STUDIES ON THE BACTERICIDAL ACTIVITIES OF THE 4-QUINOLONES Thesis submitted by Bernadette Maria Anne Howard for the degree of Doctor of Philosophy in the University of London. The Microbiology Section, June 1991 Department of Pharmaceutics, The School of Pharmacy, University of London. ProQuest Number: U049390 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 U049390 Published by ProQuest LLC(2017). 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 1 ABSTRACT The 4-quinolones act primarily on the A subunit of DNA gyrase coded by the gyrA gene. The nalA mutation in gyrA was found to abolish mechanism B which is the first evidence that mechanism B may reside within gyrase. The coumarins novobiocin and coumermycin inhibit the B subunit of DNA gyrase. The sensitivities of the Escherichia coli KL16 nalR mutants to both coumarins were investigated. New novobiocin-resistant (nov*) and coumermycin-resistant (couR) mutants of E.coli KL16 and Staphylococci warneri were isolated and their sensitivities to ciprofloxacin, novobiocin and coumermycin determined. Both species showed incomplete cross-resistance. Other workers have found that the 4-quinolone antibacterials do not seem to interact with the coumarin antibacterials when minimum inhibitory concentration tests were used to judge interactions. However, when bactericidal interactions between the 4- quinolones and novobiocin or coumermycin were studied in this thesis with E.coli KL16 and Staphylococci significant antagonism of the 4-quinolones by the coumarins was found in all combinations tested. These results agreed with in vivo findings, which hence disagreed with the in vitro results of other workers. SOS DNA repair did not repair damage caused by nalidixic acid, while recombination repair did repair damage caused by the drug. Ciprofloxacin- ofloxacin- and norfloxacin-induced damage, however, showed some differences as regards these DNA repair systems. Nalidixic acid possesses only one mechanism of bactericidal activity, termed A, ciprofloxacin and ofloxacin possess mechanisms A, B and C, while norfloxacin possesses mechanisms A and C. Mechanism B, which does not require protein synthesis or RNA synthesis nor bacteria capable of dividing, was found to operate when drug concentrations reached the co-operative binding concentrations with supercoiled DNA. The effect of the 4-quinolones and coumarin antibacterials on DNA supercoiling was investigated by alkaline or in situ lysis. In-situ lysis was found to be more appropiate than alkaline lysis for such investigations. 4-quinolones are known to exhibit post antibiotic effects (PAE’s). E.coli KL16, Staph, aureus, Klebsiella pneumoniae and Streptococcus pyogenes were investigated for PAE’s with ciprofloxacin, ofloxacin and a new cephalosporin, cefdinir. PAE’s were found with all three drugs in Staph, aureus and Strep, pyogenes. However in E.coli PAE’s were only found with ciprofloxacin or ofloxacin and were generally absent with Klebsiella pneumoniae. 3 ACKNOWLEDGEMENTS I would like to express my deepest thanks and appreciation to Professor J.T. Smith for his supervision, encouragement and support over these last 5 years. I would also like to acknowledge my gratitude and thanks to: Mrs. K. Wood and the technical staff of the Microbiology Section for their expertise and assistance; Dr Phillipe Mazodier and colleagues at Institut Pasteur, Paris for their friendship and kindness; Dr. Clement Lewin as a colleague and friend for his much appreciated support and advice; colleagues past and present for their enthusiasm and support; to the Blatchley family and Turner family who cared and supported me through my illness and all my friends and family for their prayers which have been answered; Miss Jayne Pascoe for her much appreciated friendship and encouragement; and finally to my parents and brother to whom this book is dedicated for their encouragement and support throughout. This thesis was funded by the S.E.R.C. "..When I applied my mind to know the wisdom and to observe man’s labour on earth-his eyes not seeing sleep day or night- then I saw all that God has done. No-one can comprehend what goes on under the sun. Despite all his efforts to search it out, man cannot discover its meaning. Even if a wise man claims he knows, he cannot really comprehend it." Ecclesiastes 8, vl6-17 4 INDEX TO CONTENTS PAGE NUMBER Abstract 1 Acknowledgements 3 Index to contents 4 Index to figures 8 Index to tables 17 Introduction 20 A The Bacterial Chromosome 20 B DNA topoisomerases 21 B.l Topology and energetics of supercoiling 21 B.2 Nomenclature of DNA topoisomerases 23 B.2.1 Type I topoisomerases 23 B.2.1.1 Prokaryotic Type I topoisomerases 23 B.2.1.2 Eukaryotic Type I topoisomerases 24 B.2.2 Type 13 topoisomerases 25 B.2.3 DNA gyrase 25 B.2.3.1 Discovery of DNA gyrase 25 B.2.3.2 Structure of DNA gyrase 26 B.2.3.3. Extraction of DNA gyrase 26 B.2.3.4 Supercoiling of DNA by gyrase 27 B.2.4 Other Type II topoisomerases 28 B.2.4.1 Prokaryotic topoisomerase II’ 28 B.2.4.2 Topoisomerase IV 29 B.2.4.3 T4 DNA topisomerase 29 B.2.4.4 Reverse gyrase 29 B.2.4.5 Eukaryotic type II topoisomerases 30 B.3 Comparison of eukaryotic topoisomerase II with 30 E.coli DNA gyrase B.4 Topoisomerases in mitochondria and chloroplasts 31 and viruses C Physiological role of Topoisomerases 31 C.l Topoisomerases and the level of supercoiling in vivo 31 C.2 Topoisomerases and DNA replication 32 5 INDEX TO CONTENTS (contd.) PAGE NUMBER Introduction (contd.) C.3 Supercoiling effects of topoisomerases on 34 transcription and gene expression D Inhibitors of DNA topoisomerases 40 D.l The coumarin antibacterials 40 D.2 The 4-quinolones 42 D.2.1 Selectivity of the 4-quinolones 44 D.2.2 Clinical use of the 4-quinolones 44 D.2.2.1 In vitro inhibitory antibacterial activity of the 45 4-quinolones D.2.3 Mechanisms of killing of bacteria by 4-quinolones 46 D.2.3.1 Kinetics and Characteristics of killing 47 D.2.3.2 Antagonism of the bactericidal effects of the 47 4-quinolones D.2.3.3 Differences in killing activity among 4-quinolones 48 D.2.3.4 Quinolone-DNA interactions 49 D.2.3.5 Inhibition of DNA synthesis 51 D.2.3.6 DNA damage 51 D.2.3.7 The SOS repair system 52 D.2.3.7 a The recA protein 52 D.2.3.7 b The lexA protein 53 D.2.3.7 c Inducible repair involving recombination functions 53 D.2.3.8 Post antibiotic effect 55 D.2.4 Quinolones and other antibacterials 56 D.2.5 Quinolones and resistance 57 D.2.5.1 Chromosomal mutations in DNA gyrase 58 D.2.5.2 Mutations affecting permeability 59 D.2.5.3 Frequency of resistance to the 4-quinolones 62 INDEX TO CONTENTS (contd.) PAGE NUMBER Abbreviations 64 Materials 67 A Liquid media 67 B Solid media 67 Antimicrobial Agents 69 Bacterial Strains 70 Experimental Methods 75 1. Agarose gel electrophoresis 75 - Chemical reagents and solutions used for alkaline 75 lysis of plasmids for gel electrophoresis - Chemical reagents and solutions for in situ lysis 78 for gel electrophoresis - Method A: Investigation of plasmids in bacteria 79 previously treated with 4-quinolones in intact cells - Protocol for extraction of plasmid DNA by alkaline 80 lysis - Preparation of Agarose gels 81 - Loading the gel 81 - Running the gel 82 - Visualisation of gel 82 - Method B: In situ lysis of large plasmids 83 - Preparation of agarose gels 83 2. Mininmum inhibitory concentrations 84 - Preparation of plates 84 - Preparation of cultures 84 3. Fractional Inhibitory Concentration Tests 85 4. Bactericidal tests 85 5. Measurement of the rate of kill by 86 4-quinolones 6. Isolation of resistant mutants 87 7. Measuring the UV survival of UV sensitive 88 bacteria 8. Post Antiobiotic Effect 88 INDEX TO CONTENTS (contd.) PAGE NUMBER RESULTS Chapter One The action of the 4-quinolones on DNA gyrase mutants. 90 Chapter Two Sensitivity and mutational resistance to the 109 coumarin antibacterials. Chapter Three Interactions between the bactericidal effects of 131 4-quinolones and other gyrase inhibitors. Chapter Four The effect of 4-quinolones on recombination and 173 SOS repair. Chapter Five Mechanisms of kill of the 4-quinolones. 190 Chapter Six Development of an in vitro assay to determine the 207 effect of gyrase inhibitors on plasmid supercoiling. Chapter Seven Post antibiotic effects of ciprofloxacin, ofloxacin 228 and a new cephalosporin, cefdinir. DISUSSION 262 1. -The effect of DNA gyrase mutations on the 262 bactericidal mechanisms of the 4-quinolones. 2. -Sensitivity and mutational resistance to the 264 coumarin antibacterials. 3. -The effects of combinations of 4-quinolones and 266 coumarin antibacterials. 4. -The role of DNA repair processes in 4-quinolone 268 lethality. 5. -The characteristics of mechanism B and C and 269 the possible involvement of co-operative binding. 6. -Changes in supercoiling in intact bacterial cells. 271 7. -The potential role of 4-quinolones and cefdinir 273 based on the post antibiotic effects. REFERENCES 276 8 INDEX TO FIGURES FIGURE TITLE PAGE NUMBER 1 Chemical structure of the coumarin antibacterials.