.90 f 0.24 Pylorus Fundus (414) 2 0.73 Pylorus
Helico
PMN
3.8 1.0 3.8 1.0 3.8 4.0 4.0 1.3
0 5.0
5.0
1.8
‘Log counts per 1 l-mm biopsyor per 2 2-mm section of stomach. bHistopathologyscores based on modified Whiteheadscale (0-5). CCladome = clarithromycin 50 mgkg days 21-34, omeprazole 10 mglkg days 21-48. dAmox/met/ome = amoxicillidmetronidazoleSot75 rngikg days 21-34, omeprazole 10 mgikg days 21-48. CSignificantly different (p S .OS) than untreated controls. fOme = omeprazole 10 mglkg days21-48.
Ewing et al.
490
Table 2 Helicobacter Burden and HistopathologyScores of Gastric Samples from Ferrets Treated with ClarithromycidOmeprazole Combinations
Mean log Helicobacter 2 SD (No. positive/total)” Mean histopathology scores, Day 49 GroupMNCPMN Region pylorus biopsy Clari/omec (214weeks) Cladornee (1/4weeks) Omef (4weeks)
Day 63
day 63b ,
‘Helico
1.21 2 1.86d 2.85 2 3.0Sd . (418) 1.93 .+ 2.24d 4.70 2 2.24d . (4/8) 5.13 2 0.30 6.40 0.52 I
1.9 1.3 2.3 Pylorus Fundus 1.6 1.0 1.0 3.3 2.3.2.8 Pylorus Fundus 3.4 4;3 Pylorus‘. Fundus 2.8 2.8 1.3
.
1.0 2.0 4.5’
.Log counts per 1 X l-mm biopsy or per 2 X %mm section of stomach. bHistopathologyscores based on modified Whitehead scale (0-5). CClari/ome (U4 weeks) = clhrithromycin 50 mgkg days 21-34, omeprazole 10 mgkg days 21-48. dSignificantly different(p .05) than omeprazole monotherapy. Cladome (U4 weeks) = clarithromycin 50 mgkg days 21-27, omeprazole 10 mgkg days 21-48. [Ome = omeprazole 10 mgilcgdays 21-48.
Length of Therapy Trial Data aresummarized in Table 2.
Triple Therapy Trial Data aresummarized in Table 3.
Recovery of Resistant Isolates from Length of Therapy Trial The MIC values for clarithromycin against the H.mustelae ATCC strain used for inoculation and isolates recovered from ferrets prior to treatment (day 21) were similar (0.008 pg/ml). After therapy (day 63),three of four ferrets with recoverable H . mustelae yielded isolates with an increase in clarithromycinMICvalues to 32 pg/ml.MICvalues for amoxicillin, metronidazole, and omeprazole remained unchanged.
Pharmacokinetic Analyses Following four daily oral dosesof clarithromycirdomeprazole(50/10 mgkg per day), the 24-h plasma C, and C, levels of clarithromycin were 5.90
of Gasfrifis
Clarithromycin Treatment EfJicacy in
491
Table Helicobacter Burden and Histopathology Scoresof Gastric Samples from Ferrets Treatedwith Clarithromycin/Metronidazole/OmeprazoleCombinations
Mean log Helicobacter f SD (No. positive/total)8 . Day 35
Day 49
Mean histopathology scores, day 63b
Day 63
PMN Region pylorus biopsy biopsy Group
MNC Helico
2.52 f 1.78d 4.70 2 2.22 Pylorus ClarVmetl 0.60 f Fundus 2.5 1.5 omec (3/12) (10112) (9112) 3.2 Omee 4.10 f 0.52 4.19 2 0.42 5.58 f 0.82 Pylorus Fundus (1112) (1112) (12/12) '
3.7 3.4 0.6 3.7 0.9 3.3 2.1
'
3.6 4.1
.Log counts per 1 l-mm biopsy or per 2 2-mm section of stomach. bHistopathology scores based on modified Whiteheadscale (0-5). CClari/met/ome = clarithromycidmetronidazole 50/75 mglkg days 21-34, omeprazole 10 mglkg days 21-48. dsignificantly different(p .OS) than omeprazole monotherapy. eOme = omeprazole mgkg days 21-48.
and 2.35 pglml plasma, respectively. Thearea under the curve (AUC,,+,) was 112.89 pg h/ml. Following a single 50-mglkg oral dose of clarithromycin given with10 mg/kg of omeprazole, the 24-h plasma C, and C, of clarithromycin were 8.01 and 1.62 pglml, respectively. The A U G wh was 83.66 pg Wml.
DISCUSSION The C/O therapy was more effectivethan for treatment of Helicobacter mustelae-induced gastritis. Amoxicillin-based therapy may not be effective inthe ferret model due to the relatively high MIC value (0.5 ml) againstH . mustelae. Two weeks of clarithromycin therapywas more effectivethan 1week of therapy. Triple therapy with clarithromycin was more effective than omeprazole monotherapy at decreasing Helicobacterburden. Successful treatment of Helicobacter-induced gastritis with clarithromycin was more difficultto achieve in ferrets than in humans. Eradication rates were generally lower and more variable (25-100%) ferrets in than in humans, even though plasma concentrations of clarithromycin in ferrets exceeded levels achieved in humans. The ferret model may best serve to compare relative efficacies of experimental therapies withinthe same drug class andto determine optimal dose schedule.
Ewing et al.
492
REFEXENCES 1. Lee A, Fox Hazel1 S. The pathogenicity of Helicobacter pylori: a perspective. Infect Immunoll993; 61:1601-1610. 2. Fox Correa P, Taylor NS,et al. Helicobacter mustelae-associated gastritis in ferrets: an animal model of Helicobacter pylori gastritis in humans. Gastroenterology 1990;99:352-361. Hardy D, Swanson R, Hensey D, et al. Comparative antibacterial activities of temafloxacin hydrochloride (A-62254) and two reference fluoroquinolones. Antimicrob Agents Chemother 1987; 31:1768-1774.
-
AzithromycidRanitidine Combined Treatment of Helicobacterpylori in Patients with Duodenal Ulcer and Chronic Gastritis: A Pilot Study B. Desnica, V. Burek;
N. Makek
University Hospital of Infectious Dbeases "DrFran MihaljeviC" Zagreb, Croatia
INTRODUCTION Peptic ulcer disease is a chronic inflammatory condition of the stomach and duodenum that affects as many as 10% of people at some time in their lives. Althoughthe disease has relatively low mortality, it results in substantial human suffering and its wide prevalence underscores the high economic cost of the .illness. The association with Helicobacter pylori as a major pathogenic.factorin peptic ulcer disease was established in1983. It is now generally accepted that H . pylori is a major causeof gastric and duodenal ulcer and antral gastritis as well.The eradication of the organism is necessary for optimal therapyof the disorder. The gold standards of establishing the diagnosis of H. pyloninfection are invasive tests which include endoscopy followed by gastric biopsy and histological demonstration of organisms, biopsy with direct direction of urease activity, and biopsy with culture of H.pylon. Excellent diagnostic sensitivity and specificity can be obtained with serologic kits for IgG anti493
Desnica et al.
494
bodies to H . pylori. Because of their technical limitations and because antibody levels decrease rather slowly following successful eradication of H . pylori infection, serologic tests may not be useful in diagnosing infections after antimicrobial therapy unless repeated during a longer follow-up period. Azithromycin, administered in combination with ranitidine, bismuth salts, or omeprazole, is well tolerated and safe in doses up to 1 g daily. The aimof our studywas to evaluate the efficacyofcombined azithromycidranitidinetreatment o,n the healing and recurrenceof chronic gastritis typeB and duodenal ulcer disease associated with H . pylori and to establish the correlation with seronegativity achieved after antimicrobial therapy.
PATIENTS AND METHODS Patients of both sexes, aged between 17 and 72 years, with clinical symptomssuchasepigastricpain,dyspepsia, heartburn, or anorexia,were enrolled in this open, noncomparative study. They were included in the study on the basisofendoscopicfindings(endoscopic appearance of duodenal ulcer, measuring between 5 and 20mmin longest dimension, or endoscopic appearance of antral erosions, spotty erythema of antral mucosa, pale areas, goose-pimple-like appearance of antral mucosa, fine spotty erythema of the body of the stomach), histologically defined gastritis (by the Sidney system), and HP seropositivity (ELISA IgG > 1.0 U). Patientswereexcluded if theyhadreceivednonsteroidalantiinflammatorydrugtherapy,corticosteroids,antimicrobialdrugs, or bismuth salts within4 weeks prior to entry or.antiulcer medications within 2 weeksprior to entry.Patientswithevidence of chronicrenal or liver disease,gastricsurgery or vagotomy,pregnancy,andchronicalcohol abuse were also excluded. After discontinuation of all previously employed therapy and serum bilirubin, AST, ALT, g-GT, AP tests performed, 44 patients who met the entry criteria and gave their written informed consent were.treated with azithromycin mg bid for 6 days and ranitidine mg bid for days. Endoscopic evaluations were done in all' patients after 1'month. A new endoscopic evaluation was performed in unhealed patientsafter 2-months of therapy with ranitidine.ELISA was repeated after and 180 days at follow-up. Histological evaluation was performed patients in with macroscopic appearanceof antral gastritis after days and in unhealedpatients after 2-months of therapy.IgG ELISA anti-HP test (DPC, USA)was performed in all patients, andIgM and IgA antibodies were determined as well, but their values were considered insignificant forthe purpose of the
AzithrornycinlRanitidine Treatment of Helicobacter pylori
495
study. Although this test is considered secure from FW interference, each specimen was re-tested by Western blotting. Patients with IgG antibody titer >1.0 U were considered elective for study. Reduction in antibody titer below 0.5 U was defined as seronegativity. Four antral biopsy specimens were assessed by hematoxylin and eosin stain and graded using the Sidney system in casesof antral gastritis. Pathologists were blindto the treatment and serological results.
RESULTS Ulcer healing was.obtained in 23/28 patients with duodenal ulcer (baseline characteristics of patients are presented in Table 1). Clinical and endoscopic cure was followed by IgG seronegativity or a dec1ine;of IgG antibodies (Fig. 1). Three patients returned to seropositivity after 6 months although they were asymptomatic. W Opatients complained of dyspepsia and abdominal discomfort in spiteof complete healing of the ulcer after 2 months of therapy and a decline of IgG antibody titer. Resolution of endoscopic, histological, andELISA evidence of antral gastritis (baseline characteristics of the patients presented in Table1) was obtained in 11/16 patients (Fig. 2). Three patients had. dyspepsia after treatment although they had a histological improvement and turned to seronegativity. W Opatients had a relapse, confirmedby ELISA, although they had undergone a 2-month treatment. relevant adverse events were noticed. Four patients complainedof mild nausea during the first week of treatment. However,bloodchemistrytestsandurinalysisrevealedno pathologic findings. Table I Baseline Characteristics of Patients
Duodenal ulcer (n = 28) Age (mean 2 SD in years) Gender (W) First duodenal ulcer No. of ulcers present (mean 2 SD) Size of largest ulcer (mean -t SD) Gastritis B (n = 16) Age (mean 2 SD in years) Gender (MiF) Intestinal metaplasia Alcohol use
43.2 2 11.5 17/11 36% 1.0 2 0.2 9.1 2 3.6 48.3 2 12.1 11/5 63% 75%
496
Desnica et al.
0 days
30 days
60 days
180 days
Figure Z Healing of duodenal ulcer.
CONCLUSION There was improvement in the healingduodenal ulcer and antral gastritis associated withHelicobacter pylori infection with combined azithromycid ranitidine therapy. The precise role azithromycin in eradicatingHelicobacter pylori should be evaluated in a study including urease activity tests and culture.
100
-
......
90
.
seropositivity ... m Endoscopic cur( IClinical cure
80 70
.c"
60
," 50 n
40
30 20 10
0 0 days
30 days
Figure 2 Healing of antral gastritis.
60 days
180 days
AzithromycinlRanitidine Treatment of Helicobacter pylori
497
BIBLIOGRAPHY Malfertheiner P et al. The year of Helicobacter pylori 1995. Curr Opin Gastroenteroll995; 11(suppl 1). Peters DH et al. Azithromycin: A review of its antimicrobial activity, pharmacokintics properties and clinical efficacy.Drugs 1992; Shonova 0, Petr P. Concomitant and independent treatment with Surnamed" and Jatrox". Its effects on Helicobacter pylon infection in vivo. Workshop on Helicobacter pylon and the New Concepts in Gastroduodenal Diseases, Prague, 1992.
Azithromycin as a Promising Part of Helicocidal Regimens 0. Shonovh, P. Petr, and
Hausner
Regional Hospitalof Southern Bohemia Budweis, CzechRepublic
INTRODUCTION Helicocidal regimens have been reported recently, targeting short-term, low-dose, and high compliance. The aim of this trialwas to assess the effect of omeprazole and azithromycin in Helicobacter pylori positive patients with peptic ulcerof duodenum and aboral stomach.
PATIENTS AND METHODS
An open, uncontrolled, noncomparative clinical trial was performed inan outpatient gastroenterology unit. Patientsof both sexes, aged18-70 years, with endoscopically diagnosed peptic ulcer of duodenum or aboral stomach, measuring at least 5 mm in size, nonmalignant (Diagnoses and of OMED classification), and the presence of H . pylori (confirmed by at least two out of three methods used), were enrolled into the study. Exclusion criteria were pregnancy, women with childbearing potential not taking adequate contraceptive measures, malignant diseases (includinggastricmalignancy),mentalincapacity,administration of antiulcer drugs within the last 6 weeks (bismuth within the last 12 weeks) and/or 498
AzithromycinRegimens in Helicocidal
499
antibiotics therapy within 6 weeks, and/or nonsteroidal anti-inflammatory drug medication within6 weeks priorto entry.
Treatment The combined therapy with azithromycin (1 X mg dailyfor 1week) and omeprazole mg for weeks)wasgiven to all patients with endoscopically diagnosed peptic ulcer (minimal size5 mm) of the duodenum or aboral stomach.
Efficacy Assessment Endoscopy, including gastric biopsy for direct microscopy, culture, and urease test, was performed before the treatment. Control endoscopy and biopsy were performed4 weeks afterthe treatment (i.e., 5 weeks after the end of antibiotic treatment) and the presence of H . pylori was assessed.
Microbiological Assessment Three biopsyspecimens,takenfromantrum of each patient ateach endoscopy, were slightly pressed onto cultivation media as described below and transported to the microbiological laboratory within h, using no special transport medium. The specimens were homogenized and divided in three equal portionsfor each test.
Direct Microscopy The modified Gram-stain method was used, with prolonged fuchsin exposure (H. pylori absorbs the dyes poorly).
Urease Test Christensen fluid medium was used. The results were evaluated and h after inoculation. A semiquantitative three-level simple scalewas used to grade positive findings froml'to
Culture All specimens were cultured on three media, for H . pylori isolation and one (blood agar)for concomitant bacterial flora.The following media were used forH . pylori isolation: (a) Brain-heart infusion (BHI), with addition (per liter): Supplement A (for Neisseria gonorrhoeae cultivation) Yeast
m1 5g
500
(ram)
Shonov& et al. Starch Horse serum 50 m1 Active 2g Sheep 5% (b) Brucella agar, with additionof supplements as above
Vancomycin, trimethoprim, colistin, and amphotericin were added to the selective medium. H . pylori plates were incubated in microaerophilic conditionsat 35°C and assessed after 5 days (time span:3-7 days). Identification of H . pylori was made by typical morphology of the colonies, oxidase, and catalase activity and Gram-stain microscopy. H . pylori presence before treatment was defined by positive findings inat least two of the three methods used. H . pylori presence after treatment was defined by positive findings in at least two the three tests or by positive culture only.
Susceptibility to Azithromycin Sensitivity of all H . pylori isolates to azithromycin was tested using the disk-diffusionmethod.Azithromycin(15 pg) testingdisks(ImunoloSki zavod Zagreb, Croatia) were employed.
RESULTS In this study, 19 adults (11 men and 8 women), aged 21-70 years (mean age: 43 years), with endoscopically confirmed peptic ,ulcer (minimal size5 mm) were included. Before treatment, H . pylori presence was confirmed in all patientsby allthree described methods. Four weeks after the treatment, the ulcer was healed in19 patients (100%) andH.pylori was eradicated in 16 patients (85%) (Table 1). No drop outs and no adverse events were recorded, either in spontaneous referenceor in active interviewing. Sensitivity testing was performed on 20 consecutively isolated H. pylori strains and 19 strains (95%) were sensitive, with an average inhibition zone of mm (176% of mm, regarded as safe marker for susceptibility). Table Z Efficacy of Azithromycin and Omeprazole inthe Treatment of H.pyloriPositive Patients with Peptic Ulcer Disease
Ulcer H . pylon eradication
No. of patients
Rate
19 16
100% 85%
501
AzithromycinRegimens in Helicocidal
CONCLUSION Combined therapy with azithromycin and omeprazole was well tolerated and effective in ulcer healing and inH . pylori eradication. The advantage of this therapy is the short duration of treatment and low dose both of which should provide good compliance.
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ANAEROBES, ENTEROCOCCI, HAEMOPHILUS INFLUENZAE
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Comparative In Vitro Activity of Five Macrolides Against Gram-Positive Cocci, Campylobacter Species, and Anaerobes M. Marina and K. Ivanova National Center of Infectious and Parasitic Diseases Sofia, Bulgaria
N. Hadjieva University Hospital “QueenJoanna” Sofia, Bulgaria
A. Urumov National Drug Institute Sofia, Bulgaria
INTRODUCTION Erythromycin, whichis an alternative to penicillininpenicillin-allergic patients and shows a good antibacterial activity against gram-positive bacteria, has some disadvantages. Several new macrolide compounds with improved pharmacokinetic properties have been introduced, such as josamycin, roxithromycin, clarithromycin, and azithromycin. The purpose of this study was to compare the in vitro activities of these macrolides against gram-positive cocci,Campylobacter sp. and some anaerobes. 505
506
Marina et al.
MATERIALS AND METHODS Bacterial Strains All tested strains were clinical isolates identified by the methods outlined in the Manual of Clinical Microbiology, 1991: gram-positive cocci 237, most ofwhichweremultiresistanthospitalstrains: S. aureus (87 strains), coagulase-negative staphylococci (92strains), and enterococci (58strains); Campylobacter sp. (156 strains): C . jejuni (112 strains), C. coli (40 strains), and C . fetus (4 strains); obligately anaerobic bacteria(134 strains, chosen among those species with greater tendency of erythromycin resistance: Bacteroides fragilis group (86 strains); Prevotella sp. (26 strains), including P . oralis, P . oris, P. intermedia, P . melaninogenica; Fusobacterium sp. (10 strains); Bilophila wadsworthia (2 strains), andClostridium sp. (10 strains).
Antimicrobial Agents The following antibiotics were tested: erythromycin, josamycin, roxithromycin, clarithromycin, and azithromycin.
Susceptibility Testing The minimal inhibitory concentration (MIC) was determined using the NCCLS-recommended agar dilution methods with Mueller-Hinton agar for the cocci and Campylobacter, and the Wadsworth agar dilution method with Brucella blood agar for the anaerobes.
RESULTS Table 1 summarizes the susceptibility of the aerobic gram-positive cocci, Campylobacter species,andanaerobicbacteria to erythromycin,josamycin, roxithromycin, clarithromycin and azithromycin. Data on the last two groups of organisms are also illustrated graphically in Figs. 1 and 2.
DISCUSSION Almostall of the testedmacrolideshadsimilar.activitiesagainst the staphylococci except for a slight predominance of clarithromycin (forboth) and josamycin (for the coagulase-negative staphylococci) (see Table 1). Clarithromycinandjosamycinweremoreactivethan the other drugs against enterococci-29% and 25%of susceptible strainsat 8 pg/ml, respectively.Azithromycinandroxithromycinwere the mostactiveagainst Campylobacter sp.; 90% of the strainsinhibited at 0.5@m1 for azithromycin and at 4 pg/ml for roxithromycin. Josamycin and clarithro-
507
Macrolides Against Gram-positive Bacteria
Table Z Susceptibility of Gram-positive Cocci, Campylobacter Species, and Some Obligately Anaerobic Bacteriato Erythromycin, Josamycin, Roxithromycin, Clarithromycin, and Azithromycin Percentage strains inhibited at (pglml)
MIC (Clglml)
Organism (No.) and Range antimicrobial agents
90%
S. aureus
Erythromycin Josamycin Roxithromycin Clarithromycin Azithromycin Coagulase-negative staphylococci (92) Erythromycin Josamycin Roxithromycin Clarithromycin Azithromycin Enterococci Erythromycin Josamycin Roxithromycin Clarithromycin Azithromycin Campylobacter sp. Erythromycin Josamycin Roxithromycin Clarithromycin Azithromycin Anaerobic bacteria Erythromycin Josamycin Roxithromycin Clarithromycin Azithromycin
OS-> 50.5” OS-> OS->
> > >
os->
>
50.5-> OS-> OS-> OS->
60
40
>
> > > > > >
OS-> 50.5->
2
0.5
os-> OS-> OS-> 50.5->
> > > >
Marina et al. %
90
80 70 60
50 40 30
20 10
0.5
1
2
4
l 64
32
128
MIC /pg/ml Figure l
Campylobacter: Inhibited strains versus MIC.
mycin were intermediate with 96% and 91%, respectively, of susceptible strains at 8 pg/ml. Erythromycin had the lowest activity and the highest percent of resistant Campylobacter strains (11.5%) at the same concentration. Josamycinandclarithromycinwere the mostactiveagainst the anaerobes-83% and 80%, respectively, of susceptible strains at 4 &ml. Erythromycin and azithromycin had similar activity and inhibited 41% of strains at this concentration. Roxithromycin showed the lowest activity, with only 16.4% of susceptible strainsat 4 pg/ml. Our results are comparable to those of other authors (1-7).
CONCLUSIONS There is a trend toward increasing erythromycin resistance in Campylobacter sp., and the new macrolides can be a good alternative for the treatment of severe campylobacteriosis. They can be used in infections
509
Macrolides Against Gram-positive Bacteria YO 100
90 80
70 60 50
40 30 20 10
0.5
1
2
4
8
l 6
32
64
128
MZC &/m1 Figure 2 Anaerobic bacteria: Inhibited strains versusM C .
caused by gram-positive cocci or anaerobes but only after in vitro testing of their activity.
REFERENCES 1. Arguedas AG, et. al, Comparativeactivity of josamycin,roxithromycin, clarithromycin and azithromycin against erythromycin resistant staphylococci. 1st Scientific Meetingof the European Society of Chemotherapy, Budapest, 1993; abstr P2. 2. Citron D, etal. Activity of ampicillidsulbactam, ticarcillidclavulanate, clarithromycin, and eleven other antimicrobial agents against anaerobic bacteria isolated from infections in children. Clin Infect Dis1995; 20 (suppl 2):S356. 3. Dubreuil L, et al. Activitt in vitro de la roxithromycine, nouveau macrolide semisynthetique envers les anaerobies stricts. Pathol Bioll986; 34:440. 4. Garcia-Rodriguez JA, Garcia-Sanchez JE, Prieto-Prieto J. Josamycin alone and in combination against anaerobic bacteria. Drugs Exp Clin Res 1982; VI11 (3):285. 5. Gomes-Garces JL, Cogollos R, Alos JI. Susceptibilities of fluoroquinoloneresistant strains of Curnpylobacterjejuni to 11oral antimicrobial agents.Anti microb Agents Chemother 1995;39542.
510
Marina et al.
6. MaskellJP,Sefton AM, Williams JD. Comparative in-vitro activity of azithromycin and erythromycin against Gram-positive cocci, Haemophilus influenzae and anaerobes. J AntimicrobChemother 1990; 25 (suppl A):19. 7. Spangler SK, Jacobs MR, AppelbaumPC.Comparison of the E test and oxyrase methods in susceptibility testing of201 Gram-negative and Grampositive anaerobes to erythromycin, azithromycin, clarithromycin and roxithromycin. 7th European Congress of Clinical Microbiology and Infectious Diseases, Vienna, 1995; abstr 171.
Prophylactic Effect of Azithromycinon Experimentally Induced Intraabdominal Infection in Rats N. Panovski,
Mil&vski, P.
N. Labaeevski
University “Sv. K i d i Metodij” Skopje, Republic of Macedonia
INTRODUCTION Azithromycin is not recommended for the treatment of intraabdominal infections becauseof the relative resistanceof common bacterial pathogens at this site mean minimal inhibitory concentration (MI(&,) is m& for Escherichia coli and 11 mgL for Bucteroidesfrugilis (the range: mgL) (1). However, azithromycin has been shown to.be effective in several in vivo models of localized infections induced withE. coli or B. frugifis (2). The purpose of the present studywas to evaluate the prophylactic effectof azithromycin, in comparison with clindamycin and ciprofloxacin, on the development of acute peritonitis and on the formation of intraabdominal abscess in an animal model using Wistar rats.
MATERIALS AND METHODS From one strain each of B . fragifis and E . coli, clinical isolates were obtained from a patient with a perforated appendix. The MICs of azithromycin, clindamycin, and ciprofloxacin were 16, and dirithromycin. Dirithromycin is easily and rapidly hydrolyzed to erythrocylamine, which accounts for much ofthe antimicrobial activity(13). We were unable the E-test in a to establish anti-H. influenzae activity of dirithromycin using carbon dioxide atmosphere. pH values before and after incubation were stable (pH 7.3). We also showed the influence of the medium (E-test MICs on PDM were lower than on I"), methods (E-test MICs were lower than agar dilution MICs), and expression of results (MICsor categories). Categories are dependent on defined breakpoints, whereasMIC, and MIC, are representative of MICdistribution.Diskdiffusionis inappropriate to test macrolides againstH. influenzae and the expression of sensitivity by categories is inadequate.
CONCLUSION H . influenzae is pharmacologically resistant to erythromycin, poorly susceptible to roxithromycin (resistant to the concentrations obtained in vivo), whereas it is pharmacologically more susceptible to clarithromycin (susceptible to the concentrations obtained in vivo) and both pharmacologically and microbiologically more susceptible to azithromycin. There are major pitfalls associated with in vitro testing but they can be overcome. The Etest is an interesting alternativeto the agar dilution method. Disk diffusion is not appropriate to test the activity of macrolides against H. influenzae.
522
Crokaert et al.
REFERENCES 1. Klein JO. Selection of oral antimicrobial agentsfor otitis media and pharyngitis. Clin Infect Dis 1994;19:823-833. 2. Early D. Purulent pericarditis causedby Haemophilus influenzae.Antimicrob Infect Dis Newslett 1994; 13:29. 3. McDonald CL, Crafton EM, Covin FA. Pericarditis: a probable complication of endocarditis due to Haemophilusinfluenzae. Clin Infect Dis 1994; 12548-649. 4. Moxon ER, Wilson R. The role of Haemophilus infruenzae in the pathogenesis of pneumonia. Rev Infect Dis1991; 13 (suppl6):S518-S527. Najm W, C", Spurgeon L. Bacteremia due to Haemophilus infections: a retrospective studywith emphasis on the elderly. Clin Infect Dis 1995; 21~213-216. 6. Scriver SR, Walmsley SL, Kau CL. Determination of antimicrobial susceptibilities of Canadian isolates of Haemophilus influenzae and characterization of their p-lactamases. Antimicrob AgentsChemother 1994; 38:1678-1680. . 7. Barry AL, Fernandez PB, Jorgensen JH. Variability of clarithromycin and erythromycin susceptibilitytests with Haemophilus influenzae in four different broth media and correlation with the standard disk diffusion test. J Clin Microbioll988; 26:2415-2420. 8. National Committeefor Clinical LaboratoryStandards. Methods for Dilution Antimicrobial Susceptibility Testsfor Bacteria That Grow Aerobically. Third Edition, Villanova, PA: Approved Standard M7-M. National Committee for Clinical Laboratory Standards, 1993. 9. Manual of Clinical Microbiology, 6thed. Washington, D C American Society for Microbiology, 1995. 10. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Disk Susceptibility Tests-Fifth Edition, Approved standard M2-AS. Villanova, PA: National Committeefor Clinical Laboratory Standards, 1993. 11. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing. Approved Standard M100-S3. Villanova, PA: National Committeefor Clinical Laboratory Standards, 1994. 12. Steigbigel NH. Macrolides and clindamycin. In: Mandell GL, Douglas RG, Bennett JE eds. Principles and Practices in Infectious Diseases, 4th ed. New York: Churchill Livingstone,1995:334-346. 13. Kirst HA, Creemer LC, PaschalJW.Antimicrobial characterizationand interrelationships of dirithromycin and epidirithromycin. Antimicrob Agents Chemother 1995; 39:1436-1441.
Macrolide Susceptibility of Isolates of Haemophilus inpuenzae, Streptococcus pneumoniue, and Moraxella catarrhalis from Patients with Community-Acquired Lower Respiratory Tract InfectionResults of an International Multicenter Study (the Alexander Project), 1992-1994 D. Felmingham, J. Linares, and the Alexander ProjectGroup University CollegeLondon Hospitals London, England Hospital de Bellvitge Barcelona, Spain
INTRODUCTION Antimicrobial choice for the empirical therapy of pulmonary infection is threatened by the increasing prevalence of resistance among communityand hospital-acquired pathogens (1). Therefore, determination of local, national, and worldwide antimicrobial susceptibility patterns, which may vary with time, is of fundamental importance. In early 1992, a prospective, international, multicenter collaborative study of the susceptibility of 523
524
Felmingham et al.
community-acquiredlowerrespiratorytractpathogens-theAlexander Project-was established. We report on the macrolidesusceptibility of isolates of H . influenzae, S. pneumoniae, and M. catarrhalis examined in the study duringthe period 1992-1994.
MATERIALS AND METHODS
Fifteen centers were enrolled into the study, including the following: London/ Belfast, UK; Toulouse/Paris,France; BarcelonaMadrid, Spain; Weingartemunich, Germany; GenodCatania, Italy; New YorkNorcester, MMortland, OWCleveland, OWJohnson City, TN, USA. Each center collected up to 400 isolates from patients with community-acquired lower respiratory tract infection and sent to them the central laboratory in London for susceptibility testing.After reidentification, minimal inhibitory concentrations (MICs) of the macrolides were determined using a microbroth inco poration techniquewith an inoculum of approximately 2 X lo4CFU (colonyforming units) contained in 50-p1 Mueller-Hinton broth, supplemented 2% v/.) and NAD (final concenwith lysed horse blood (final concentration tration 10 m a ) for isolates of H . influenzae and S. pneumoniae. Breakpoint MICs publishedby the NCCLS (2) were used for qualitative interpretation of susceptibility.
RESULTS The MICs of the macrolides for4155 isolates of H . influenzae were distributed unimodally (Table 1). No significant differences in mode MICs were observed among isolates fromthe 3 years or among centers. No high-level resistance was detected. All strains were inhibitedby 5 4 mgL of azithromycin and 97.2% of the isolates were susceptible to clarithromycin at 5 8 m&. No interpretativeerythromycin MIC breakpoints are published bythe NCCLS; therefore, no comment can be made regarding this compound. A total of 1193 isolates of M. catarrhalis were tested. No significant differences in mode MICs were observed among isolates from the 3 years or among centers (Table 2). High-level macrolide resistance was not detected. However, examination of the distribution of MICs for this species shows a trailing downslope of increasing MIC which may indicate incremental changes in susceptibility in a small proportion of isolates. A total of 2829 isolates of S. pneumoniae were tested.The prevalence of high-level macrolide resistance varied widely from country to country, being highest in France and Spain the in European Union with evidenceof increasing prevalence in one center in Italy (Genoa) and one in the United States (Cleveland, OH) (Table 3). Cross-resistance to all three macrolides
Macrolide Susceptibility
Bacteria in RTI
525
526
et
Felmingham
al.
Table 2 Distribution of Macrolide MIC (m@) for 1193 Combined EU and USA Isolates ofM.catarrhah-Alexander Project 1992-1994
No. of isolates inhibited at 0.25 0.12 50.06" Antimicrobial
Erythromycin Clarithromycin Azithromycin
0.5
154 989 1167
865 176 14
143 4 18 3 4
22 3 6
4 1
2
3
2 3 1
1
1
-
'In 19!Z and 1993,0.06 m& was the lowest concentration tested. In 1994, mode MICs were as follows: erythromycin,0.12 m&; clarithromycin, 0.06 m&; azithromycin, m&.
was apparent and was associated with p-lactam resistance. However, this association was notcomplete. Thus, in Toulouse and Genoa, erythromycin resistance among penicillin-susceptible strains (28.8% and 12.3% in 1994, respectively) was considerably higher than in Barcelona (1.5% in 1994), where some of the highest rates of penicillin resistance were found, indicating other selective pressures. Table 3 High-Level Macrolide Resistance Among 2829 Isolates of S. pneumoniae-Alexander Project 1992-1994 '
Center
UK:
2.3
London (1) Belfast (2) France: Toulouse (3) Paris (4) Spain: Barcelona (5) Madrid (6) Germany:Weingarten (7) Munich (8) 58 Genoa Italy: (9) Catania (10) USA: YorkNew (11) 38 14 Worcester 43 (12) Portland 2 (13) 37 49 Cleveland 26 (14) Johnson City 12 8 (15)
No. of isolates tested
% Resistance
1992
1993
1994
1992
1993
1994
128 38 89 80 267 2
105 45 152 73 228 52
1.6 0 27 17.5 9
2.8 0
2 1.4 3 79
110 42 147 42 11.4 263 51 16 17 128 10 51
31
18
0.9 0 45.6 31 18.6 45.1 6.3 0 14.1 0 2 2.6 5.6
60 44 70
46
-
1.7 0
25 45.2 19.2 0 -a
5.2
-
-
0 0 0 0 10.8 4.1 8.3 8.7
-
Note: High-level resistance breakpoint MIC greater than or equal to 4 m& erythromycin. 'No data, % calculated only when total isolates examined 210.
Macrolide Susceptibility of Bacteria inRTI
527
CONCLUSIONS High-level macrolide resistance was not detected among the isolates of H. influenzae and M. catarrhalis tested. Establishmentof MIC breakpoints to guide clinicians inthe choice of any one of the marolides, rather than another, for the treatment of infections caused by H . influenzae can only be achieved by considering controlled clinical response data for defined infections. The breakpoints referredto in this study, or lack of them inthe case of erythromycin, may not be appropriate. High-level cross-resistance to the macrolides among isolatesof S. pneumoniae is wellestablished worldwide, but the prevalence varies greatly depending on geographical originof strains and, to some extent, with the passage of time. Although strongly associated with penicillin resistance, macrolide-resistant penicillin-susceptible strains occur with high prevalence in some countries.
REFERENCES 1. Felmingham, D. Antibioticresistance. Do we need new therapeutic approaches? Chest 1995; 108:70S-78S. NationalCommittee for ClinicalLaboratory Standards. PerformanceStandards for Antimicrobial Susceptibility Testing. Fifth Informational Supplement Document M100-SS. Villanova, PA, NCCLS; Vol. 14, No. 16.
Resistance of Common Respiratory Pathogens to Erythromycin and Azithromycin Milan CiZman University Medical Centre Ljubljana Ljubljana, Slovenia
Ana Zlata Drag& and Katja Seme Institute of Microbiology Ljubljana, Slovenia
Andreja OraZem and Metka Paragi Institute of Public Health Ljubljana, Slovenia
INTRODUCTION
Macrolides account for 10-15% of the worldwide oral antibiotic market(1). In the period 1991-1994,macrolide/azalideprescriptions for nonhospitalized patients in Slovenia increased from 4.8% to 12.7% of all antibiotic prescriptions, or from 0.89 to 1.87 defined daily doses (DDD) per 1000 inhabitants per day. A DDD for erythromycin is 1 g and that for azithromycin (AZM) is g. The greatest increase in prescriptions was documented for AZM (from 0.0703 to 0.7997 DDD/1000 inhabitantsper day) (2). A correlation between erythromycin consumption and erythromycinresistant pathogens has been observed in some countries (3,4). to This le 528
Respiratory Pathogen Resistance to Erythromycin and Azithromycin
529
examine the frequency of erythromycin and AZM resistance in common respiratory pathogensin Slovenia.
MATERIALS AND METHODS Throat swabs were obtained from inpatients and outpatients with acute respiratory tract infection treated at the Department of Infectious Diseases, University Medical Centre Ljubljana. Bacterial isolates were performed at the Institute of Microbiologyof the Faculty of Medicine in Ljubljana. Additional strains of S. pneumoniae isolated from the respiratory tract (predominantly upper) or sterile body fluids and H.injluenzue isolated from sterile body fluidsof patients in different Slovenia hospitals were included in the study as well. Susceptibility tests were performed at the Institute of Public Healthof Slovenia. Antimicrobial susceptibility testing of S. pyogenes, group B, C, and G streptococci, S. pneumoniae, H. injluenzae, H. parainjluenzae, and B . catarrhalis was performed routinely using NCCLS disk-diffusion methods (5). Erythromycin (15 pg) and AZM (15 pg) susceptibility testing disks (Becton Dickinson Microbiology Systems, Cockeysville, USA) were employed. S. pyogenes, S. pneumoniae, and B . catarrhalis isolates were cultured on Mueller-Hinton agar supplemented with 5% defibrinated sheep blood (100-mm plates). For H.injluenzae and H . parainfluenzae isolates 100-mm agar plates of Haemophilus Test .Medium (Oxoid, Basingstoke, B. England) were used. The agar plates (except those inoculated with catarrhah) were incubated at 35°C in 5-7% CO, for 18-24 h before measuring the zones of inhibition. The organisms were reported as either susceptible, moderately susceptible, or resistant to erythromycin andAZM according to thefollowing zone diameter interpretative standards: Moderately Susceptible susceptible Resistant organisms organisms organisms 14-22 Erythromycin 513 218 Azithromycin The Fisher exact test and the chi-square test were used for statistical analysis. p-Values below .05 were considered to indicate statistical significance.
RESULTS The nearly triple increase in macrolidehzalide prescriptions recorded in 1991-1994 did not have a major impact on the resistance rates of common
&fman et al.
530 Table Z In Vitro Antimicrobial Resistance
Clinical Isolates to Erythromycin
1994
No. of isolates tested Pathogen S. pyogenes S. agalactiae
Group C streptococci Group G streptococci S. pneumoniae Invasive strains Noninvasive strains H . injluenzae Invasive strains Noninvasive 1.9 strains H . parainfluenzae B . catarrhalis
112 10 12
1995
% of resistant isolates
No. of isolates tested
10
13
11
11
4.8
16.6b 108 5.2 4.2
0 0
0.74
0.6
10 62 19 28
p-Valuea
4.1
21 242
92
25 61
96 0.09 4 ' 17
%
resistant isolates
0.50 0.65
8
0.12
0.60 0.68
.Incidence of resistance, 1995 versus 1994.
b7% moderately susceptible isolates. CModerately susceptible isolates.
respiratory pathogensto erythromycin andAZM in the year 1994 (Tables 1 and 2). Aninsignificant (p = .12-.74) increasein the resistance of S. pneumoniae, S. pyogenes, and B . catarrhalis to erythromycin was observed in the year 1995.
DISCUSSION The increase in macrolide/azalide prescriptions in Slovenia did not have a nificantimpact on the resistance ofcommon respiratorypathogens to erythromycin andAZM during the 4-year period our survey. The results are in agreement with the data of Barry and Jones(6), who found that the introduction a numberof antibiotics in U.S. hospitals was not followed by substantial changes in the incidence of resistant strains 3-5 years later. In Slovenia, macrolide/azalide consumption in 1994 was below the rates re-
Respiratory Pathogen Resistance to Erythromycin and Azithromycin
531
T&& 2 In Vitro Antimicrobial Resistance of Clinical Isolates to Azithromycin 1994
Pathogen S. pyogenes S. agalactiae Group C
streptococci Group G streptococci S. pneumoniaeb H . influenza8 H . parainfluenzae B. catarrhalis
No. of isolates resistance tested
1995 % of
No. of
isolates
isolates tested
% of resistant isolates
3.2
12
93 4 17
13
11
0
92
21 39 14 12
0
112
108 25 61
4.2
p-Valuea
0
nIncidence of resistance, 1995 versus 1994. bNoninvasive strains.
ported by anumber of countriesfor the previousdecade. In 1983, erythromycin was consumed atrate a of 4.1 DDD/1000inhabitants per day in the United States and 2.3 DDD/1000 inhabitants per day in France, whereas in 1989, a consumption rate 3.0 DDD/1000 inhabitants per day was recorded in Finland Despite the high erythromycin consumption inthe 5% of United States, no more than of S. pneumoniae isolates, less than S. pyogenes isolates, and a very small proportion of Moraxella catarrhalisisolates were resistantto erythromycin duringthe 1980s (8). In contrast, about 4% of S. pyogenes iso20% of S. pneumoniae isolates in France and upto 4 lates in Finland showed resistanceto erythromycin, although the estimated usage of macrolides in thosetwo countries was comparatively low (3,9). The correlation between antibiotic use and bacterial resistance is not straightforward, however, and many other parameters besides antibiotic consumption, such as infection control practices, presence or absence of resistance genes, selectivity and transmissibilityof these resistance genes, and antibiotic dosages, warrant consideration (10).
CONCLUSION Close monitoringof antibiotic prescriptions and frequent determinationof the susceptibility common respiratory pathogens to macrolides is needed in the era of macrolide renaissance.
532
REFERENCES 1. Kirst HA. New macrolides. Expanded horizons for and old classof antibiotics. J Antimicrob Chemother 1991; 28:787-790. 2. Institut za varovanje zdravja republike Slovenije. Ambulantno predpisovanje v letu 1994, I1 del. Zdrav Var 1995; zdravil v Sloveniji in zdravstvenih regijah 39 (suppl5):6-13. 3. Seppala H, Nissinen A, Jiirvinen H, et al. Resistance to erythromycin in group A streptococci. N Engl J Med 1992; 326:292-297. 4. Westh H. Influence of erythromycin consumptionon erythromycin resistance in Staphylococcus aureus in Denmark. APUA Newslett 1995;13:1-4. 5. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Disk Susceptibility Tests. Fifth Edition Approved standard M2-A5. Villanova, PA: National Committeefor Clinical Laboratory Standards, 1993. 6. Barry AL, Jones R. Bacterial antibiotic resistance before and after clinical application in the United States. BullNY Acad Med 1994; 63:217-230. 7. Col NF, O’Connor RW. Estimating worldwide current antibiotic usage:report of Task Force. Rev Infect Dis 1987;9 (suppl3):S232-S243. 8. Jacobi GA. Prevalence and resistance mechanisms of common bacterial respiratory pathogens. Clin Infect Dis 1994;18:951-957. 9. Acar JF. Bun-Hoi AY. Resistance patterns of important gram-positive pathogens. J Antimicrob Chemother 1988; 21 (suppl C):41-47. 10. PCchere JC. Antibiotic resistance is selected primarily in our patients. Infect Control Hosp Epidemiol 1994;15472-477.
Efficacy of Clarithromycin and Azithromycin at Human Pharmacological Dosage Against Experimental Haemophilus injluenzae Pulmonary Infection J. D. Alder, M. J. Mitten, A. Conway, A. Oleksiew, K. Jarvis, L. Paige, and S. K. Tanaka Abbott Laboratories Abbott Park,Illinois
INTRODUCTION The macrolide antibiotics clarithromycin and azithromycinare commonly used for the treatment of respiratory tract infections. These two drugs have modest minimal inhibitory concentration (MIC) values against Hemophilus influenzae, and the serum C, concentrations oftendo not attain the MIC value. Clinically,H . influenzae strains with different MIC valuesappear to respond as a uniform population to clarithromycin or azithromycin therapy due in part to intracellular concentrationof the macrolides. However, the NCCLS susceptibility breakpointfor clarithromycin 8 &ml) arbitrarily bisects the population at approximately whereas the breakpoint for azithromycin 4 pg/ml) covers99.5% of the isolates. rat model of pulmonary H . influenzae infection was used to test clarithromycin and azithromycin efficacyat pharmacological dosages. The
533
al.
et
534
Alder
Table Z Plasma and Lung Concentrations of Clarithromycin and Azithromycin in Rats Following Oral Dosing Plasma concentrations concentrations Lung C-
Cm,
AUG-24
Druddosage (Pdml) (Pdml) Clarithromycin mgkg mgkg Azithromycin 25 mdkg mgkg
Cm, cm, (PdN (Pdml)
AUG24
0.06
Note: Clarithromycin 100 mgikg represent near human pharmacologicaldosage in rat; 150mg/ kg represents a slight (1.3-2x) overdosage. Azithromycin 25 mgikg represents dosage moderately higher (2.5X) than attained in human; 100mg&g represents a large overdosage (4-5x).
Table 2 Efficacy of Clarithromycin and Azithromycin Versus H.infruenzae Rat Lung Infection
H.injluenzae Druddosage Clarithromycind mgkg 100mg/kg Azithromycine mg/kg mg/kg Untreated
H.in.uenzae
H.injluenzae
(log count/lung)a
(log count/lung)b
(log count/lung)e
f
f f
f f
f f
f
f
f
f
f
f
f
~
f
~ ~ ~ _ _ _ _____ _ _
Note: Clarithromycin yielded5.31-5.69 log reduction at 150 mgikg,and 3.51-4.11 log reduc-
tion at 100 mgikg. Azithromycin yielded 1.06-4.12 reduction log at 25 mgikg (50 mgikg day 0) and 0.4-2.33 log reduction at 12.5 mgikg(25 mgikg day 0). 'Mean log count f SD; H.influenme 43095 clarithromycin MIC = 4, azithromycin MIC = 1. bMean log count f SD;H.influenme 1435 clarithromycin MIC = 8, azithromycin MIC = 2. CMean log count f SD;H.influenzue 43095 clarithromycin MIC = 8, azithromycin MIC = 2. dclarithromycin dosage bid days0-2. CAzithromycin dosage qd days 1-2; 2X strength dosage day 0.
Pharmacology Dosage of Clarithromycin and
Azithromycin
535
Efficacy of Clarithromycin and Azithromycin Versus H.influenzae Rat Lung Infection Table
H.influenzae Drugdosage (log
count/lung)8
H.influenzae H.influenzae count/lung)b (log counflung)” (log
Clarithromycind mgkg mg/kg
2 f
f 2
f 0.00
f f
f 2 f
2 2 f
f
Azithromycine mg/kg mg/kg
Untreated
f
Note: Clarithromycin yielded3.18-6.33 log reduction at 150 mgkg, and 1.39-5.30 log reduction at 100 mgkg. Azithromycin yielded0.65-2.51 log reduction at25 mgkg (50 mgkg day 0) and 1.07-1.29 log reduction at12.5 mgkg (25 mgkg day 0). .Mean log count SD;H . influenzae 3598 clarithromycin MIC = 8, azithromycin M C = 2. bMean log count f SD;H.influenzae 3643 clarithromycin MIC = 16, azithromycin MIC = 4. CMeanlog count f SD;H . influenzae 3558 clarithromycin M C = 16, azithromycin MIC = 4. dClarithromycin dosage bid days0-2. CAzithromycindosage qd days 1-2; 2 x strength dosage day0.
goal was to determine the relation between clarithromycinefficacy and H . influenzae MIC.
MATERIALS AND METHODS The MIC values against H . influenzae isolates were determined by broth dilution using standard NCCLS guidelines. A single oral dose pharmacokinetic trial was conducted in g Sprague-Dawley rats. The rats were bled and 24 h postdosing. Drug concentration was determined by bioassay using Micrococcus leuteus as the detecting organism. For the efficacy trials, rats were inoculated intratracheally with logs of H . influenzae in molten agarose. All treatments were initiated 5 h postinfection with dosages to model clinical pharmacokinetics. Clarithromycin was administeredPO, bid on days Azithromycin was administered PO, qd, strength dosage day0, and 1 X strength dosage days 2. Lungs were harvested h after the last dosage. Bacterial burden was determined by dilution platingon chocolate agar.
RESULTS The results of the pharmacokinetic trialare shown in Table The results of the efficacy trials are summarized in Tables and
536
Alder et al.
DISCUSSION H . infruenzae pulmonary infection was established rats in using strains with clarithromycin MIC valuesof and azithromycin MIC values of 1-4. Clarithromycin or azithromycinwas administered to rats on schedules and dosages to produce human pharmacological concentrations in plasmaand tissue. At dosagesthat produced pharmacological concentrations in plasma, clarithromycin (C- = pg/ml; area under the plasma concentration versus time curve (AUC) = pg Wml) produced greater reductions of H . infruenzae in lung tissuethan azithromycin (C,,,= =.0.74 pg/ml; AUC = pg Wml). Clarithromycin was effective against H . infruenzae strains with MIC values of Clarithromycin therapy produced up to a log reductions in bacterial burden against H . infruenzae strains with MIC= which is greater than the established MIC breakpoint. The lung concentrationsof clarithromycin (C- = 43.4 pg/ml) and azithromycin (Crn== 22.7 pg/ml) werein great excess of the MICvalues of all H . infruenzae strains. Clarithromycin therapy demonstrated efficacy against H . infiuenzae strains with MIC values of indicatingthat successful therapy was independent of NCCLS breakpoint considerations.
MYCOBACTERIA AND OTHER INFECTIONS IN HIV-INFECTED PERSONS
This Page Intentionally Left Blank
Clarithromycin 500 mgBID as Prophylaxis for MAC Disease-A Follow-up Review John T. Sinnott, A. Holt, Sally H. Houston, Gary Bergen, Pamela Sakalosky, JulieA. Larkin, and Richard Oehler University of South Florida College of Medicine Tampa, Florida
BACKGROUND In the last several years, the as prevalence of Mycobacterium avium(MAC) infection amongAIDS patients has increased, antiretroviral and prophylactic therapies have been introduced to delay the onset of AIDS-defining events andto prolong survival (1). Clarithromycin is a macrolide antimicrobial agent which is highly potent against a varietyof aerobic and anaerobic gram-positive and gramnegative organisms with activity equal to or greater than that of erythromycin for most strains tested.It has also demonstrated antimicrobial activity against MAC in vitro [minimal inhibitory concentration (MIC) = 0.254 pg/ml] and in vivo. Clarithromycin has been shownto effectively inhibit the intracellular replication of MAC within human macrophages (2), as well as to decrease and eradicate MAC from the blood of AIDS patients (3Y4).
Clarithromycin is approved for treatment and for prevention of disseminated MAC(DMAC).SuccessfulprophylaxisofDMACinfection 539
tY
a
-3
m
&
m
E
d
8
a
cl
m m
a 4
Sinnott et al.
542
with clarithromycin therapy could have a significant impacton the morbidity and mortalityof AIDS patients.
STUDY OBJECTIVE The objective was to evaluate retrospectivelythe efficacy of clarithromycin in preventing the onset of disseminated MAC infection in patients with
METHODS Study Design Retrospective studyof patients with AIDS Clarithromycin mg bid
Evaluation Parameters Patient demographicdata Baseline and during prophylaxis CD, counts Number of MAC infection-free days Occurrence of adverse events
RESULTS The mean CD, countat or prior to initiation of clarithromycin was cells/mm3(N= (range During clarithromycin prophylaxis, one patient received concurrent clofazamine, three received concurrent ethambutol, and two received concurrent rifabutin. The mean duraton on clarithromycin (as of September was days (range days), with no symptomatic evidence or blood culture evidenceof DMAC infection. No untoward side effects werenoted. Although diarrhea was present in several patients before therapy had begun and developed on therapy in others, it did not warrant drug discontinuation. Eleven patients died of AIDS-related complications and all were MAC-free at timeof death, with a mean of days of prophylaxis (range: days). Six patients continue to receive clarithromycin prophylaxis witha current mean CD, count of cells/mm3 (down froma mean of cells/mm3) and remain MAC-free for almostyears.
Clarithromycin as Prophylaxis for MAC Disease
543
CONCLUSION Clarithromycin is of benefit in preventing DMAC infections and is well tolerated in persons with AIDS. Clarithromycin at a doseof 500 mg twice daily appears to be efficacious in preventing disseminated MAC infections and shouldbe considered a good potential agent for MAC prophylaxis.
REFERENCES 1. Horsburgh Jr CR. Mycobacterium avium complex infection in the acquired immunodeficiency syndrome.N Engl J Med 1991; 324:1332-1338. 2. Gikas A, Perrone C, Truffot C, et al. Inhibition of Mycobacterium aviumInintracellulare (MAIC) by antimicrobialsinhumanmacrophages.29th terscience Conferenceon Antimicrobial Agents and Chemotherapy, Houston, 1989; abstr 885A. 3. Dautzenberg B, Legris S, Truffot C, Grosset J. Double-blind studyof efficacy of clarithromycin versus placebo in Mycobacterium avium-intracellulareinfection in AIDS patients. J AmThorac SOC 1990; 141:S615. 4. Data on file. Abbott Laboratories, Abbott Park, IL, 1992.
Clarithromycin Prophylaxis for Disseminated Mycobacterium avium Infection
,
T. M. File, Jr., D. C. Claypoole, S. J. Longstreth, D. J. Signs, W.H.Ruby, and A. S. Indorf Summa Health System Akron, Ohio
INTRODUCTION Disseminated Mycobacterium aviumcomplex (DMAC) is a common AIDSrelated opportunistic infection and increases morbidity and mortality in AIDS patients. One prospective studyof over lo00 patients with advanced HIV infection revealed a40% risk of becoming bacteremic withMycobacterium avium complex after 2 years (1).Previous trials have demonstrated that rifabutin (300 mg PO daily) can reducethe incidence of DMAC infection by approximately 50% (2). The effect on survivalwas less clear, however. Clarithromycin has been shown to be effective in vitro against the Mycobacterium avium complex A recent study by Pierce et al. of 682 patients treated in the United States or Europe to evaluate the efficacy of clarithromycin demonstrated a statistically significant reduction in DMAC as well as a survival benefit in patients treated with clarithromycin versus placebo (4). Further clinical experience in the community will be helpful further in evaluating the utility of clarithromycin. The aim of this study wasto evalu544
Clarithromycin Prophylaxis
DMAC
545
ate the efficacy and safety of clarithromycin as prevention for DMAC in HIV-infected patients in a community setting.
PATIENTS AND METHODS Charts of patients followed at an HIV unit associated with a teaching community hospital were retrospectively reviewed. All patients who received clarithromycin 500 mg bid for MAC prevention were included (including two patients who also received rifabutin at some time concomitantly with clarithromycin). Blood cultures were not routinely done prior to clarithromycin.
RESULTS Fourteen patients were identified who received clarithromycinfor prophylaxis. Patient characteristics and summary of clarithromycin useare shown in Tables1and 2, respectively. The mean CD4 countat or prior to initiation of clarithromycin was 60 cells/mm3( n = 14; range: 2-262). Elevenpatients had CD4 counts of 4 0 0 cells/mm3. The mean duration of clarithromycin was 340 days (range: 132-720). lhelve patients (86%) remained free of DMAC, including 10 patients who diedof other causes. lho patients developed DMAC (at 135 and 167 days, respectively); both hadlow CD4 at the time of clarithromycin initiation (5 and 23 cells/mm3, respectively). adverse effects secondaryto clarithromycin werereported.
DISCUSSION The United States PublicHealthService/InfectiousDiseaseSociety of America Guidelines currently recommended that prophylaxis for DMAC should be considered for HIV-infected adults and adolescents who have a CD4 count less than 75/p1(5).Current data indicate that clarithromycin is effective for this purpose. We found that 12/14 patients (86%) remained free of DMAC during our evaluation-including 10 who died of other causes. patients developed DMAC(at 135 and 167 days, respectively) after initiation of clarithromycin. Both had low CD4 counts (5 and 23, respectively)andpossiblyhadasymptomaticinfection atthe time of clarithromycin initiation. For this reason, the guidelines recommend that DMAC should be ruled out (by a negative blood culture) before prophylaxisis started. Susceptibility data werenotavailable for either of the isolates fromour two patients who developed DMAC.
File et al.
546 Tabk I
PatientCharacteristics
baseline Agelgender Patient
018
47M
10
KS
Cryptococcus 6oM 4
60
CMV
retinitis, PCP 5
5 m PCP
Concurrent therapyb
TMP-SXT, Anti-Retro, IJW, Flu Anti-Retro, Flu, W - S X T Anti-Retro, TMP-SXT Anti-Retro, ganciclovir, Flu, Clinddprimaquin Flu W-SXT,
Flu TB, m, nephropathy KS, PCP PCP, KS PCP, Thrush
59/M
Candidiasis 4uM
5
10 58
CMV retinitis Thrush
"P-SXT, Flu, Anti-Retro Anti-Retro, pentamidine "P-SXT,
Flu Anti-Retro, Flu, W - S X T , rifabutin TMP-sm, Anti-Retro, Flu TMP-SXT, ganciclovir, Flu Anti-Retro, dapsone, Flu, rifabutin Anti-Retro, TMP-SXT, Flu
'opportunistic infections prior to baseline. Toncurrent therapy while receiving clarithrornycin (Anti-Retro = antiretroviral agents; Flu = fluconazole;TMP-SXT = trimethoprimhlfarnethoxazole).
Clarithromycin Prophylaxis
547
for DMAC
n
0
%
0
0
N
File et al.
548
CONCLUSIONS Clarithromycin was well tolerated in these patients. Clarithromycin appears to be effective in preventing DMAC disease. DMAC was observed two in patients who had advancedHIV at the time of initiation of clarithromycin.
REFERENCES 1. Nightingale SD, Byrd LT, Southern PM, et al. Incidence of Mycobacterium avium-intracellulare complex bacteremia in human immunodeficiency viruspositive patients. J Infect Dis 1992; 1651082-1085. 2. Nightingale S, Cameron DW, Gordin FM, et al. ' b o controlledtrials of rifabutinprophylaxisagainst Mycobacteriumavium complexinfection in AIDS. N Engl J Med 1993; 329:828-833. 3. Fernandes PB, Hardy DAYMcDaniel D, Hanson CW, Swanson RN. In vitro and in vivo activities of Clarithromycin againstMycobacterium avium. Antimicrob Agents Chemother 1989; 33:1531-1534. 4. Pierce M,Crampton S, Henry D, Craft C, Notario G . The effect of MAC and its prevention or survival in patients with advanced HIV infection. 35th Interscience Conferenceon Antimicrobial Agents and Chemotherapy, FranSan cisco, 1995; abstr LB-18. 5. USPHS/IDSA Guidelines for the Prevention of Opportunistic Infections in Persons Infectedwith Human Immunodeficiency Virus. Morbidity and Mortality Weekly Report 1995; 44:No. RR-8.
+
Azithromycin + Pentamidine Pyrimethamine in the Prophylaxisof Opportunistic Infections in IHV-Positive Patients withCD4 Count Less Than 200 G. Barbarini, G. Garavelli,
P. Alcini
IRCCS S. Matteo-University of Pavia Pavia, Italy
G. Barbaro University La Sapienza Roma, Italy
B. Division of Infectious Diseases0 O . R R . Foggia ,Italy
A. Lucchini Ser. T. of Gorgonzola Gorgonzola,Italy
Lopez Ser. T. of Abbiategrasso Abbiategrasso,.Italy
Del Buono Ser. T. of Voghera Voghera, Italy Ed0
Ser. T. of Vigevano Vigevano,Italy
549
550
Barbarini et al.
INTRODUCTION Pneumocystis carinii pneumonia (PCP) and toxoplasmic encephalitis are two of the most common opportunistic infections that define the diagnosis of AIDS. In Italy, PCP occurred in 1983-1989,the asAIDS-defining diagnosis before primary PCP prophylaxis was standardized. in about 65% of all cases, After 1989 toxoplasmic encephalitis became the AIDS-defining diagnosis in about 25% of all cases, and todaythe appropriate prophylaxis against this opportunistic infection is also not standardized. W Odrugs have proved effective in primary prophylaxis against PCP [TMP-SMZ (trimethoprid sulfamethoxazole) and aerosolized pentamidine] and they are extensively used today (1).The combination of pyrimethamine and sulfadiazinerepresents the most effective therapyfor cerebral toxoplasmosis; pyrimethamine plus clindamycin has been also reported as successful (2). The risk of a primary episodeof PCP in HIV-positive patients presenting withthan less200 CD4 cells is about 70% and the risk ofcerebral toxoplasmosis about is 30% if they have antibodies against T . gondii. Obviously, the primary preventionof these opportunistic infections is desirable for high-risk patients. Azithromycin, a new azalide(3) with a long half-life and high and sustained tissue concentration without toxicity, has been shown to concentrate in phagocytic cells (4), which could act as a vector to deliver azithromycin to the site of infection. On the basis of studies in animals, azithromycin was considered a candidate for the prevention of cerebral toxoplasmosis in HIV-positive patients and it was included in many primary prophylaxis protocols (5). It was also demonstrated that azithromycin can reduce rates of Mycobacterium avium complex (MAC) infections (6) by about 50% and it may be efficacious against cryptosporidiosis: (7): These two opportunistic infectionsare common in HIV-positive patients presenting with less than CD4 100 cells.
OBJECTIVE The primary endpointof our work wasto verify if primary prophylaxis with azithromycin plus pyrimethamine plus aerosolized pentamidine may reduce the incidence of PCP and cerebral toxoplasmosis in asymptomatic HIV-infected patients presenting with 100-200 CD4 cells. The secondary endpoint was to prove if long-term therapy with azithromycin is safe and tolerable for patients because there is very limited experience with longterm administration.
SUBJECTS AND METHODS Ninety asymptomatic HIV-positive patients (68 males and 22females) presenting with 100-200 CD4 cells and positive toxoplasma serology wereen-
Azithromycin + Pentamidine Table I
+ Pyrimethamine
H N Patients
551
Prophylaxis Dosage Groups
Group 30 Subjects (22 M and 8 F) Mean age: 31 years (range: 20-39) Mean CD4 cells count: 152 (range: 121-194)
Aerosolized pentamidine:300 mg monthly Pyrimethamine: 50 mg daily orally Azithromycin: 500 mg daily orally Group 2 30 Subjects (23 M and 7 F) Mean age: 28.5 years (range:20-35) Mean CD4 cells count: 139 (range: 111-186)
Aerosolized pentamidine:300 mg monthly Pyrimethamine: 50 mg daily orally Azithromycin: 500 mg every 2 days orally Group 3 30 Subjects (23 M and 7 F) Mean age: 29.3 years (range: 23-37) Mean CD4 cells count: 146 (range: 121-187)
Aerosolized pentamidine:300 mg monthly Pyrimethamine: 50 mg daily orally Azithromycin: 500 mg daily for 3 days, followedby 2 drug-free days rolled from June 1994 to March 1995, in a multicentertrial (Pavia, Roma, Foggia, Gorgonzola, Abbiategrasso, Vigevano, and Voghera). All subjects received 50 mg pyrimethamine orally dailyand 300 mg aerosolized pentamidine monthly. Azithromycin was administered orally at three different dosages: (1) mg daily (30 subjects), (2) 500 mg every 2 days (30 subjects), and 500 mg daily for days followed by two drug-free days (Table 1). Thepatients were followedeither to termination of the study (6 months) on 30 September 1995,or longer.
RESULTS All enrolled patients completed at least 6 months of therapy; 25 were treated for 1year and8 for morethan 1year. Therapy was well tolerated in Groups 2 and In Group 1, no patient was able to tolerate the daily administration of azithromycin longer than months, at which time they reverted to thedosage used in Group 2. With this change,the drug was well
Barbarini et al.
552
tolerated and no further stoppage for gastric intolerance was reported. No clinically significant changes in liver function testsor hematology parameters were observed as a consequenceof azithromycin plus pyrimethamine treatment. We did not observe any PCP, MAC, or toxoplasma infection; among treated patients we diagnosed one CMV infection (Group 2), one Kaposi’s sarcoma (Group 2), one esophageal candidiasis (Group 2), and one lymphoma (Group
DISCUSSION Our preliminaryexperiencewith pyrimethamine-azithromycin-pentamidine prophylaxis suggeststhat it is well tolerated if azithromycin is not administered daily and may effectively prevent for 6 months the development of PCP; cerebral toxoplasmosis, and MAC infection in asymptomatic HIV-positive patients with 100-200 CD4 cells. Because longer controlled trials are warranted to establish the definitive efficacy of this prophylaxis, starting fromJune 1995we are conducting a controlled trial in asymptomatic HIV-positive subjectswith 100-200 CD4 cells with pyrimethamine, azithromycin, and pentamidine, and a controlled trial on HIV-positive subjects with >l00 CD4 cells with pyrimethamine, azithromycin, pentamidine, plus rifabutin.
REFERENCES 1. Centers forDiseasesControl.Recommendationsforprophylaxisagainst Pneumocystis carinii pneumonia for adults and adolescents infected with human immunodeficiency virus.MMWR 1992; 41:l-ll. 2. Danneman B, McCutchan JA, Israelski D, et al. Treatment of Toxoplasmic encephalitis in patients with AIDS. A randomized trial comparing pyrimethamine plus clindamycin to pyrimethamine plus sulphadiazine. Ann Inter Med 1992; 116:33-43. 3. Girard AE, Girard D, Englisha R, et al. Pharmacokinetic and in vivo studies with azithromycin, a new macrolide with an extended half life and excellent tissue distribution. 4. Gladue RP, Snider ME. Intracellular accumulation of azithromycin by cultured human fibroblasts. Antimicrob Agents Chemother 1990; 34:1056-1060. 5. Chang HR. Thepotential roleof azithromycin in the treatment on prophylaxis of Toxoplasmosis. Intern J STD AIDS 1996; 7 (suppl1):18-22. of 6. Inderlied CB, KolonoskiPT, Wu M, Young LS. In vitro and in vivo activity azithromycin against the Mycobacterium avium Complex. J Infect Dis 1989; 159:994-997. Intern J STD AIDS 1996; 7 (suppl 7. Hoepelman IM. Human cryptosporidiosis. 1):28-33.
Ix SPECIAL PATHOGENS
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Human and Animal Bite Wounds: Bacteriology, Macrolide Susceptibility, and Therapeutic Potential Ellie J. C. Goldstein and Diane M. Citron Santa Monica-UCLA Medical Center and theUCLA School of Medicine Los Angeles, California
INTRODUCTION The bacteriology of infected bite wounds is diverse and includes aerobic and anaerobic organisms from veterinary, environmental, and skin sources (1-6). Human and animal bites can result in infectious complications including cellulitis, septic arthritis, and osteomyelitis (1,5). Annually they account for approximately 1% of emergency department visits and numerous physician office visits and hospital admissions, often as a result of infection and related problems. Streptococci, Staphylococcus aureus, Staphylococcus intermedius, Pasteurella multocida, Capnocytophaga canimorsus, and oral anaerobes of the Prevotella and Porphyromonas species are frequently isolated from animal bite wounds (Fig. Streptococci, S. aureus, Haemophilus influenzae, Eikenella corrodens, and oral anaerobes are often isolated from human bite wounds. Empirical treatment of infected human and animal bite wounds should include broad coverage against these common pathogens. Erythromycin has been used in the penicillin-allergic patient as an alternative therapy, but it has relatively poor activity against certain bite 555
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Pasteurella Streptococci
SIOphylococcus Fusobacterium Bacteroides Porphyromanas Prevotella Peptostreptococci Enterococci Klebs~e//a/En~r Others
Figure I Approximate frequency of various bacteria growing in infected cat and dog bite wounds. (Adapted fromRef. 1.)
would isolates such as P . multocida, E. corrodens, and many oral anaerobes (6). Previous studies of the activity of the macrolides clarithromycin androxithromycinand the azalideazithromycinagainstcommonbite wound isolates have been reported (2-4). This study reviews and assesses their comparative therapeutic potential and effectiveness as alternatives to erythromycin.
MATERIALS AND METHODS Bacterial strains were isolated from wounds causedby the bites of dogs, cats,andhumans,as wellas other animalsandsaved at -70°C’ for various periods of time in the R. M. Alden Research Laboratory culture collection. Isolates were taken from frozen stock and transferred twice to assurepurityandadequacy of growth.Agardilutiontestingwas performedaccording to NCCLSguidelines;Brucellabloodagarwasused for mostisolates.Antimicrobialagents,azithromycin,clarithromycin, erythromycin, and roxithromycin, were suppliedby the respective manufacturers and reconstituted according to the manufacturers’ instructions into serial twofold dilutions. The plates were inoculated using a Steers replicator and incubated for 24-48 h in the appropriate atmosphere. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of an antibiotic that yielded no growth, a barely visible haze, or one discrete colony.
Bite Wounds: Bacteriology, MacrolideSusceptibility,Therapeutics
557
Table l Activities of Erythromycin, Clarithromycin, Roxithromycin, and Azithromycin Against Clinical Bite-Wound Isolates(MI%, pglml) Species (No. of isolates) ERYTH CLARa
ROXb
2.w
Streptococcur and Enterococcus spp. Staphylococcus aureus Coagulase-negativeStaphylococcus spp. Pasteurella spp. EF-4 Eikenella corrodens Miscellaneous gram-negative bacilli Peptostreptococcus spp. Fusobacterium spp. Prevotella, Porphyromonas, and Bacteroides
AZITH
NIAd 0.5
NIA
NIA NIA
NIA NIA 0.25
“IC,values against Streptococcus spp. (23),S. aureus (13), P. multocidu (16), EF-4 (13), E. corrodens (16), Peptostreptococcus spp. (12), Fusobacterium spp. (20), and Prevotella and Porphyrornonas spp. (29). (From Ref. 3.) bMIC, valuesagainst S. aureus (17), P . multocidu (22), E. corrodens (12), miscellaneous gram-negative bacilli (U), Fusobacterium spp. (H), and nonpigmented (18) and pigmented (16) Bacteroides. (From Ref. 4.) =Does notinclude Enterococcus spp. dNot available. =IncludesEF-4 (7), Actinobacillus actinomycetemcomituns (U), and Haemophilus spp. (3). Source: Adapted from Refs. 2-4.
RESULTS The results of the susceptibility studies are shown in Table 1 as MIC, values. Erythromycin demonstrated limited activity againstP . multocida, E . corrodens, Peptostreptococcus, and Fusobacterium species and limited activity against staphylococci. Clarithromycin more was active than erythromycin against P . multocida (MC, 52.0 and E . corrodens, as well as Prevotella, Porphyromonas,and Bacteroides species, but showed poor activity against Peptostreptococcus and Fusobacterium species. Roxithromycin was less active than erythromycin against all species on a weight basis. Azithromycin was more active against P. multocida ( M I C 4 2 . 0 ml), E . corrodens, and species of Prevotella, Porphyromonas, and Bacteroides and was 4-16-fold more active than erythromycin against Peptostreptococcus and Fusobacterium species.
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DISCUSSION Whereas p-lactam agents remain attractive for the primary therapy of human and animal bite wounds, our in vitro data suggest that the new azalide, azithromycin, has improved in vitro activity comparedto erythromycin against a broad spectrum of bite wound pathogens. Azithromycin and clarithromycin have similar activities against P. multocida, E . corPrevotella and rodens, and EF-4. Althoughbothwereactiveagainst Porphyromonas species, clarithromycin showed a lower MIC, (0.25 pg/ml versus 1.0 pg/ml). Azithromycin, but not clarithromycin, had improved activity againstPeptostreptococcus species andFusobacterium species. Azithromycin appearsto merit further clinical evaluation in the treatment of bite wounds.
REFERENCES 1. Goldstein EJC, Citron DM, NesbitCA, Talan DA, the Emergency Medicine Animal Bite Infection Study Group. Prevalence and characterization of anaerobic pathogens from 50 patients with infectedcat and dog bites. Proceedings of the Society for Anaerobic Microbiology (British), 1995. (in press). 2. Goldstein EJC, Citron DM, Vagvolgyi AE, Finegold SM. Susceptibility of bite wound bacteria to seven oral agents including RU-985, a new erythromycin: Considerations in choosing empiric therapy. AntimicrobAgents Chemother 1986; 29556. 3. Goldstein EJC, Nesbit CA, Citron DM. Comparative in vitro activities of azithromycin, Bay y 3118,levofloxacin,sparfloxacinand 11 other oral antimicrobial agents against 194 aerobic and anaerobic bite wound isolates. Antimicrob Agents Chemother1995; 39:1097. 4. Goldstein EJC, Citron DM. Comparative susceptibilitiesof 173 aerobic and anaerobic bite wound isolates to sparfloxacin, temafloxacin, clarithromycin, and older agents. Antimicrob AgentsChemother 1993; 37:1150. 5 . Goldstein EJC. Bite wounds and infection. Clin InfectDis 1992; 14:633. 6. Goldstein EJC, Citron DM, Richwald GA. Lack of in vitro efficacy of oral forms of certain cephalosporins,erythromycinandoxacillinagainst Pasteurella multocida. Antimicrob AgentsChemother 1988; 32:213.
Azithromycin in the Treatmentof Pertussis in Children: A Pilot Study A. Bate and N. Kuzmanovit University Hospitalof Infectious Diseases "Dr. Fran MihaljeviC" Zagreb, Croatia
T.ZmiC PLNA d.d. Pharmaceuticals Division Zagreb, Croatia
INTRODUCTION National epidemics of pertussis have been controlled in Croatia for over three decades (since 1958) by the universal immunization of infants and children. The vaccination program for pertussis used in 1991 and 1992 resulted in 87.4% and 86.7% coverage, respectively (1). Because of the presence of unvaccinated and incompletely immunized children as well as infants up to months, outbreaks and regional epidemics continue to occur. Thus, pertussis is still a noticeable disease in Croatia with 310 cases reported to the Epidemiologic Service, Croatian National Institute of PublicHealthin1994(2).Erythromyciniscurrentlyrecommendedin the treatment of pertussis.Although a 14-dayerythromycincourseis very effective, the relatively low tolerability and inconvenient dosing of erythromycin prompt further efforts to improve antibiotictreatment of pertussis. 559
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The pilot study was conducted to assess efficacy and safety of azithromycin inthe treatment of pertussis in children.
PATIENTS AND METHODS
Fifteen children of both sexes aged months with symptoms and signs consistentwithpertussis-whoopingcough,presenceofleukocytosis (> with lymphocytosis-were included in the study. Clinical diagnosis of pertussis was confirmed by presence Bordetellapertussis of in culture from nasopharyngeal or pharyngeal swabsor nasal suction aspirates. Only children whose parents provided an informed consent were enrolled. Patients with hypersensitivity to macrolides, severe renal or hepatic impairment, or gastrointestinal tract disturbances which could affect drug absorption were excluded, as were patients who received any antibiotic within h prior to entering the study. Azithromycin (Surnamed, Pliva)was administered as anoral suspension once daily for5 days: mgkg on day followed by 5 mgkg on days Azithromycin was taken h before or 2 h after a meal. All patients were hospitalized. Clinical responseto therapy was assessed at days and after the start of therapy. It was classified as a success (completeor partial disappearance of all baseline signs and symptoms of infection) or failure (no change or worsening of initial condition). Bacteriological examinations were performedat baseline andat days and after the start of therapy. Samples for bacteriological culture were obtained with nasopharyngeal swabs, pharyngeal swabs, and nasal suction aspirates. All specimenswereculturedonRegan-Lowemedium.Bacteriologicalfindings were classified as elimination, elimination with relapse, or persistence of pathogen. All adverse eventsthat occurred duringtreatment were recordedand followed up. Adverse events were scored for severity, duration, and relationship to treatment. Laboratory safety tests (hematology, blood biochemistry, and urinalysis) were undertaken at baseline and at days and after the initiation of treatment.
RESULTS Fifteen hospitalized children, boys and girls, aged months (mean age: months) were included in the study. All children had acute an form of pertussis. B . pertussis was isolated in of patients included inthe study. In all of them, at least one specimen was positive (Table Clinical response to azithromycin therapy is presented in Table
Azithromycin for Children Pertussis in
561
Table Z Baseline Bacteriological Findings in Children with Pertussis
Specimen No. of patients ~~
Nasopharyngeal swab
Nasal suction aspirate
Pharyngeal swab
~
-a
positive specimen positive specimens positive specimens
+ + + +
Total ~~
~
'Absence of B. pertussis in culture. bPresence of B. pertussis in culture. positive In examined specimens.
Eight children missed the final checkup visit (at day 21), probably because they were free of symptoms. The results of the bacteriological findings are presented in Table Seven days after initiation of azithromycin therapy, all bacteriological cultures(taken from nasopharynx,pharynx, and nasal suction aspirate) were negative. At the end of the follow-up period, no relapses or reinfections were observed. Tolerance of azithromycin was very good. side effects were recorded. Laboratory parameTable 2 Clinical Response to Azithromycin in the Treatment of Children with Pertussis
No. of patients Pretreatment Symptoms General Condition Good Moderately altered Cough Weak Moderate Intensive Whooping Cyanosis Vomiting
8
Day 7 (n=15)
Day
Day (n=7)
5
6 8
9
9
6 9 7
2
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Table 3 Eradication of B. pertussis in ChildrenTreated with Azithromycin
No. of patients Positive culture for B. pertussis Nasopharyngeal swab Pharyngeal swab Nasal suctionaspirate
Pretreatment (n=15)
Day 7 (n=15)
Day 14 (n=14)
13 3 8
0
0
Day 21 (n=7)
ters were within reference limits with the exception of a slight and transient increase in liver enzymes in6 children.
DISCUSSION Azithromycin showed excellent bacteriological efficacy with eradication rate of B . pertussis within 7 days after the start of therapy. Assuming the natural course of pertussis, the reported clinical results could be also considered as successful. The drug was well tolerated. Although the incidence of liver function abnormalitieswas higher than usuallyreported for azithromycin, a causal relation to azithromycin is uncertain, as the majority of patients (10 of 15) already had slightly elevated baseline ALT values. According to our experience, a slight increase of liver enzymes is not uncommon in children with pertussistreated with erythromycin.
CONCLUSION Related to highefficacy,goodtolerability,and short dosageregimen, azithromycin could be a promising agent in the treatment of pertussis in children.However, the observedresultsshouldbeverifiedinalarger comparative study.
REFERENCES 1. Bejuk D, Begovac BaCe A, KuzmanoviC N, Aleraj B. Culture of Bordefeffa pertussis from three upper respiratory tract specimens. Pediatr Infect Dis 1995; 14 (l):@-65. 2. BorEiC B, DobrovSak-Sourek V. The impact of compulsory vaccination against some diseases on their incidence in Croatia. In: Hajsig, D, ed. Vaccinology Today and Tomorrow. Zagreb: Croatian Society for Microbiology, 19954246.
The Useof New Macrolides in Experimental Brucella melitensisInfection R. Lang,D. Torten, B. Shasha, and Ethan Rubinstein Sheba Medical Center and Meir Hospital, Sackler Schoolof Medicine Tel-Aviv,Israel
INTRODUCTION The most effective and least toxic therapy for brucellosis remains an unsettled issue.The frequently used combinations of tetracycline and streptomycin or tetracycline with rifampin usually achieve excellent results. Nevertheless, a variable percentage of failures and recurrencesare reported (relapse rates 8-12%). The new azalides present an opportunity for single-drug therapy for brucellosis, with hopes of shortening the duration of therapy and diminishingthe number of antibiotics administered.
MATERIALS AND METHODS The Brucella melitensis standard smooth strain 16M was used. The strain was inoculated into nonstudy mice to confirm viability and pathogenicity and purify the strain. The organism was cultured brucella agar (Difco) at with 5% CO, to the logarithmic growth phase and stored at until use.
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Susceptibility Testing MICs and MBCs Minimal inhibitory concentrations (MICs) were tested both with the broth microdilution method in microtiter trays and with the agar dilution method usingbrucellaagarcontaininggradedconcentrations of antibiotics at inocula of 105 CFU (colony-forming units) per milliliter. Agarplate results were used to finalize MICs. Minimal bactericidal concentrations (MBCs) were determinedby plating on brucella agar MIC values plus samples from two higher concentrationwells.
Animals Adult white male ICR miceaveraging 20-40 g were used. Mice were housed according to therapeutic groups and were provided with food and water adlibitum.
Inoculation The B . melitensis smooth strain 16M was grown in brucella broth for 4872 h to the logarithmic growth phase. The culture was adjusted (on the basis of viable counts) to yield 4 x 10 to 8 x lo8 CFU/ml. Inoculation was performed by injecting 0.5m1 of culture containing2x105: to 4X lo5organisms in saline, intraperitoneally(IP). Mice were then randomly assignedto treatment and control groups.
Therapeutic Groups Followinganincubationperiod of 7days,animalswereadministered azithromycin clarithromycin (50 or 75 mgkg per day) for 7 or 14 days. Both were administered IP in one (azithromycin) or two divided (clarithromycin) daily doses. Treatment was started 1 week after inoculation. Animals were randomizedto be studied for primary cure or relapse, as well as for control groups.
Primary Cure Mice were sacrificed 7 days after the last antibiotic dose to assure clearance of the drugs which might cause false-negative culture results. Therapy was continued for 7or 14 days.
Relapse Group Azithromycin, 50 mgkg per day, was givenfor 1 or 2 weeks, and75 mgkg per day for 2 weeksto 12,15, and 14 animals, respectively. Clarithromycin,
Macrolides melitensis in B.
Znfection
565
120%
mcm
S
80%
c
60%
LOG
.y
20%
0% Figure I
+ LOG cm 4.46
Cllarithrom 50 mgKgl
Cure rate 7 days following completion of a 7-day course of therapy.
50 mgkg per day, was givenfor 1 and 2 weeks and 75 mgkg per day for 2 weeks to 14 animals in each group.
Assays At the conclusion of the scheduled therapy period, treated and control mice were weighed and sacrificed under ether anesthesia, and their spleens were aseptically removed, weighed, and homogenized in 1.0 m1of sterile saline. The homogenates (0.1 ml) were diluted in saline at decimal dilutions and plated brucella agar. Plates were incubated at with 5% CO, for 48-72 h and B. melitensis colonies were counted. Each procedure was performed in triplicate andthe results averaged and expressed as a decimal logarithm.
Data Evaluation Cure was defined assterilization of spleens and reductionof the log CFU of Brucellae cultured fromthe nonsterile homogenized spleens.
Lang et al.
566
STATISTICAL ANALYSIS The cure rate was evaluated statistically by use of the chi-square test. A comparativeanalysis of meanlog CFU was performedbetweenmice treated with the different regimens andnontreated mice.
RESULTS Therapeutic Outcome by Groups Primary Cure: One of 26 untreated animals sacrificed parallel to thethera-
peuticgroupswascured(cure rate: 3.8%). All animals treated with azithromycin 50 mgkg per day, for 1week or for 2 weeks (15 in each group) were cured. This represents a100%cure rate (Figs. 1 and 2). Following 1 week of treatment with clarithromycin 50 mgkg per day, 10of 15 animals were cured (66.6% curerate), and following 2 weeksof the same regimen, 11 of 13 mice were cured (cure rate: 84.6%). The log CFU of Brucellae
120% LQGCFU
100%
k E
80%
60% 40%
20%
0% 50 mg/Kg/day
50 mg/Kg/day
Figure 2 Cure rate 7 days following completion of a 14-day course of therapy.
Macrolides in B. melitensis Infection
567
120%
100%
S
80%
W
.c,
60%
a 40%
20%
0% SO mg/Kg/;lay
50 mg/Kg/day
Figure 3 Cure rate 4 weeks following completion of a 7-day course of therapy.
isolated fromthe spleen, averaged 3.24 for animalstreated for 1 week and for animals treated for 2 weeks. Relapse groups: Tivo of nine (22.2%) control animals were cured. In the azithromycin 50-mgkg per day group, 11 of 12 animals sacrificed 4 weeks after completion 7 daysof therapy were cured(91.6% cure rate) and 14 of 15 animals treated by the same dose for 14 days were cured (cure rate: 93.3%). The mean log CFUs of infected spleens were1.6 and 2.3, respectively. In the azithromycin 75-mgkg per day group 13 of 14 animals were cured at the relapse end point (92.9%), and the log CFU of the single infected animalwas 1.6. (See Figs. and 4.) In the clarithromycin 50-mgkg per day l-week therapeutic group, 4 of 14 had sterile spleens (28.6% cure rate). The mean log CFU of the infected animalswas 3.48. In the clarithromycin 50-mg/kgper day 2-week therapeutic group, all14 animals were infected4 weeks after completion of therapy (0% cure rate). The mean log CFU was 3.56. Clarithromycin 75 mgkg per dayfor 14 daysresulted in no cure (100% infection)when animals were sacrificed 4 weeks after terminationof therapy. The mean log CFU was 3.46.
h n g et al.
568
120% LOG
100%
8.
80%
W
E
60%
40%
20%
0% AzithromycinClarithromycin
Figure 4 Cure rate 4 weeks following completion of a 14-day course of therapy.
DISCUSSION AND SUMMARY The nearly 100%efficacy of azithromycin 50 mgkg per day in the primary cure of brucellosis following1and 2 weeks of therapy, demonstratedin the present experiment, has not yet been reported with any single antibiotic agent. This therapeutic effect was maintained for 1month and prevented relapses, an effect hitherto undescribed. In view of these results and the therapeutic failures with other macrolides and fluoroquinolones, plusthe drug’s known safety and tolerance profiles,we suggest a possible role for azithromycin in the treatment of human brucellosis with special emphasis on the potentialmanagement of brucellosisininfantsandpregnant women.
X NONANTIBACTERIAL EFFECTS
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In Vivo Effectof Azithromycin Subinhibitory Concentrations on the Mortality of Experimental Pseudomonas aeruginosa Sepsis M. N. Marangos, M. E. Klepser, D. P. Nicolau, Charles H. Nightingale, and Richard Quintiliani Hartford Hospital Hartford, Connecticut
INTRODUCTION Pseudomonas aeruginosa remains one of the most prevalent and clinically significant pathogens involved in nosocomial infections. Owing to its synthesis of extracellular products (e.g., exotoxin A, proteases, cytotoxins, hemolysins), P . aeruginosa has the ability to colonize, break down physical barriers, impair host defenses, and damagethe host. Previous in vitro and in vivo studies have shown that different antibiotics inhibitthe expression of the exoenzymes of P . aeruginosa Molinari et al. have demonstratedthat macrolideshzalides as a class share the potential to downregulate the expression of these virulence factors in vitro at sub-MICs(minimalinhibitoryconcentrations). Of thisclass,azithromycin,anazalide,exhibited the highestinhibitoryeffectonenzyme expression (3,4). 571
Murungos et al.
572
The aim of this study was to investigate the effect of azithromycin sub-MICs on mortality in an experimental P . ueruginosu sepsis model.
MATERIALS AND METHODS Antibiotics The following antibiotics were used: azithromycin (&er Inc., New York, and ceftazidime (Eli Lilly& Company, Indianapolis, IN).
Bacteria Ten clinicalisolates of P.ueruginosu acquired atour institution were evaluated for the production of exoenzymes.
Susceptibility Testing The MIC and MBC (minimal bactericidal concentration) were determined for eachantimicrobial by the microdilutiontechniqueusingcationsupplemented Mueller-Hinton (MH) broth (Difco Laboratories, Detroit, MI).
Determination of Exoenzyme Production Enzyme activities and pyocyanin production were qualitatively evaluated employing separate agar plates by spotting a 10-p1 inoculum from broth cultures. Lecithinase production was tested by adding 10% (v/v) egg yolk enrichment to tripticase soy agar plates (Difco). A white precipitate around or beneath the inoculum spot indicated lecithinase activity. Elastase activity was measured on nutrient agar plates containing 1%elastin. Plates were incubated at 37°C for 48 h and then transferredto room temperature for 3 additional days. Clearingof the opaque medium aroundthe inoculum spot was taken to indicate elastase activity. DNAse production was detected on DNAse plates. After 72 h of incubation at 37"C, the plates were flooded with 1N HCl and a clear zone around the growth area manifested DNAse activity. Hemolysins were detected after 72 hof growth at room temperature on MH agar plates containing5% sheep red cells. Gelatinase production was determined on MH agar plates containing 0.4% gelatin. After incubation, the plates were flooded withsaturated ammonium sulfate and examined for zones of clearingaround the inoculum.Pyocyaninand mucoid production were evaluatedby examining the color and consistency of the colonies on MH agar plates after 48 h of incubation.
Effect of Azithromycin on Mortality of P. aeruginosa
573
Animals Swiss Webster mice (20-25 g) were obtained and caredfor according to the guidelines providedby the U.S. Department of Health andHuman Services.
Determination of the Minimum Lethal Dose Groups of15-30mice were infected intraperitoneally with a range of inocula in a 3% mucin solution. A total of 0.5 m1of organism suspension was injected and the minimum lethal dose (MLD) was the dose that resulted in 100% mortality within36 h of inoculation.
Determination of the Protective Dose One hour followingthe intraperitoneal inoculationof P . aeruginosa at the MLD, groups of 10 mice were injected with 0.2 m1 of ceftazidime subcutaneously (SC) in various dosing regimens. The dosing regimen that produced a survival rate of 25-40% on day 6 following inoculation wasto be selected asthe regimen in the comparative trial. Ceftazidimeat 375 mgkg SC 2 dosesat 4-h intervals protected25% of the animals fromthe MLD.
Comparative Survival Study inoculum of lo7 CFU (colony-forming units) in a 3% mucin solution was determined to be the MLD and was injected intraperitoneally. Onehour postinoculation,mice were randomizedto receive ceftazidime(CFZ, 375 mgkg SC 2 doses) aloneor in combination withAZ (20 mgkg SC 1dose). llvo other groups received either AZ alone or no treatment (control). All mice were observed, and mortality was recorded every 12 h over a 72-h period. In addition, another group of 24 mice received a single 2O-mgkg SC dose of AZ todetermine the pharmacokinetic profileof the agent. Animals were sacrificed at times between 0.25 and 6 h postdose. Serum concentrations were determined using a validated high-performance liquid chromatographic assay.
RESULTS One of the 10 isolatesthat displayed activity of all enzymes was selected for the in vivo study.The MIC/MBC values for this nonmucoid isolateto AZ and CFZ were 32/64 and 4/4 pg/ml, respectively. As shown in Table1, all animals in the untreated control and AZ alone groups died within 24 hof the intraperitoneal inoculation with P . aeruginosa. However, as displayed in Fig. 1, a significant(p < .01 usingthe logrank test) decrease in mortality
Marangos et al.
574
Tabk l Percent Survival After Treatment with CFZAlone, AZ Alone, AZ, or No Treatment (Control)
+
Timea (h)
60 Control AZ Alone CFZ Alone + AZ
0
0 0
'Postinoculation.
rate occurred during the first h postinoculation in animalstreated with CFZ plus AZ compared to CFZ alone. This beneficial effectof AZ treatment wasno longer evident atthe 48-h observation period. Serum concentrations of AZ obtained from animals receiving the single 20-mgkg SC dose revealed a mean peak concentration of 1.47 pg/ml at 0.25 h postdose and a mean concentration of 0.14 &m1 at 6 h postdose. Thus, serum concentrationsof AZ were well belowthe MIC (32 pg/ml)of the test organism.
I
0
12
24
36
48
60
72
T h e (hours)
Figure l Percentage survival after treatment with CFZ alone and in combination with AZ.
Effect
Mortality of P. aeruginosa
Azithromycin on
575
DISCUSSION When given alone in the P . aeruginosa peritonitis-sepsis model, azithromycin does not improve survival overuntreated controls and providesno measurable antibacterial effect. However, when azithromycin is given at sub-MICs with ceftazidime, there appears to be a significant reduction in mortality rate compared with animalstreated with ceftazidime only. These observations suggest that the suppression of pseudomonal exoenzymes by azithromycin, as reported by Molinari et al., might be responsible for the improvement in survival when the animals are treated with the combination The protective effect shownby azithromycin also might berelated to factors other than exoenzyme. suppression (e.g., the host's defense tem). Further studies are required to clarify the protective mechanisms. .
CONCLUSION
Our data indicate a potential adjunctive role for azithromycinthe in treatment of P. aeruginosa infections despite a lack of specific activity for this pathogen. Our observations underscore the complexity of the interactions among the organism, sub-MICs .of azithromycin, and the need for additional studyto elucidate the mechanism responsiblefor these observations.
REFERENCES 1. Grimwood K, To M, Rabin HR, Woods, DE. Inhibition of Pseudomonas aeruginosa exoenzyme expression by subinhibitory antibiotic concentrations. Antimicrob Agents Chemother 1989; 33:41-47. 2. Kita E, Sawaki M, Oku D, Hamuro-'A, Mikasa K, Konishi M, Emoto M, Takeuchi S, Narita N, Kashiba S. Suppression of virulence factors of Pseudomonas&eruginosa by erythromycin. J. AntimicrobChemother1991; 27: 273-284. 3. Molinari G, Guznlan' A, Pesce A, Slchito GC. Inhibition of Pseudomonas aeruginosa virulence factors by subinhibitory concentrationsof azithromycin and other macrolide antibiotics. J Antimicrob Chemother 1993; 31:681-688. 4. Molinari G, Paglia P, SchitoGC.Inhibition of motility of Pseudomonas aeruginosa and Proteus mirabilis by subinhibitory concentrations of azithromycin. Eur Clin Microbiol Infect Dis 1992; 11:469-71.
Clarithromycin Reduces Cl-Dependent Transepithelial Potential Difference in Tracheal Mucosa of Anesthetized Rabbits J. Tamaoki, H. Takemura, E. Tagaya, Y. Takeda, K. Konno Tokyo Women’sMedical College Tokyo, Japan
INTRODUCTION Long-term administrationof eiythromycin is effective inthe treatment of diffusepanbronchiolitisprobablythroughactions other than its antimicrobial properties (1). Although the mechanism of the effect is uncertain, immunomodulatoryactiononinflammatorycells,inhibition of mucus secretion and inhibition of airway epithelial Cl transport and subsequentwatersecretion havebeenproposed.However, the effect ofnewlydevelopedmacrolides on Clsecretionisunknown and, previous in vitro findings do not necessarily reflect ion transport function in vivo because of the lack of innervation and blood supply. Therefore, to determine whether the newmacrolideinhibitsClsecretionin vivo,we studied the effect of clarithromycin(CAM) on Cldiffusion potential difference (PD) (4) across the tracheal mucosa in anesthetized rabbits; 576
Reduction
Cl-PD in Tracheal Muscosa
with Clarithromycin
577
MATERIALS AND METHODS Measurement of PD of Tracheal Mucosa Male Japanese white rabbits were anesthetized, and a polyethylene tube was canulated 5 mm above the carina, through which artifical ventilation was performed. Cartilage rings of the upper trachea were incised transaxially and the surface of the membranous portion was fully exposed. The exploring bridge was placed the on surface of the tracheal mucosa(5), and contactwith the tracheal surface was ensured by continuous perfusion (0.3 mumin) through the bridge with Krebs-Henseleit (KH) solution. The perfusion reservoir was connected to a calomel half-cell via a polyethylene tube filled with KH solution in agar. The reference bridge, a 21-gauge needle that contained KH solution in agar, was inserted intothe subcutaneous space of the right anterior chest wall. Each bridge was connected by a calomel halfcell to a high-impedance voltmeter, and the electrical signal (transmembrane PD) was monitored.
Effect of CAM on Cl-PD
To measure Cl-PD of the tracheal mucosa, superfusing KH solution was switched to one that contained amiloride (10-4M), a Na channel blocker. This maneuver decreasesPD, and the remaining PD (Cl-PD) is an index of epithelial cellular and paracellular paths available for Cl diffusion. When C1PD became stable, the amiloride-containing solution was changed to a similar solution that contained CAM and 10-4M), or CAM (10 m a g ) was administered throughthe jugular veinby a bolus injection. To determine the dose-response relation, several doses of CAM (1,3, 10, and30 m a g , IV) or its vehicle (saline) were administered in a cumulative manner; in every case, the next dose of CAM was given 5 min after the response of Cl-PD to a given dose plateaued. To test the effects of other antibiotics, erythromycin (EM), aminobenzyl penicillin (ABPC), cefazolin(CEZ), or amikacin (AMK) at 10 mg/ kg was intravenously administered, andthe change in Cl-PD was continuously recorded. RESULTS demonstrated inFig. 1, the baseline value of in vivo PD of rabbit tracheal mucosa was 17.8 -t 1.9 mV (n =14), lumen negative. Application of amiloride to KH solution reduced PDto 11.0 -t 1.2 mV (n = 14), which isreferred to asCLPD.Subsequentapplication ofCAM ator M did not significantly alter the Cl-PD. Onthe other hand, intravenous admin-
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Tamaoki et al.
20 -
-
-\,
CAM (perfusion)
n a. 10
-
CAM (i.v.)
1 min
Figure I Representative tracing of rabbit tracheal potential difference (PD) in vivo. Addition of amiloride(AML, M) to Krebs-Henseleit solution perfusing the tracheal mucosa decreased PD, which referred to as Cl diffusion PD (Cl-PD). When Cl-PD became stable, clarithromycin (CAM) was added to the perfusion media at lod5M or administered by bolus injection from jugular vein at m a g .
*** ***
"
Control
1
'0
CAM ( m g W
Figure 2 Dose-dependent effect of clarithromycin (CAM) on Cl diffusion potential difference (Cl-PD)of rabbit tracheal mucosain vivo. CAM was intravenously administered in a cumulative manner, the andplateau valueof Cl-PD in response to each dosewas determined. Values are expressed asthe decrease in Cl-PD fromthe baseline values. Data are means SE; n = 9 for each point. **p < ***p < significantly different from control values.
*
Reduction
579
Cl-PD in Tracheal Muscosa with Clarithromycin
Table l Effects of Antibiotics on Cl Diffusion Potential Difference Across Rabbit Tracheal Mucosa
Cl diffusionpotential difference
(mV) Before Control CAMa EMa ABPCa CEZa AMP
After
f
2
f
f f
f f
*
f 0.5
f f
Difference f
* f f
*
Note: Each drug was intravenously administered at10 m a g . In the control experiment, the same volume of saline alone was given. Values are means2 SE; n = 8 for each group. ***p < .001,significantly different from valuesfor placebo. C A M , clarithromycin; EM, erythromycin; ABPC, aminobenzyl penicillin; CEZ,cefazolin; A M K , amikacin.
istration of CAM (10 m a g ) rapidly decreased Cl-PD from 10.8 2 0.7 to 6.9 0.4 mV (p < .001, n = 7) within 2 min. As shown in Fig. 2, intravenous CAM reduced Cl-PD in a dose-dependent fashion, the maximal decrease in Cl-PD fromthe baseline value andthe apparent dose requiredto produce a half-maximal effect (EC,,) being 5.6 2 0.9 mV (p < .001, n = 9) and 2.7 mg/kg, respectively, whereas administration of the vehicle alone had no effect. Effects of several antibiotics (10 m a g , IV)on Cl-PD are shown in Table 1. Each CAM and EM dose decreased Cl-PDby 5.1 2 mV (p < .001, n =S) and 5.6 2 0.7 mV (p < .001, n = 8), respectively, but other antibiotic agents did not significantly alter Cl-PD.
DISCUSSION Our present studieson the transepithelial bioelectricproperty provide indirect evidence that CAM may inhibit Cl secretion across rabbit tracheal mucosa in vivo. This notion is based on the finding that Cl-PD, apotential difference between the submucosal space and the mucosal surface of the trachea recorded under open-circuit conditions the presence in of amiloride, was decreased by CAM. The inhibitory effect on Cl-PD was likewise observed with another macrolide antibiotic agent EM, but other types of antibiotic suchas ABPC,
Tamaoki et al.
580
and AMKhadnoeffect.Thesefindings are inaccordancewith previous observationsin vitro suggesting that the effect may be specific for macrolides. Because the inhibition of C1 secretion resultsin the reduction of subsequent movement of water toward the lumen, the effect of CAM may be associated with the inhibition of water secretion, thereby causing a decrease inthe amount of airway secretions.
CONCLUSION Macrolide antibiotics specifically decrease Cl-PD of tracheal mucosa in vivo, an effect that mayresultin the corresponding decrease in water secretion from the submucosa toward the airway lumen, thereby possibly implicating one of the mechanisms ofthe efficacy of macrolides on patients with chronic airway diseases who have copious amounts of sputum.
REFERENCES 1. Kadota J, Sakito 0,Kohno S , et al. A mechanism of erythromycin treatment in patients with diffuse panbronchiolitis. Am Rev Respir Dis 1993; 147:153-159. 2. Goswami SK, Kivity S, Marom Z. Erythromycin inhibits respiratory glycoconjugate secretion from human airways in vitro. Rev Respir Dis 1991; 141~72-78. 3. Tamaoki J, Isono K,Sakai N, et al. Erythromycin inhibits Cl secretion across canine tracheal epithelial cells.Eur Respir J 1992; 5234-238. 4. Roszkowski K, Beuth J, KO HL, et al. Comparative study onthe macrolides erythromycin and clarithromycin: antibacterial activity and influence on immune responses. Zbl Bakt 1990; 273518-530. 5. Boucher RC, Bromberg PA, GatzyJT. Airway transepithelial electric potential in vivo: species and regional differences. J Appl Physioll980; 48:169-176.
Inhibition of Adherence of KZebsieZla pneumoniue Strains to Intestine-407 Cell Lines by Roxithromycin S. Favre-Bonte, C. Forestier, A. Darfeuille-Michaud, C. Rich, J. Sirot, and B.Joly Universitt!#Auvergne-Clermont I Clermont-Ferrand,France
INTRODUCTION Klebsiella pneumoniue accounts for 10% (among the Enterobacteriacae family) of nosocomial infections in intensive care units. It is responsiblefor urinary and respiratory tract infections, septicemia, and meningitis (1). Epidemiological studies have shown that infection is precededby intestinal colonization (2). Mucosal colonizationby bacteria is always linked to adhesion processes K.pneumoniue adherence can be reproduced in vitro using intestinal cells in culture. This study was designedto determine the effect of subinhibitory concentrationsof roxithromycin on the adhesion of K.pneumoniue strains to Intestine-407 cell lines.
MATERIALS AND METHODS Bacterial Strains K . pneumoniue CF504 and K . pneumoniue LM21 were obtained from clinicalisolatesinvolvedinnosocomialinfections(collection
of J. Sirot). 581
582
et
Favre-Bonte
al.
K . pneumoniae CF504 adheres with a diffusepattern in which the adhesion CF29K is involved (4).K . pneumoniae LM21 presents an aggregativepattern characterized by the formation of a bacterial clusteron the intestinal cell surface (5). Cell Cultures The Intestine-407 (Int-407) cell line derived from human embryonicjejunum and ileum was used. The cells were cultured in Eagle medium supplemented with 10% heat-inactivated fetal calf serum.
Adherence Assays Bacteria were suspended in the cell culture medium containing 2% D mannose. lo8 bacteria were addedto confluent monolayers of Int-407 cultured in 24-well plates and incubated for 3 h at 37°C. After incubation, each well was rinsedthree times in phosphate-buffered saline and released by the addition of 0.5% Triton X-100. Adherent bacteria were quantified by plating appropriate dilutions on Luria Bertani agar medium.
Adhesion Inhibition Tests The inhibitory effectof roxithromycin was studied following two protocols.
Protocol A: Roxithromycin was added to the culture medium of the bacteria. Bacteria weregrown for 18 h at 37°C in brain-heart infusion medium containing subinhibitory concentrations of roxithromycin. The bacteria were then centrifuged, washed, and allowed to adhere as described earlier. ProtocolB: Roxithromycin was added to the culture mediumof the Int407 cells. Bacteria were suspendedin the cell culture medium containing subinhibitory concentrationsof roxithromycin and addedto the Int-407 cells. Adherence assays were performed as above.
RESULTS The results are summarizedinFig. 1. Whenadded to the cell culture medium (protocol B), roxithromycin inhibits adhesion of K.pneumoniue CF504 from 80% for minimal inhibitory concentration (MIC)/2 to 46.5% for MIC/8 (Fig. 1A). This result is similar forK . pneumoniue LM21 (Fig. lB), for which adherence inhibition ranges from 96.75% (MIC/2)to 42% (MICB). According to protocol A (when roxithromycin was addedto the culture mediumof bacteria), adherenceof the K.pneumoniae strains is diminished by more than 50% only for MIC/2 (Figs. 1A and 1B).
Adherence of K.pneumoniae to Intestine Cell
Lines
583
A. Adherence of K. pneumoniae CF504
l
Control
MIC12
MIC14
MlC10
MIC116 MIC132
B. Adherence of K. pneumoniae LM21
l 100
5
80 60
(I)
40
20
0 Control
MIC12
MIC14
MlC18
MIC116 MIC132
Adherence of Klebsiella pneumoniae strainsinthepresence of subinhibitory concentrationsof roxithromycin.CFU = mean numberof bacteria adhering per c m 2 of tissue culturewell. live assays were performedin duplicate.
Figure l
DISCUSSION significant adherence inhibition ofK.pneumoniae by subinhibitory concentrations of roxithromycin was obtained only whenthe drug is added to cell culture medium containing the bacteria (protocol B). Furthermore, this effect occurs independently of the type of adherence factor because it is the same with K.pneumoniae CF504 (which presents a diffuse adherence
584
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pattern) and with K. pneumoniue LM21 (which presents an aggregative adherence pattern). Therefore, we speculate that the effect could be related to a modification of the surface or the metabolism of eucaryotic cells by roxithromycin. We obtained similar results when the adherence assays were performed with Caco-2 cellsthat produced microvilli after 15 days of culture (6). Thus, adherence inhibition of K.pneumoniae by roxithromycin is not limited to bacteria adhering to Int-407. Moreover, because microvilli produced by Caco-2 cells have the same properties as human enterocytes, we can postulate that adherence inhibition also could be produced in vivo. Thus, it may be possible that roxithromycin could prevent colonizationof the gut by K . pneumoniae.
CONCLUSIONS Whenadded to Int-407cells(Protocol B), roxithromycinhasa strong inhibitory effect (more than 40%) on the adherence of two K.pneumoniue strains at concentrations ranging fromMW2 to MIC/8. This effectof subinhibitory concentrationsof roxithromycin could be related to a modification of the INT-407 cells because it occurs when roxithromycinis added in the culture medium of the Int-407 cells and it is independent of the pattern of adhesion. Because the same effect was obtained with cultured Caco-2 cells (which have the same properties as human enterocytes), it can be postulated that roxithromycin could prevent the in vivo adherence of K. pneumoniae to enterocytes, whichis the initial step in the gut colonization.
REFERENCES Markowitz SM, Veazey JM, Macrino FT+ Mayhall CG, Lamb VA. Sequencia1 outbreaks of infection due to Klebsiella pneumoniaein a neonatal intensive care unit: implication of a conjugative R plasmid. J Infect Dis De Champs C,Sauvant MP, Chanal C, Sirot D, Gazui N, Malhuret R, Baguet JC, Sirot J. Prospectivesurvey of colonization and infectioncaused by extended-spectrum-P-lactamase-producingmembers of the family Enterobacteriaceae in an intensive care unit. J Clin Microbiol Finlay BB, Falkow S. Common themes in microbial pathogenicity. Microbiol Rev Darfeuille-Michaud A, Jallat C, Aubel D, Sirot D, Rich C, Sirot J, Joly B. Rplasmid-encoded adhesive factor Klebsiella in pneumoniae strains responsible for human nosocomial infections. InfectImmunoll992; 60:44-55.
Adherence
K.pneumoniae to Intestine Cell Lines
585
5. Favre-Bontk S, Darfeuille-Michaud A, Forestier C. Aggregative adherence of Klebsiella pneumoniae to human Intestine-407 cells. InfectImmunol1995; 63~1318-1328. 6. Joly B, Darfeuille-Michaud A, Rich C, Sirot D, Sirot J, Cluzel R. Inhibition of Klebsiella pneumoniae adherence to human intestinalcells by roxithromycin. 19th ICC, Montrkal, 1995.
Clinical and Immunological Studyof Roxithromycin on Infectious Diseasesin Obstetrics and Gynecology K. Izumi, H. Mikamo, K. Kawazoe,
T. Tamaya
Gifu University Schoolof Medicine Gifu, Japan
INTRODUCTION Evidence accumulated in recent years has shown that macrolide antibiotics have a wide varietyof pharmacological effects.In particular, the activities of erythromycin (EM) have been extensively studied, demonstrating that EM reduces the motility and the production of oxygen species in polymorphonuclear leukocyteswhile it enhances phagocytosis by macrophages and cytokine productionin leukocytes It also has beenreported that along-termadministration ofEM reduces the severity of bronchial hyperresponsiveness in bronchial asthma Roxithromycin (RXM), one ofnew macrolides,hasbeenused therapeuticallybecauseitseffects are similar to those of EM. In this study, we investigated the effect of long-term administration of RXM on clinicalresponsesandimmuneresponses(changes of neutrophil count and Interleukin-8 level) in patients with chronic intrauterine infection or pyometra. 586
Roxithromycin on Infectious Diseases in OBIGYN
587
MATERIAL and METHODS Subjects Eighteen patients with intrauterine infections diagnosed by transvaginal ultrasound tomography, uterine tenderness, and bacterial presence and 5 healthy womenweresubjected to the study.Nowomanreceivedany antimicrobial drug before this trial.
Drug Administration One hundred fifty milligrams of RXM was administered orally once a day for months to the infected patients.
Sampling Method disposable Nelaton catheter connected to a microsyringe was inserted into the uterine cavity, bluntly or after cervical dilatation. Sequentially, 5-m1 aliquots of phosphate-buffered saline (PBS) were instilled, followed by immediate aspiration,which wasdone twice. Bacteria were cultured and identified inthe aspirate fluid.
I a 300
200
5
-
100 0
0
Normal
Pyometra
Figwe I Neutrophil percentage (right) and Interleukin 8 (left) in the lavage fluid of the uterine endometrial cavity.
588
Izumi et al.
Assessment Each patient was completely evaluated clinicallyfor temperature, subjective (pelvic pain) and objective (uterine tenderness) findings, white blood cell count ( W C ) , erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP). The clinical responses were classified as excellent, good, and poor. Excellent or good were defined as resolution or improvement, respectively, of all clinical symptoms and laboratory findings at the end of treatment and during the postantibiotic follow-up period. Either inapparent or incomplete clinicalresponse to therapy or relapsewasconsideredpoor. For bacteriological evaluation, bacteriological response was graded as follows: (a) eradicated, (b) decreased, (c) unchanged, and(d) replaced.
Investigation Materials Neutrophil counts and Interleukin-8 (IL-8) levels were determined in lavagefluid of the uterineendometrialcavityobtained from 5 healthy women and the 18 infected patients.
'
I
betore
Figure 2 Neutrophil percentage (right)and Interleukin 8 (left) in the lavage fluid of the uterine endometrial cavity obtained frompatients with pyometra beforeand
after roxithromycin treatment.
Roxithromycin on Infectious Diseases in OBIGYN
589
0
Neutrophil In the lavagefluid (%)
Figure Correlation between the count of neutrophils accumulated and the level of Interleukin-8 inthe lavage fluid.
RESULTS Of the 18 patients, 16 (88.9%) experienced excellent or good improvement, showing significant improvements in WBC count, ESR, and CRP levels. %o of 18 (ll:l%) or 10 of 18 (55.6%) patients showed bacterial eradication or decrease, respectively. The results of neutrophil counts and Interleukin-8 determination in lavage fluid fromthe uterine endometrial cavity are summarized in Figs. 1 and 2. There was a close correlation between the neutrophil count and IL-8 levels in the lavage fluid from infected patients (Fig.
DISCUSSION Our study demonstratedthat patients with intrauterine infection had significantly higher percentages of neutrophils in pretreatment endometrial lavage fluids than did normal women. The neutrophil percentagewas significantly decreasedwith RXM treatment.
Izumi et al.
590
The accumulation of neutrophils in the inflammatory uterine cavity predicts neutrophil oxidative and proteolytic products,which are capable of producing endometrial damage in the uterine cavity(4,5). number of novel chemotactic cytokines are becoming increasingly recognized as important participants that contribute to the migration of specific inflammatory cells fromthe peripheral blood to inflammatory sites.Recent observations have demonstrated that each chemotactic cytokine carries a specificity for the individual movement of immune/inflammatory cells. IL-8 has been identified as a neutrophil chemotactic factor (6). These results indicatethat RXM decreases uterine endometrial cavity inflammation through a reduction in neutrophil migration to the inflammatory sitesand is effectiveon chronic intrauterine infection.
REFERENCES Kadota J, Sakito 0, Kohno S , Sawa H, Mukae H, Oda H, Kawakami K, Fukushima K, Hiratani K, Hara K. A mechanism of erythromycintreatment in patients with diffuse panbronchiolitis.Am Rev Respir Dis Yagyu Y. Analysis of the long-term treatment with erythromycin in chronic lower respiratory tract infections: I. Effects on human polymorphonuclear leukocyte functions. J Nara Med Assoc Mikami M.Clinical and pathophysiological significance of neutrophil elastase in sputum andthe effect of erythromycin in chronic respiratory diseases. Jpn J Thorac Dis 4. Nelson S, Summer WR, Terry PB, Warr GA, Jakab GJ. Erythromycininduced suppressionof pulmonary antibacterial defences: a potential mechanism of superinfection in the lung.Am Rev Respir Dis 5. Hirata T, Matsunobe S, Matsui Y, Kado M, Mikiya K, Oshima S. Effect of erythromycin on the generation of neutrophil chemiluminescencein vitro. Jpn J Thorac Dis Kunkel SL, Standiford, T, Kasahara K, Strieter RM. Interleukin-8 (IL-8): The major neutrophil chemotactic factor in the lung. Exp Lung Res
XI CLINICAL STUDIES: OTITIS MEDIA
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Five-Day Treatment of Acute Otitis Media in Children with Clarithromycin Dimitris A. Kafetzis, Theodore Bairamis, Dimitra Dinopoulou, Stamatina Vlachou,and Nicholas Apostolopoulos University of Athens “A. Kyriakou” Children’s Hospital Athens, Greece
INTRODUCTION Acute otitis media is common a infectious disease and is a major reason for seeking medicalattention during early childhood. Duringthe preantibiotic era, resolution required myringotomyor followed spontaneous perforation of the tympanic membrane, whereas severe intracranial complications developed in approximately of cases. In recent decades, antibiotics are usually prescribed for a 10-day treatment period, and complications have been almost eradicated with an incidence of lessthan 0.15% (1). Seven-day antibiotic therapy also has beenwell accepted and proven effective In some studies, however, it has been shown that even ashorter duration of therapy could be as effective(3,4). We report the results of an open, randomized clinical study comparing the efficacy and safety of clarithromycin with cefuroxime-axetil, each given orallyfor 5 days forthe treatment of pediatric patients suffering from acute otitis media.
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Kafetzis et al.
594
PATIENTS AND METHODS Patients were seen in the outpatient clinic of the “P. & A. Kyriakou” Children’s Hospital in Athens and were eligible for the study if they had a diagnosis of acute otitis media with fever > 38”C, otalgia, or irritability, with otoscopic findings ofan erythematous bulging and opacified tympanic membrane. To be considered eligible for the study, a parentof each patient had to give hisher informed consent. Patients were excluded from the study if they had received any antibiotic treatment during the last 2 weeks before enrollment, had an underlying disease that might affect the pharmacokinetics of the medication or the outcome of the study, had a perforated tympanic membrane or if complication of otitis mediawas present atthe time of enrollment. Each patientwas randomly assignedto either antibiotic for 5 days and was reevaluatedat the end of treatmentandfollowed for 30days. Wpanocentesis was performed when patients failed to respond to treatment or if there was arecurrence of the infection. In all patients, tympanometry was performed priorto treatment and posttreatment. Forty patients were enrolledin the study andtheir demographic characteristics are shown in Table1.
RESULTS
No significant differences were found between the treatment groups. Patients in both treatment groups were similar with regard to their medical histories, physical examination results, and baseline otoscopy and tympan metry results. A clinical response to therapy could be determined for all Table I
Patient Demographics Cefuroxime-axetil Clarithromycin
No.of Patients
20
Males Females Age median (months) Range Mean weight (kg) Dosage (mgkg) (every 12h) Prior history of otitis media episodes 1episode >l episodes
12
20 11
21 6-48 12.1 7.5
22 6-52 12.4 15
4
5 12
Treatment of Acute
OtitClarithromycin is with
595
Tub& 2 ClinicalResults Cefuroxime-axetil Clarithromycin 17 14
Cured clinically Posttreatment pathologic tympanometry results Failed clinically Recurrence of infection Side effects Nausea
16
14 4 2
1
patients. Two and three patients in the clarithromycin and cefuroximeaxetil study group, respectively, required an extensionof therapy in order to achieve a successful result. Clinical results are shown in Tables2 and
DISCUSSION Treatment of uncomplicated acute otitis media with amoxicillinklavulanate for 5 days has been found to be as effective as 10 days (4). Similarly, we have found that a 7-day treatment with either clarithromycin or cefuroximeaxetil is effective in almost 95% of cases (5). This study demonstratesthat both clarithromycinor cefuroxime-axetil are effective for 5-day treatment as well. Results showthat clarithromycin and cefuroxime-axetil achieved clinical cure ratesof 85% and SO%, respectively, when administered to children suffering from clinicallyor tympanoscopy-documented casesof acute otitis media. Persistence of middle ear fluid at posttreatment tympanometry did Tab& 3 Treatment of Clinical Failures Cefuroxime-axetil Clarithromycin Clinical failures Isolated organism
Recurrences Isolated organism Amoxicillidclavulanate Amoxicillidclavulanate Treatment
Extension of treatment:2 Treatment changed: H.influenzae:
Extension of treatment: 4 Failed after 10 days: 1 H.influenzae: 1 S. pneumoniae: H . influenzae + Strept. pneumoniae: 1
H.influenzae:
H . influenzae:1 Strept. pneumoniae: 1
596
Kafetzis et al.
not differ betweenthe two groups(14 and 14 patients, respectively). Additionally, although five patients (two and three cases in each study group, respectively) requiredfurther treatment in order to achieve a95% satisfactory result, the decision to continue treatment beyond 5 days on the fifth treatment day was reasonable and effective.
CONCLUSION 5-day regimen of clarithromycin for treatment of uncomplicated acute otitis media is safe and effective. Patients should be evaluated on the fifth day of therapy to determine whether additional therapy is warranted.
REFERENCES 1. Bass J W , Cashman TM, Frostad A L , et al. Antimicrobials inthe treatmentof acute otitis media.A second clinical trial. Am J Dis Child. 1973; 125:397-403. 2. Chaput de Saintonge DM, LevineDF, Templae Savage et al. Trialof threeday and ten-day courses of amoxicillin in otitis media. Br Med J 1982; 284: 1078-1081. 3. Meistrup-Larsen KI, Sorensen H, Johnson NJ, et al. ?kro versus seven days penicillin treatment for acute otitis media. Acta Otolaryngol (Stockh) 1983; 96:99-104. 4. Hendrickse WA, KusmieszH, Shelton S, et al. Five versusten days of therapy for acute otitis media. Pediatr Infect Dis J 1988; 7:14-23. 5. Kafetzis DA, Malaka-Zafiriou C , Bairamis T, et al. A comparison of the safety and efficacy of clarithromycin suspension and cefuroxime axetil suspension in the treatment of acute otitis media. Unpublished.
Azithromycin Versus Amoxicillin for Acute Otitis Media Prophylaxis Nicola Principi, Paola Marchisio, Emanuela Sala, Luisa Lanzoni, and Stefania Sorella University of Milan Milan, Italy
INTRODUCTION Recurrent acute otitis media (AOM) is common in infants and young children. Because of the morbidity and possible long-term sequelae, prevention is the main goal. Among the various approaches (chemoprophylaxis, immunoprophylaxis, surgery, and control of environmental risk factors), chemoprophylaxis is still considered the best medical option. However, there is some concern about the possible role of chemoprophylaxis in the emergence of resistanceamongrespiratorypathogens. In our previous study (l),we demonstrated that once-daily administration of low-dosage amoxicillin for 6 months, is as effective as trimethoprim-sulfamethoxazole (TMPBMX) and superiorto placebo in reducingthe occurrence of AOM: of otitis-prone childrentreated either with amoxicillin or TMP/SMX developed AOM compared to of patients given placebo. However, compliance with such a long-term administration canbe a problem, especially in younger children. Intermittent prophylaxis theoretically could be more suitable, but several studies(2-4) have demonstratedthat intermittent administration of 597
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Pnncipi et al.
recommended drugs such as amoxicillin is less effective than a continuous prophylactic regimen.A possible alternative could the be use of long-acting antibiotics. Azithromycin, an azalide compound structurallyrelated to the macrolides, is characterizedby a broad spectrumof activity which includes all the bacteriausuallyencounteredinAOMandpeculiarpharmacokinetics which allows high concentrations in polymorphonuclear leukocytes and respiratory fluids (including middle ear effusion) for a long period after a single dose. Our study compares the efficacy and safety of prolonged administration of continuous low-dosage amoxicillin with that of intermittent azithromycin in a groupof otitis-prone children.
PATIENTS AND METHODS Children aged 9 months to 6 years with a history of recent recurrent AOM, defined as three or more AOM in the preceding 6 months, were included. Children were randomly assignedto (1) amoxicillin, 20 mgkg per day, (2) azithromycin, 10 mgkg followed by 5 mgkg once a week, or azithromycin, mgkg once a week. All patients weretreated for 6 months and were examined at entry and subsequently at intervals of4-6 weeks and whenever they developed symptoms upper respiratory tract illness or suggesting AOM.At each visit, an interval history was obtained, and pneumatic otoscopy and tympanometric testing were performed.At entry into the study, at completion of the third month, andat the end of the treatment period, a nasopharyngeal swab was obtained to detect the presence of S. pneumoniae, H.influenzae,Moraxella catarrhalis,and beta hemolytic streptococcus and their resistance to p-lactams andto azithromycin. Occurrence of AOM duringthe 6-month periodwas calculated for all groups.
RESULTS Patient characteristicsare summarized in Table1. The number of children enrolled in the 5-mgkg per week azithromycin arm is lower than the other two groups because the inclusion was prematurely interrupted due to the high incidence of AOM in this group: Overall, 55.5% of the children had new episodes of AOM and 419 (44.4%) had two episodes of AOM in the first months of prophylaxis and were thus discharged fromthe study and no longer analyzed. The occurrence of AOM duringthe 6-month study period is reported in Table2: Azithromycin 10 mgkg once a week was superior to amoxicillin in preventing AOM. Both drugs were safe and well tolerated. In none of
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Azithromycin Versus Amoxicillin for AOM Table I
Patient Characteristics Azithromycin (5 mg/kg once
Azithromycin (10 mg/kg once
Amoxicillin week) a week) a
No. of patients Sex Male Female Age 9 months-3 years >3-6 years Initial ear status No OME OME bilateral OME monolateral Season at entry October-March April-September Day care attendance Yes
No Most recent AOM 31 days
70
9
74
39 (55.7%) 31 (44.3%) 49 (70.0%) 21 (30.0%)
3 (33.3%) 6 (66.7%) 2 (22.2%) 7 (77.8%)
43 (58.1%) 31 (41.9%) 43 (58.1%) 31 (41.9%)
17 (24.2%) 40 (57.2%) 13 (18.6%)
1(11.1%) 6 (66.7%) 2 (22.2%)
40 (54.1%)
52 (74.3%) 18 (25.7%)
8 (88.9%) 1(11.1%)
61 (82.4%) 13 (17.6%)
40 (60.6%) 26 (39.4%)
7 (77.8%) 2 (22.2%)
50 (73.5%) 18 (26.5%)
53 (75.7%) 17 (24.3%)
4 (44.4%) 5 (55.6%)
49 (66.2%) 25 (33.8%)
15 (20.2%) 19 (25.7%)
Note: No statistical difference between azithromycin 10 mgkg once a week and amoxicillin.
Table 2 Occurrence of Acute Otitis Media During 6-Month Study Period
No. of children with AOM No. of episodes of AOM Mean No. of episodes per patient
Amoxicillin (20 mgkg per day) (n=70)
Azithromycin (5 mgkg per week) (n=9)8
Azithromycin (10 mgkg perweek) (n=74)
22 (31.4%)
5 (55.5%)
11(14.8%)b
28
7
16
0.4
0.7
0.21
T h e 5-mgkg once-a-week azithromycin group was prematurely interrupted (and not further analyzed)because of thehighoccurrencerate of AOM: Childrenwerethenrandomizedonly to amoxicillin or azithromycin 10 mgkg. bp < .05 versus amoxicillin and azithromycin5 mgkg once a week.
Principi et al.
600
Table 3 Nasopharyngeal Colonization According to Prophylactic Regimen
Baseline nasopharyngeal culture Amoxicillin Negatives (n=60)(92.3%)
End of prophylaxis nasophar. culture
End of prophylaxis nasophar. culture
(negative)
(positive)
54
6 S . pyogenes H.influenzae 2 (1 R to azithromycin)
Strep beta hemol group 1C Positive (n=5)(8.7%)
5
0
S. pneumoniae S. pyogenes 1
H.influenzae 3 Azithromycin Negative (n=62)(93.9%) 4Positive (n=4)(6.1%)
61
1 (S. pyogenes) 0
S. pneumoniae 1
H.influenzae .Negative or positivefor S. pneumoniae, H . influenzae, S. pyogenes, and Moraxella catarrhalh. Only resistanceto D-lactams and azithromycinis shown.
the two prophylactic regimens was any remarkable modification of the nasopharyngeal flora demonstrated, both regardingthe prevalence of the bacteria and their susceptibility to p-lactams and macrolides (Table 3).
CONCLUSION Intermittent administration of azithromycin 10 m a g per week (but not at 5 m a g per week) is more effective than continuous low-dosage(20 m a g per day) amoxicillin in preventing AOM in children with recent recurrent AOM. As far as the possible role of chemoprophylaxis in determiningthe emergence of resistant bacteria,we did not note any substantial modification in the small proportionof children harboring nasopharyngeal pathogens. Thus, azithromycin appearsto be suitable for the prophylaxisof acute otitis media in children with a recent historyof recurrent acute episodesof otitis media. In particular, azithromycin could be suggested in (1) those children, suchas infants, inwhom compliance with a continuous long-term treatment could be problematic, (2) in children with p-lactam allergy, (3) when amoxicillin-resistant, new macrolide-susceptible bacteria are highly prevalent inthe population.
Azithromycin Versus Amoxicillin for AOM
601
REFERENCES Principi N, Marchisio P, Massironi E, et al. Prophylaxis of recurrent acute otitis media and middle ear effusion. J Dis Child Peterson L, Peterson KE, Peterson LR. Intermittent antimicrobial prophylaxis for recurrent acute otitis media.J Infect Dis Berman S, Nuss R, Roark R, et al. Effectivenessof continuous vs intermittent amoxicillin to prevent episodes of otitis media. Pediatr Infect Dis J 4.
Heikkinen T, Ruuskanen 0, Ziegler T, et al. Short-term use of amoxicillinclavulanate during upper respiratory tract infection for prevention of acute otitis media. J Pediatr
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XII CLINICAL STUDIES: BRONCHITIS
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Azithromycin for the Treatmentof Community-Acquired Bronchitis: A Large Multicenter Efficacy-Tolerance Trial F. Raffi Centre Hospitalier Rkgional Universitaire de Nantes Nantes, France
INTRODUCTION Antibiotic therapymay be needed for persistent acute bronchitis (AB) and acute exacerbations of chronic bronchitis(AECB), particularly in high-risk patients. Azithromycin is a new azalide (nitrogen-containing macrolide) antibiotic andmight proveto be useful as first-line treatment in these indications. The open, multicenter study described was performed the withobjective of evaluating the efficacy and safety of azithromycin when administered once-daily for 5 days in everyday practice in a large adult population suffering fromAB or AECB. Particular attention was paidto rare adverse events.
METHODS Patients included were adult outpatients presenting with either persistent AB or AECB based on the following criteria:
A B : Persistent progression with mucopurulent sputum accompanied by a risk factor for bacterial secondary infection such as heavy 605
smoking (more than packdyear) or chronic disease (alcoholism, heart disease, arterial disease, diabetes, autoimmune disease, obesity, etc.). AECB: Mucopurulent or purulent sputum and increase in dyspnea and/or volumeof sputum (Anthonisen typesI and I1 criteria). The exclusion criteria were the lack of informed consent, concomitant pneumonia, acute sinusitis, stage V dyspnea (not excluding minor deviations), cystic fibrosis, bronchial dilatation, and concomitant administration of another antibiotic. If concomitant pneumonia was suspected, a chest x-ray was prescribed andthe result documented withinthe first 48 h following inclusion. Patients received500 mg of azithromycin (2 250-mg capsules) on the first day and then 250 mgof azithromycin daily for the next 4 days. Patients hadto be seen again at day 2 1 and day 14 for the final evaluation.
ASSESSMENT CRITERIA Assessment of Efficacy in Patients with AB Recovery with complete disappearance of symptoms was defined by a combination of the following four criteria: cessationof all expectoration, disappearance or return to previous state of dyspnea, disappearanceof fever (if initially present), and disappearanceof cough. The result was regarded as an improvement when physical symptoms improved frequent but coughing or mucous or mucopurulent sputum persisted. Treatment was regarded as a success in patients whoeither recovered or showed considerable improvement. Treatment was considered a failure when the final evaluation revealed continued coughing, severe fits of coughing, purulent sputum, temperature equal to or greater than 38"C, or if the lack of anyimprovement or the aggravation of symptoms during the trial had warranted a change in therapy.
Assessment of Efficacy in Patients with AECB Recoverywasdefinedby a combination of the following four criteria: disappearance of purulence of sputum, disappearanceor decrease (return to previous state) in dyspnea, disappearance of fever (if initially present), and decrease of sputum or cessation of expectoration. An improvement was defined as a decrease in physical symptoms but without the disappearance of all clinical signs or a return to the previous state. Failure was defined asthe persistence or an increase inthe volume of purulent sputum or signs of infection requiring an alteration in antibiotic therapy.
Azithromycin for Bronchitis
607
Assessment of Safety All patients who received at least one capsule of azithromycin were included inthe safety assessment which was based on clinical data or questioning, together with any additional tests requested for documentation. The analysis included 7947 patients entered between 1 September 1994 and 15 May 1995 and distributedon the basis of diagnosis as follows: A B , 4817 cases (60%); and AECB, 3130 cases (40%).
RESULTS Efficacy was evaluated (Table 1) In terms of the total sample sizeof patients included (“intention to treat”); that is, n = 7904 (4791 AB and 3113 AECB) In terms of the samplesize of patients whocompliedwith the protocol(“perprotocol”)[i.e.,n=7286(4444ABand2842AECB)], after excluding 661 patients forthe following reasons (severalreasons might be involved for any given patient): mucopurulent or purulent sputum absent at day (n= 87), dyspnea absent at day (n=42),type I11 AECB accordingto the Anthonisen classification (n= 46), radiological diagnosisof infectious pneumoniaor,pleural abnormality (n= 50), final evaluation too early (less than 12 days after day ) (n= 430), and data missing for the global evaluation of efficacy (n= 43).
EVALUATION OF SAFETY The “intention-to-treat” analysis (entire patient population) showed that 97.2% (7728) did not exhibit any adverse event (A.E.). The A.E.s in2.8% of the patients included diarrhea(0.7%), nausea ( O S % ) , gastralgia (0.5%), and abdominal pain(0.2%). Of the rare A.E.s, rash was noted in 0.06% of cases (n=6) and dizziness in 0.06% of cases (n=5). Fewer than 0.5% of patients (38/7947)had to discontinue the trial because of an A.E. An analysisof safety inthe various subgroups showedthat more than 4% of patients with one of the following risk factors presentedat least one A.E.: renal or hepatic impairment (8.3%), history of cardiorespiratory decompensation diabetes (5.6%), heart disease, valvular disease or arterial disease (4.3%), alcoholism (4.2%), o r obesity (4.1%). Patients over 70 years of age did not constitute a subgroup at risk in terms of adverse reactions by comparisonwith the general population: 2.7% of patients over 70 presented at least one A.E. (the same frequencyas in the general population).
608 Tabh
Evaluation of Efficacy “Intention-to-treat” analysis
“Per protocol” analysis
AB Success rate Recovery Improvement Failure rate AECB Success rate Recovery Improvement Failure rate
%
CONCLUSION The trial was carried out in routine practical conditions and confirmedthe satisfactory results obtained using 5 days of once-daily azithromycinin the treatment of AB and AECB in adults A success rate of 96.6% and 95.6% was achieved in compliant patients with AB and AECB, (Anthonisen typesI and II), respectively. A large numberof patients were studied, thereby allowing an accurate estimate of the incidence of adverse events and confirmingthe absence of rare adverse events. Gastrointestinal symptoms werethe most frequent A.E.s, but still accounted for less than 2%. A combination of a low incidence of A.E.s and once-daily treatment for 5 days contribute to goodcompliancewith treatment and the successof azithromycin in these indications.
REFERENCES Balmes P, Cerc G , Dupont B, et .al. Comparative study of azithromycin and amoxicillidclavulanic acid in the treatment of lower respiratory tract infections. Eur J Clin Microbiol Infect Dis Feldstead SJ. Azithromycin LTRI Study Group. Double blind comparison of azithromycin and amoxicillin in the treatment of lower respiratorytract infections. Proceedings of the 6th International Congress of Infectious Diseases,
Safety and Efficacy of Clarithromycin Compared with Cefpodoxime Proxetil in the Treatment of Acute Exacerbation of Chronic Obstructive Pulmonary Disease P. IRophonte and J. P.Chauvin Hospital Rangueil Toulouse, France Abbott France Laboratories Rungiv, France
INTRODUCTION Chronic obstructive pulmonary disease (COPD) is characterized by a reduc-' tion of the expiratory output together with clinical symptoms associated with chronic bronchitis. Infection is a frequently encountered problem in the everyday treatment of this disorder. Infection in patients with COPD entails an immediate riskof pulmonary decompensation and potential aggravation of chronicbronchitis.Rapidactionin the form of antibiotic therapy is recommended, particularly in the face of worsening clinical symptoms (dyspnea and increase a n d or purulent sputum).At the present time, p-lactams are the treatment of choice. Various studies have revealedthat in approximately 50% of cases, a bacteriological profile exists that includes Huemophilus influenzae or Strep609
610
Ltophonte and Chauvin
tococcus pneumoniae and, in a few cases, Moraxella catarrhalis. For the remaining 50% of cases, no microbiological confirmation is apparent, although Mycoplasma pneumoniae or Chlamydia pneumoniae infection cannot be ruled out, given that little is known about the incidence of such microorganisms in this disorder. As clarithromycin is active against microorganisms that are responsible for lower respiratory tract infections, it should be considered inthe treatment of these infections.
STUDY OBJECTIVE The objective was to compare the safety and efficacy of clarithromycin with cefpodoxime proxetil in the treatment of acute bacterial exacerbationsof COPD as administered under everyday general practice conditions.
PATIENTS AND METHODS Study Design Multicenter, open-label, comparative randomized study. lbelve regionalcoordinatingpneumologistsandinfectiousdiseasespecialistsactedaslocalscientificadvisors to 80 general practitioners. Efficacy wasevaluated onthe basis of the nature of patient sputum ondays12-15(success = absendmucus;failure = purulent/ mucopurulent) and resolution of signs ofexacerbation (e.g., aggravation of dyspnea, increase in sputum).
Inclusions Male or female outpatients aged 18-80 years. Presenting COPD: History of chronic bronchitis (cough and sputum every day for a continuous periodof months for 2 consecutive years) Confirmedbronchialobstruction,practicallyirreversiblewith &stimulants Demonstrating an acute exacerbation of COPD at time of inclusion definedby Modification of sputum color or consistency indicative of acute bacterial infection (e.g., change to yellow/green color, increased tenacity of sputum) andthe presence of at least one of increased volume of expectoration worsening of dyspnea No use of antibiotics within 15 days priorto the study.
ry
Clarithromycin Versus Cefpodoxime Proxetil for COPD Table I
611
Evaluations Visit 1 Evaluation (day
1)
Medical
Visit 2 Day
X X X
Physical examination Clinical examination
Clinical pulmonary evaluation Prescription for standard Cytobacterial examination Clinical response Adverse drug events
(days 12-15)
Day
X X X
X X X X
X X
X X
.Telephone consultation; if necessary, the investigator could request patient consultation. Welephone consultation to determine clinicalevolution (especially infectious relapse).
suspicion of active tuberculosis, severe respiratory failure, associated parenchymal infection, or signs of respiratory decompensation that could necessitate hospitalization. hypersensitivity to p-lactams or macrolide antibiotics. severe renal or hepatic impairment. Written informed consent.
Drug Administration 332 patients were randomizedto receive 1or 2 treatments, each of which was to be taken by mouth for a 10-day treatment period. Treatments were as follows Clarithromycin (nclar): 250 mg 2 bid (n =51 179) Cefpodoxime proxetil (Cefodox): 100mg 2 bid (n = 153)
Evaluation The visits are summarized in Table1.
RESULTS Demographic Data The study included 332 patients with a mean age of 60 years (sex ratio M/F = 1.8). The mean history of the pulmonary disease was up to 10 yearsfor 55% of patients.
Ltophonte and Chauvin
612
Table 2 Clinical success at Days 12-15 by Treatment Group
proxetil Clarithromycin Cefpodoxime Success Failure Missing data
1361168 (81.0%) 321168 (19.0%) 11
115/149 (77.2%) 341149 (22.8%) 4
Clinical success defined as absenceof mucus expectorate.
The Anthonisen score atday 1was equal to 1in of patients in both groups. Spirometric data: the average VEMS was at 60% of the theoric value. There was nodifferencein the two groupsconcerningdemographic data and infectionstatus of enrolled patients.
Clinical Evaluation Table 2 shows that the clinical success defined as the absence of mucous expectorate on days 12-15 was not significantly different in the two groups. Regardingclinicalfailure, there werefewerclarithromycinpatients who required anew course ofantibiotic (7.9% for clarithromycin group, 15% for cefpodoxime proxetil,p = .0048).
Adverse Events Most adverse events were mild to moderate in severity in each treatment group. Excluding taste perversion reported exclusively in the clarithromycin group, there was no statistically significant difference in the two groups.
DISCUSSION
In this clinical trial, analysis of lung respiratory tract sampleswas not very helpful for therapeuticdecisions. The high frequency of poor quality expectorated sputum or the presence of normal respiratory flora makes cytobacterial examination of expectorated sputum less reliable. Treatment of acute exacerbationof chronic bronchitis in patients with chronic obstructive pulmonary disease still remains basedtheonmost likely etiologic organisms in COPD. This study concludedthat the clinical efficacy of clarithromycin compared to that of a third-generation cephalosporin is equivalent inthe treatment of COPD although clarithromycin incurs lower cost.
Clarithromycin Versus Cefpodoxime Proxetil for COPD
613
CONCLUSIONS Tbice-daily treatment with 500 mg of clarithromycin resulted in a high rate of clinicalsuccesscomparable to that obtained with twice-daily 200 mg of cefpodoxime proxetil in the treatment of acute exacerbationof chronic obstructive pulmonary disease. Overall, both drugs werewell tolerated, although the incidence of drug-related effectswas higher in the clarithromycin group due to the increased frequencyof taste perversion. Fewer clarithromycin patients required a new course of antibiotic therapy (p = .048).
REFERENCES 1. Anthonisen NR. Antibiotic therapy in exacerbations of chronic obstructive pulmonary disease. Ann Intern Med 1987; 106:196-204. 2. Guay DRP, Craft JC. Comparative safety and efficacy of clarithromycin and of ampicillin inthe treatment of out-patients with acute bacterial exacerbation chronic bronchitis. J Intern Med 1992; 231:295-301.
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XIII CLINICAL STUDIES: PNEUMONIA
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Randomized Comparative Trial of the Safety, Efficacy, and Cost of Intravenous Cefuroxime Plus Intravenous Erythromycin Versus Intravenous Cefuroxime Plus Oral Clarithromycin in the Therapy of CommunityAcquired Pneumonia D. Skupien, A. Margulis, Kaczander, S. Jaworski, A. Eklebeny, J. Pypkowski, and M. J. Zervos Wayne State University School of Medicine and William Beaumont Hospital Royal Oak, Michigan
INTRODUCTION Pneumonia occurs in overthree million persons per year and accountsfor 500,000 hospitaladmissions. It is the fourth leading cause of death in persons overthe age of 65 in the United States and the number 1cause of death from infectious diseases. The cost of hospitalization alone exceeds 1.5 billion dollars per year. Despite diagnostic efforts, the detection of all causative pathogens is limited. It is often not possible to obtain a sputum specimen, or cultures do not yield a specific organism; therefore, etiology is only known 50% of the time in community-acquired pneumonia (CAP). 617
618
,
et
Skupien
al.
Frequently encountered pathogens suchMycoplasma as pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, and respiratory viruses are not detected at allbyGram stain and routine culture. According to a concensus statement by the AmericanThoracicSociety, it isbecauseof these diagnostic limitations that initial empiric therapy of a second- or third-generation cephalosporin and a macrolide with activity against common pathogensisnecessary(1).Erythromycinisoftenincludedwith cefuroxime as empiric therapy because of its excellent coverage of M . pneumoniae and L. pneumophila, but it is considered ineffective in vitro against H . influenzae. Clarithromycin, a newer macrolide, also includes coverage for C . pneumoniae and H . influenzae, common pathogens in CAP patients (2). The present studywas undertaken to determine a cost-effectivetreatment regimen for CAP. We also sought to evaluate in a randomized, comparative study the safety and efficacy of cefuroxime plus erythromycin versus cefuroxime.plus clarithromycin in the treatment of CAP.
PATIENTS AND METHODS .
,
Patients
Between August 1994 and June 1995, patients admitted to William Beaumont Hospitalwith a primary diagnosis of community-acquired pneumonia were eligible for inclusion in this study. [William Beaumont Hospital is a 975-bed community teaching hospital in Royal Oak, Michigan with over 40,000 admissions annually. In 1994,863 patients with an admission diagnosis of community-acquired pneumonia weretreated at William Beaumont Hospital with an average (hospitalization) length of 14.2 days.] Adult hospitalized patients with diagnosis of community-acquired bacterial pneumonia met inclusion criteriaby presenting with a newor radiographically demonstrated changing infiltrate associatedwith fever, cough, and physical findings compatible with pneumonia, which could include purulent sputum production and leukocytosis (leukocytes > 11O , OOcm3). Patients must have been suitable candidates for oral antibiotic therapy based on the presence of a functioning gastrointestinal tract. Exclusion criteria includedthe following:evidence of sepsis or septicshock,empyema,lungabscess, or aspiration pneumonia, neutropenia (< 1500/mm3 WBC) or an immunocompromising condition, anyother infection which necessitates use of concomitant systemic antibiotics, a historyof hypersensitivity to macrolide or p-lactam antimicrobials, or prior presumably effective intravenous antibiotic therapy for more than24 h. No females who were planning pregnancy or wereactivelypregnant or breastfeedingwereincludedin the study population. Informed written consent was obtained from all patients.
Comparative Study
619
CAP
Therapy Patients were chronologically randomized to a nonblinded dosing schedule to receive cefuroxime 750 mg intravenously every 8 h plus erythromycin 500 mg intravenously every 6 h (cefuroxime/erythromycin)or cefuroxime 750 mg intravenously every8 h plus clarithromycin 500 mg every 12 h orally (cefuroximeklarithromycin). Both groups were subsequently convertedto clarithromycin 500 mg PO every12h.No other antibiotics were given during or after administration of the study drug. The duration of antibiotics, time of conversion to oral therapy, all other forms of therapy, and hospital discharge were at the discretion of the attending physician caring for the patient.
Bacteriological Investigations In patients able to produce sputum, Gram stains of sputum smears and sputum cultures were performed. Bacteriologic blood cultures were obtained on all patients; Gram stains were reviewed and correlation with culture results was needed for the organism to be considered a pathogen. Identification of pathogen@) and antibiotic susceptibilities (erythromycin, cefuroxime,andclarithromycin)weredetermined by the microdilution technique, Microscan (WalkAway Baxter, West Sacramento, CA), using NCCLS standards.
Evaluation of Efficacy and Safety Clinical response to treatment was determined by the following criteria: cure, as definedby the disappearance of clinical symptomatology andtheir continued absenceat the end of a 1-2 week patient follow-up period, and faiZure as defined byno significant improvementor clinical worsening while on the study treatment. All adverse events were recorded along with severity, and outcome and determined causality to the study medication. Microbiological efficacy was determinedby using the following criteria: eradication, defined as the elimination of the principal pathogen(s) at the endof therapy and continued absence through 1-2 weeks of patient follow-up, and persistence, defined asthe continued presenceof the principle pathogen at the end of therapy. Statistical comparisons were made using Student’s t-test for continuous variables, and Fisher’s exact test or Chi-square analysis withthe Yates correction was used for dichotomous variables.
Evaluation of Cost Cost comparisons were made between the two groups basedon duration of treatment, drug acquisition costs, and intravenous administration costs.
620
Skupien et al.
Drug acquisition costs of intravenous cefuroxime were $17/day, intravenous erythromycin $7/day, and oral clairthromycin $5/day. Other antibioticsnecessary to treat the studied episode of pneumonia were also considered in the total costs incurred by both groups. Antibiotic costs were providedby the hospital pharmacy and determined based on unit-dose drug acquisition costs. Intravenous administration charges were estimated at $Ydose. Hospitalization charges were estimated at $120/day.
RESULTS A total of 60 eligible patients were included in this study. Thirty patients were randomly assigned to receive cefuroxime and erythromycin andto30 receivecefuroximeandclarithromycin,withconversion to oral clarithromycin in both groups. Patients inthe two treatment groups were similar with respect to demographics and underlying disease history (Table 1). Table 2 summarizes the bacteriological findings in the two groups. Overall, the most frequently isolated organisms were H.infruenzae (10%) and S. pneumoniae no pathogen was isolated in 25% and 31.7% were unableto produce sputum. Adverse events occurred in 16 patients (Table Ten cefuroxime/ erythromycin patients (33.4%) experienced adverse events, six cefuroximel clarithromycin patients (20%) experienced adverse events. The most frequent adverse events were gastrointestinal disturbances (five cefuroxime/ erythromycin patients, three cefuroxime/clarithromycinpatients). Phlebitis was seen intwocefuroxime/erythromycinpatients. One cefuroxime/ clarithromycin patient experienced an allergic reaction to cefuroxime. Three Table Z Demographic and Clinical Characteristics of Patients in theTwo Study Groups at Presentation ~~
group Characteristics
evaluable No. of patients 30 No.of males/females 16/14 (range) yearsin age Mean No. (%) receiving prior abx. No. (%) of underlying disease: COPD Cardiovascular disease Genitourinary Diabetes Total
Cefuroxime/erythromycin (n=30) 30 12/18 (21-93) 70 (13.3%) 5 (16.7%) 4 8 (26.7%) 3 (10%) 9 (30%) 20 (66.7%)
~
~
Cefuroxime/clarithromycin group (n=30)
71 (31-93)
11(36.7%) 4 (13.3%) 1(3.3%) 15 31 (100%)
roup
621
Comparative Study of CAP Table 2 Bacteriologic Findings (Numberof Strains Isolated) inthe Two Study Groups
Cefuroxime Cefuroxime plusplus clarithromycin erythromycin Organism Streptococcus pneumoniae flaemophilus injluenzae Staphylococcus aureus Klebsiella pneumoniae Moraxella catarrhalis Klebsiella ozaenae Enterobacter spp. Proteus spp. Serratia marcescens Pseudomonas aeruginosa Normal oral flora Unable to produce sputum
group
0
1
Table 3 Outcome of Therapy and Clinical Course
Cefuroxime plus erythromycin group Clinical outcome No. (%) cure No. (%) failure Bacteriologic outcome No. (%) Eradication . No. (%) Persistence No. (%) Indeterminate Mean duration (range) of fever in days Mean duration (range) of leukocytosis in days Adverse events No. (%) gastrointestinal No. (%) allergy No. (%) phlebitis No. (%) ICU admissions No. (%) death .Second to cefuroxime.
Cefuroxime plus clarithromycin group
622
al.
Skupien et
cefuroxime/erythromycin patients and two cefuroxime/clarithromycinpatients expired but deaths were not related to the study medication. One cefuroxime/clarithromycinpatient was withdrawn from study for the following reasons: failure, side effects, and antibiotic resistance. One cefuroximel clarithromycin patient was withdrawn from study because of the isolation of a resistant Enterobacter cloacae. Table illustrates the difference in the cost of treatment of the two groups. The cefuroxime/clarithromycingroup shows a shorter duration of treatment and lower drug acquisition costsfor both study medicationsand additional antibiotics. In the cefuroxime/erythromycin group, intravenous erythromycin was associatedwithincreasedintravenousadministration costs, as well asthe use and costof nonstudy antibiotics.
DISCUSSION The present study was designedto compare the efficacy, safety, and cost of two antibiotic regimens for the treatment of community-acquired bacterial pneumonia in persons requiring hospitalization for acute care intravenous therapy yet suitablefor oral antibiotics. Demographic features andclinicalandlaboratoryfindingsin our study population were similarin the two groups and to those described in earlier reports on community-acquired bacterial pneumonia Adprior antibiotic treatment with no response vanced age (mean age 2 and underlying cardiorespiratoryor metabolic disease were common. Of the studied patients, (three in cefuroxime/erythromycin,two in cefuroxime/clarithromycin)died. This figure is comparable to the mortality reported in patients with CAP in other studies Overall successful treatment rates of for cefuroxime/clarithromycin group, and for cefuroxime/erythromycin group were observed. The rate of eradication of the pathogen was similar the in two groups and respectively). Serious side effects and allergy were infrequent in both groups. The cefuroxime/erythromycingroupincurredmorecost than the cefuroxime/clarithromycin group. The financialburden of the use of erythromycin inthe cefuroxime/erythromycin group is apparent not only in the actual costof drug but also in its intravenous administration costs. The cefuroxime/erythromycin group also spent more on the use of other antibiotics and their administrationdue to more treatment failures, side effects, and antibiotic resistance. The final cost analysis shows that less was
Comparative Study of CAP Table 4 Cost Analysis of Two Study Groups
Cefuroxime plus Cefuroxime plus clarithromycin erythromycin group (n=30) group (n=30) Duration of hospitalization in days mean (range) Duration of cefuroxime in days mean (range) Duration of erythromycin in days mean (range) Duration of clarithromycin in days mean (range) Drug acquisition costs Cefuroxime Erythromycin Clarithromycin Other antibiotics failures, side effects, antibiotic resistance Intravenous administration costs Erythromycidcefuroxime Nonstudy antibiotics Total antibiotic costs
7.6
7.5 4.1 (2-12)
NA 10.2 (3-12) $2397 $840 $1410 $1974
$2091
$3900 $840
$1950 $360
$11,361
$6,777
NA $1530 $846
spent in the cefuroxime/clarithromycingroup, representing a40% savings over the cefuroxime/erythromycin group. The new oral macrolide antibiotics provide a cost-effective alternative to.standard treatment in elderly patients with underlying disease suitable for oral antibiotic treatment. We also concludethat for some patients hospitalized with community-acquired pneumonia, only a short course of intravenous antibiotic therapy isnecessary. The. resultsof this study show that both intravenous cefuroxime plus intravenous erythromycin intraand venous cefuroxime plus oral clarithromycin are effective and safe for the treatment for community-acquired bacterial pneumonia, but cost savings were demonstrated with cefuroxime plus clarithromycin.
ACKNOWLEDGMENT This study was supported in part by the William Beaumont Hospital Research Institute and by Abbott Laboratories, Abbott Park, Illinois.
Skupien et al.
REFERENCES 1. Berntsson E, Blomberg J, Lagergard T, Trollfors B. Etiology of communityacquired pneumonia in patients requiring hospitalization. Eur J Clin Microbiol 1985; 4:268-272. 2. Bently DW. Bacterial pneumonia in the elderly: clinical features, diagnosis, etiology, and treatment: Gerontology 1984; 30:297-307. 3. Berk SL, Wiener SL, Eisner LB, DuncanJW, SmithJK. Mixed Streptococcus pneumoniae and gram-negative bacillary pneumonia the in elderly. South Med J 1981; 74:144-146. 4. Dorff GJ, Rytel MW, Farmer SG, Scanlon G. Etiologies and characteristic features of pneumonias in a municipal hospital. Am J Med Sci 1973; 266: 349-358. 5. Ebright JR, Rytel MW. Bacterial pneumonia inthe elderly. J Geriat Soc 1980; 27:220-223. 6. Griffith DE. Pneumonia in chronic lung disease. Infect Dis Clin North 1991; 5467-484. 7. Garb JL, Brown RB, Garb JR, lhthillRW. Differences in etiology of pneumonias in nursing homes and community patients. Am J Med Assoc 1978; 240: 2169-2172. 8. Garibaldi RA. Epidemiology of community-acquired respiratory tract infections in adults. Am J Med 1985; 78(suppl6B):32-37. 9. Karnad A, Salvador A, Berk SL.Pneumonia causedby gram-negative bacilli. Am J Med 1985; 79(suppl lA):61-67. 10. Klimek JJ, Ajemian E, Fontecchio S, Giacewski J, Nemas B, Jimenez L. Community-acquired pneumonia requiring admission to hospital. J Infect Contr 1983; 11:79-82. 11. MacFarlane JT, Finch RG, Ward MJ, MacRae AD. Hospital study of adult community-acquired pneumonia. Lancet1982; ii:255-258. 12. Sullivan RJ, Dowdle W B , Marine MW, Hierholzer JC. Adult pneumonia in a general hospital. Arch Intern Med 1972; 129:935-942. 13. Verghese A, Berk S. Bacterialpneumoniain the elderly.Medicine1983; 62~271-285. 14. White RJ, Blainey AD, Harrison W,Clark SKR. Causes of pneumonia presenting to a district general hospital. Thorax 1982; 36:566-570. 15. Karalus NC, Cursons RT, LengRA, Mahood CB, Rothwell RPG, Hancock B, Cepulis S, WawataiM,Coleman L. Communityacquiredpneumonia: aetiology and prognostic index evaluation. Thorax 1991; 46:413-418. 16. Fang GD, Fine MJ, Orloff J, Arisumi D, Yu VL, Kapoor W, Grayston JT, Wang SP, Kohler R, Muder RR, Yee YC, Rihs JD, Vickers RM. New and emerging etiologiesfor community acquired pneumonia with implications for therapy. Medicine 1990; 69:307-316. 17. Donowitz GR, Mandell GL. Empiric therapy for pneumonia. Rev Infect Dis 1993; (SUPPI 5):40-51. 18. Larsen RA, Jacobson JA. Diagnosis of community-acquired pneumonia: experience of a community hospital. ComprehensiveTher 1984; 10:20-25.
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19. Farr BM, Sloman AJ, Fisch UT. Predicting death in patients hospitalized for community-acquired pneumonia.Ann Intern Med 1991; 115:428-436.
BIBLIOGRAPHY Engle JC, Lifland PW, Schleupner CJ. Comparison of ceftazidimewithcefamandole for therapy of community-acquired pneumonia. Antimicrob Agents Chemother 1985; 28:146-148. Fekety FR, Caldwell J, Gump D, Johnson JE, Maxson W, Mulholland J, Thoburn R. Bacteria, viruses, and mycoplasmas in acute pneumonia in adults. Am Rev Respir Dis1971; 104:499-507. Fiala M. A study ofthe combined roleof viruses, mycoplasmas in acute pneumonia in adults. Am J Med Sci 1969; 257:44-51. Finch R, MacFarlane JT, Selkon JD, Watson J, White RJ, Winter JH, Woodhead MA. Guidelines for the management of community-acquired pneumonia in adults admitted to hospital. Br J Hosp Med 1993; 49:346-350. Foweraker JE, Cooke NJ, Hawkey PM. Ecology of Haemophilus influenzae and Haemophilus parainfluenzaein sputum and saliva and effects of antibiotics or their distribution in patients withlowerrespiratory tract infection. Antimicrob Agents Chemother 1993; 37:804-809. Lode H. Initial therapy of pneumonia. J Med 1986; 80 (suppl5C):70-74. McCabe WR, Jackson GG. Gram-negative bacteremia. I: etiology and ecology, Arch Intern Med 1962; 110:847-855. Murray PR, Washington JA. Microscopic and bacteriologic analysis of expectorated sputum. Mayo Clin Proc 1Y5; 50:339-344. National Committeefor Clinical Laboratory Standards. Performance Standards for Clinical laboratory Standards. Approved Standard M7-A2. Villanova, PA: NCCLS, 1990. National Committeefor Clinical Laboratory Standards. PerformanceStandards for Microdilution Susceptibility Tests. Approved Standard M2-A4. Villanova, PA: NCCLS, 1990. Rhind GB, Gould GA, Ahmad F, Croughan MJ, Calder MA. Haemophilus parainfluenzae and Haemophilus influenzae respiratory infection: comparison of clinical features. Br Med J 1985; 291:707-708. Wallace RJ, Niefield SL, WatersS, Waters B,Awe RJ, Wiss K, Martin RR, Greenberg SB. Comparative trial of cefonicid and cefamandole in the therapy of community-acquired pneumonia. Antimicrob Agents Chemother 1992; 21: 231-235. WeberDJ,CalderwoodSB,KarchmerAW,Pennington JE. Ampicillinversus cefamandole as initial therapy for community acquired pneumonia. Antimicrob Agents Chemother 1987; 31:876-882.
Azithromycin in the Treatment of Community-Acquired Pneumonia K. Golec, M. Rzeszutko-Grabowska, D. Bukowska-Nierojewska District Hospital No. 2 Rzesrbw, Poland
INTRODUCTION Most cases of community-acquired pneumonia (CAP) in adults can be treated effectively without hospitalization. Considering the variety of causative pathogens and the necessity of immediate administration of antimicrobial therapy,the selection of an appropriate drug forthe treatment of CAP is often based on empirical premises. The drug of choice should be well tolerated, easily administered, and highly effective against all common pathogens involved in CAP. Azithromycin, an azalide antibiotic with distinct pharmacokinetic and pharmacodynamic properties, seems to comply with these requirements and offers a considerable -advance inthe outpatient treatment of CAP. The aim of the study wasto assess the efficacy of 5day azithromycin therapy inthe management of CAP in adults.
PATIENTS AND METHODS Patients of both sexes withCAP were included in an open, noncomparative study. CAP was defined as an acute illness with typical clinical manifes626
Azithromycin in Treatment
CAP
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~3
Failure (5%)
(95%) Figure Z Clinical efficacyof azithromycin in the treatmentof community-acquired
pneumonia. tations, radiologicalfindingsindicatingpneumonia,andhematological changes in peripheral blood suggesting bacterial infection (leukocyte count >lO,OOO/mm3 with >60% neutrophilic granulocytes). Bacteriologicaltests (sputum culture with identification of isolates and drug-sensitivity determination according to NCCLS standards) were performed on all patients. Azithromycin was given once daily for 5 days, 500 mg on the first day, followed by 250 mg on the subsequent 4 days. Clinical examination and bacteriological tests were repeated and 9-10 daysafter the beginning of treatment.
RESULTS A total of 40 patients, aged 15-85 years, were included inthe study. In 38 of them (95%), no clinical symptoms of disease were found on days 9-10 (Fig. 1).In cases, significant clinical improvement with disappearance of fever was obtained by the third day of treatment. Baseline sputum culture was positive for respiratory tract pathogens in 32 patients. Isolated pathogens and their eradication ratesare presented in Table1. In three patients Escherichia coli coexistedwith Streptococcus pneumoniae. In 26/32patients, the pathogen was eradicated from sputum within days, in 3 cases within 7 days, and in1case within 9 days after the initiation of azithromycin therapy. In eight patients, sputum specimens were not suitable for evaluation due to contamination with oral organisms. These patients might have had infection with organisms that do not grow on routinely used media (e.g., Chlamydia sp. or Mycoplasma sp.). However, in all of them, the treatment was successful. There were two treatmentfailures:apatientwith Huemophilus paruinfluenzae who was hospitalizeddue to generalized deterioration and a second patient who needed additional treatment dueto persistence of the
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Table Z Bacteriological Efficacy Azithromycin in the Treatment of Community-Acquired Pneumonia
n eradicated / n isolated
Pathogen
17/17 515 414 U2 112 111 111 011 111 111
Streptococcus pneumonia@ Escherichia colia Haemophilus inffuenzae Moraxella catarrhalis Klebsiella pneumoniae Streptococcus agalactiae Staphylococcus aureus Haemophilus parainjluenzae Pseudomonas aeruginosa Serratia spp.
(94%)
Total ~~~
~
.In three patientsE. coli coexisted with S. pneumoniae.
initial pathogen (Klebsiella pneumoniae).Azithromycin was well tolerated and only one patient complained of slight dizziness.
CONCLUSION The results indicate that azithromycin, given once daily for 5 days, is clinically and bacteriologicallyeffective and well tolerated in the treatment of community-acquired pneumonia.
Azithromycin: 3-Day Versus 5-Day Dosage Regimen for Community-Acquired Pneumonia in Children B. Ficnar andN. Huzjak Pediatric University Hospital Zagreb, Croatia
I. Klinar and M. Matrapazovski Pliva d.d. Pharmaceuticals Division Zagreb, Croatia
INTRODUCTION Azithromycin,anazalideantibiotic,isan appropriate choice for the initial treatment of community-acquired pneumonia because it is active against all common causative pathogens (i.e. , Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae). In contrast to other oral wide-spectrum antibiotics,the unique pharmacokineticproperties of azithromycin enable simple and short dosage regimens, once daily for or 5 days, a particular advantage in pediatric practice. The aim of thisstudy was to compare the efficacyandsafety of 3-day and 5-day azithromycin courses inthe treatment of community-acquired pneumonia in children.
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Ficnar etal.
PATIENTS AND METHODS open, randomized, multicentric study was conducted in 24 study centers, placed in 16 Croatian towns, between February 1994 andJune 1995. The study protocol was approved by local ethics committees. Children of both sexes, aged 6 months to 12 years, with signs and symptoms consistent with pneumonia [cough and/or auscultatory findings (rales or evidence of pulmonary consolidation)with or without fever or leukocytosis (blood leukocyte count >lOX106/L with neutrophils or >7% band forms)] were included. Clinical diagnosis of pneumonia was confirmed by the presence of new infiltrate on chest x-ray. Before inclusion, informed consent was obtained from the patient's parent. Patients with hypersensitivity to macrolides, severe renal or hepatic impairment, gastrointestinal tract disturbances which affect drug absorption, acute viral infection and fibrocystic disease were excluded as were immunocompromised patients and patients who received any antibiotic within 24 h prior to entering the study or depot-penicillin in the past 2 weeks. Azithromycin (Surnamed, Pliva) was administered orally, once daily, 1 hbefore or 2 hafterameal.Patientswererandomized to receive azithromycin for 3 days, 10mgkg daily (3-day group)or for 5 days, 10mg/ kg on day 1,followed by 5 mgkg from days2-5 (5-day group). Clinical examination was performed at baseline and 72 h, 6 days, 10 days, and 3 weeks afterthe start of treatment. Chest x-ray was performed at baseline and optionally 10 days after the start of treatment. When possible, an appropriate specimen for microbiological studies (sputum, endotracheal aspirate, pleural fluid, or blood) was obtained at baseline, 72 h, and 10 days after the start of treatment. Pneumonia due to Mycoplasma pneumoniae wasidentifiedby at least a fourfold rise in serum titer of complement-fixing antibodies. Clinicalresponsewasdefined cure (complete disappearance of clinical symptoms of pneumonia within 5 days after the start of treatment with partial or complete regression of infiltrate on control chest x-ray)or failure (allother outcomes). Bacteriological responsewas defined as eradication of initial pathogen, presumed eradication (posttreatment cultures were not performed due to the complete .resolutionof clinical signs and symptoms), persistence,or relapse. All. adverse events were recorded at each visit and classified according to severity 'and causative relationship to the study drug. Laboratory safety tests (hematology, biochemistry) were undertaken at baseline and 10 days after the start of treatment.
Azithromycin: 3-Day Table I
Versus 5-Day Regimen for CAP
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BaselinePatientCharacteristics 3-Day azithromycin
Total no. of patients Femalelmale Mean age (years) Age range Mean weight (kg) Weight range (kg)
70 38/32 6.6 8 months-l2 years 22.1 9-74
5-Day azithromycin 85 41/44 9 months-l2 years 23.8 9-61
Clinical and bacteriological response between the two groups was compared by Cochran-Mantel-Haenszel statistics based on Ridit scores and the incidence of side effects and laboratory abnormalities by Fisher’s exact test (two-tailed). Differences were considered to be significant if p