THE ISOLATION, CHARACTERISATION AND CLASSIFICATION OF BACTEROIDES UREOLYTICUS AND ITS ASSOCIATION WITH NON-GONOCOCCAL URETHRITIS A thesis submitted for the degree of*DOCTOR OF PHILOSOPHY . by ©W ELIAS ALISTAIR FONTAINE Division of Sexually Transmitted Diseases Clinical Research Centre, Harrow in collaboration with Department of Microbiology University of Surrey March, 1936. ProQuest Number: 10798461 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10798461 Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 This thesis is dedicated with great affection and love to MY PARENTS FRONTISPIECE Colonial appearance of NGU-associated strains of Bacteroides ureolyticus (a) grown on formate-fumarate anaerobic blood agar, on primary isolation. (b) grown on plain, Columbia base blood agar. SUMMARY The primary aim of this study was to determine whether anaerobes played a significant role in the aetiology of non-gonococcal urethritis (NGU). Chlamydia trachomatis and Ureaplasma urealyticum are responsible for about 50-60% of cases of NGU, so it is plausible that anaerobes may play a role in those cases with no known aetiology. The occurrence of the anaerobe Bacteroides ureolyticus in the genito-urinary tract of men with and without disease was considered, and microbiological and clinical studies performed in conjunction with the recognised aetiological agents, demonstrated a striking association between this anaerobe and NGU. The primary cultivation of B. ureolyticus on non-enriched blood agar was practicable despite its fastidious metabolism, in particular a formate-fumarate energy-requirement for growth. Several.improved media were developed: namely, a transport medium of low redox-potential, liquid and enrichment media, solid media and a selective agar medium. Results of the various media are presented. The transport medium was effective in maintaining the viability of anaerobes during transportation, and together with the sampling procedure, produced optimal recovery from swabs. The selective medium containing vancomycin, polymyxin and tri­ methoprim was equally effective in facilitating the recovery of B. ureolyticus from clinical specimens. Methods for the identification of Gram-negative anaerobes in general are described. They were based on biochemical tests, antibiotic- disc resistance tests, and on tests of tolerance to bile salts and dyes. Such a scheme enabled the identification of unknown anaerobes. However, the definitive identification of several strains of B. ureolyticus posed a few problems when using a large number of diagnostic tests, mainly because of the heterogeneity of the species. Methods of classification V by numerical taxonomy, and characterisation aids such as ultrastructure and the electrophoretic pattern of major cel1-proteins are described. Serological studies showed that at least 30% of chlamydia-negative cases of NGU were probably due to B. ureolyticus, and organ culture studies demonstrated toxin-mediated damage to mucosal epithelia. The significance of B. ureolyticus as a potential pathogen and probable aetiological agent in NGU is discussed. ACKNOWLEDGEMENTS The work presented in this thesis formed part of a study assigned to me as Senior Research Officer at the Clinical Research Centre, Harrow. I am greatly indebted to Dr David Taylor-Robinson (Divisional Head, Sexually Transmitted Diseases) and Dr D.A.J. Tyrrell (formerly Head of the Division of Communicable Diseases), for their initial encouragement and advice to carry out this work in the Division. I express my sincere appreciation to Professor C.A. Armstrong-Esther under whose direction the project was set up, and to Professor J.E. Smith and Dr T.N. Bryant, for their advice and encouragement in developing this study to encompass numerical taxonomic classification. In particular, I am especially indebted to Dr Bryant for his indefatigable interest and zeal in performing all the computations on the University of Surrey Prime 750 Computer System. I am also grateful to Dr Michael Butler who took over final responsibility for the .submission of the thesis and provided invaluable encouragement in its completion. My most grateful thanks go to Mrs Heather A. Davies of the Clinical Research Centre for her stimulating interest, cooperation and kindly considerations, and whose collaborative efforts in this study, helped to produce the excellent electron micrographs. To Dr S.P. Borriello of Communicable Diseases, I offer my special thanks for his advice and guidance in some aspects of this work. I should also like to express my sincere thanks to the following: Dr R.J. Owen and Mr A.J. Taylor of the Central Public Health Laboratory (NCTC) for the elucidation of DNA base composition and hybridisation; Dr D.G. Smith of the Department of Botany & Microbiology, University College (London) for aspects relating to "twitching" motility; Dr N.F. Hanna and Dr E.D. Coufalik (formerly of this Division) , for their painstaking efforts in the collection of clinical specimens for this study. Finally, I offer my gratitude to those not mentioned, but vfao helped in some way or other. The clinical aspects of this work were approved by an Ethics Committee, C.R.C. The work described in this thesis is all my own . except where otherwise stated. V l l l CONTENTS page SUMMARY iv ACKNOWLEDGEMENTS vi TABLE OF CONTENTS viii LIST OF FIGURES xix LIST OF TABLES xxiii GENERAL INTRODUCTION AND SUBJECT REVIEW SECTION ONE 1. Introduction 2 1.1 A short history of sexually-transmitted diseases 4 1.2 The indigenous flora of the male urethra 7 1.3 Infections of the male genital tract and causes 11 1.3.1 Chlamydia trachomatis ' 14 1.3.2 Mycoplasmas (Ureaplasma urealyticum) 15 1.3.3 Trichomonas vaginalis 16 1.3.4 Candida albicans 17 1.3.5 Herpes and Donovanosis .17 1.3.6 Anaerobes 18 1.4 The Bacteroidaceae as members of the microflora of the lower genital tract 19 1.5 Growth requirements of Bacteroides species and anaerobic Gram-negative 'corroding' bacilli 21 1.6 Techniques of anaerobic culture 24 page MATERIALS AND METHODS SECTION TWO DEVELOPMENTAL METHODS FOR MICROBIOLOGICAL STUDIES AND CLINICAL SAMPLING OF THE MALE URETHRA 2.1 Development of methods for the isolation of anaerobes from the urethra of men with NGU 28 2.1.1 Transport media 29 2.1.2 Bacteriological swab 29 2.1.3 Growth media 30 2.1.4 Anaerobic jars and catalysts 30 2.1.5 Bacterial strains and testing procedure 32. 2.2 A preliminary survey of clinical sampling methods 33 2.2.1 Transport media 33 2.2.2 Anaerobiosis with steel-wool premoistened with copper sulphate . 34 2.2.3 Sampling swabs 34 * 2.2.4 Specimens from patients 35 2.2.5 Anaerobic chamber methods 35 2.2.6 Conventional bench methods 36 2.3 Experiments to develop improved media for use in further clinical sampling studies 36 2.3.1 Transport media 37 2.3.2 Liquid media 38 2.3.3 Enrichment media 39 2.3.4 Solid media 40 CONTENTS (continued) page 2.3.5 Selective media 41 2.3.6 Sensitivity testing of antimicrobials for incorporation in selective medium 41 2.3.7 Minimum inhibitory concentration of antimicrobials for use in selective medium 42 2.3.8 Evaluation of a new selective medium 43 2.4 The development of methods for the identification of anaerobic Gram-negative bacilli 45 2.4.1 Culture media and growth factors 49 2.4.2 Storage and maintenance of cultures 50 2.4.3 Anaerobic incubation of test cultures 50 2.4.4 Tests for characterisation of strains 50 (colony morphology, cell morphology, pigment 50 production, capsule formation, beta-haemolysis 51 . and motility 51 l-ipase, lecithinase, oxidase, catalase and urease 52 production . 52 indole production, aesculin hydrolysis, dextran 53 and gelatin hydrolysis 53 gelatin liquefaction, hydrogen sulphide production, 54 nitrate reduction and decarboxylation of amino acids 54 constitutive enzymes and fermentation of carbohydrates 55 carbon utilisation, sensitivity to antimicrobials, 56 inhibition by bile salts/dyes, reduction of dyes) 57 xi CONTENTS (continued) page SECTION THREE CLINICAL INVESTIGATION AND MICROBIOLOGICAL STUDIES 3.1 Clinical and microbiological studies 60 3.2 Clinical evaluation and sampling 61 3.3 Treatment of patients after sampling 63 3.4 Isolation and identification of NGU-associated strains of Bacteroides ureolyticus 64 SECTION FOUR CLASSIFICATION 4.1 Numerical taxonomic study of NGU-associated strains of Bacteroides ureolyticus 67 4.1.1 Sources of strains 69 4.1.2 Temperature of incubation 69 4.1.3 Aerotolerance of strains' 70 4.1.4 Maintenance of strains 70 4.1.5 Culture media * 70 4.1.6 Tests and testing conditions 71 4.1.7 Colonial characteristics of strains 71 4.1.8 Characteristics in liquid culture 71 4.1.9 Biochemical and physiological tests 72 (growth in milk medium, stimulation of growth by 73 nitrate, formate-fumarate requirement for growth, 73 metabolic end-products by gas liquid chromatography) 73 / \ J L- CONTENTS (conti.nued) page 4.1.10 Coding of characters 74 4.1.11 Cluster analysis 75 4.1.12 Computation and identification programs 76 4.2 Ultrastructure-characterisation