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Løc Ç to To+Z Lw'le Bound by 1 0285379 .E Bbey Bookblndlng Co Løc ç to to+z lw'lE Bound by 1 0285379 .E bbey Bookblndlng Co. I ló CathaysTenace, Cardllf Ct2{ {HY Soüth l{â1e3, U.K, Tel: (019) 10395881 ililf I I iltil I lltf I il I ilfilil ilIil UWIC IEARNING CEI'ITRE TIBRARY DIVI$ION.[IÅNDAFF WESTËRI.N AI'ENUE CAROIFF Y cr5 2YË Char acteri s ation of B urkh o I d eri a c ep a ci a from clinical and environmental origins. Paul Wigley BSc. (Hons) University of Wales Institute Cardiff Submiued for the degree of Doctor of Philosophy Discipline : Microbiolo gy September 1998 ABSTRACT Burkholderia cepacia isolates were obtained from the sputum of cystic fìbrosis patients or isolated from the environment. The isolates were characterised phenotypically by determining antibiotic resistance profiles and their ability to macerate onion tissue, and genetically by PCR ribotyping and macro-restriction analysis (genome fingerprinting). The replicon (chromosomal) organisation was determined by pulsed field gel electrophoresis @FGE) and the presence of plasmids was also investigated. Plasmid transfer by conjugation was also investigated. A high degree of genetic and phenotypic variation \ryas found both within and between clinical and environmental populations of B. cepaciø. Environmental isolates were generally less resistant to antibiotics but showed greater ability to macerate onion tissue. Genetic characterisation showed little evidence of acquisition ofB. cepacia from the environment by CF patients, but revealed strong evidence supporting person- to-person transmission ofB. cepacia in the CardiffCF centre and other European centres. Two or more chromosomes were found in 93 Yo of B. cepacla isolates tested, and were also found in other closely related species suggesting such organisation may be common inþ-2 proteobacteria. Plasmids, often in excess of 100 kb, were found to be harboured in 52 Yo of isolates, though were more commonly found in CF isolates (65 % of isolates) than environmental isolates (23 % of isolates). Plasmid transfer by conjugation was demonstrated into, between and from,B. cepacia strains. Evidence of plasmids encoding antibiotic resistance was also found I CONTENTS PAGE ABSTRACT I CONTENTS 2 PUBLICATIONS I TABLE OF FIGURES 10 ACKNOWLEDGMENTS 11 LIST OF ABBREVIATIONS 13 CHAPTER 1 General Introduction L4 l.l Burkholderia cep øcía t4 l. 1. I History andtaxonomy t4 1.1.2 Characteristics of B. cepacia l9 1.1.3 Genetic organisation ofB. cepacia 20 1.1.4 Clinical importance ofB. cepacia 23 1.2 Cystic fïbrosis and microbiology of CF 26 1.2. 1 Cystic fìbrosis 26 1.2.2 Microbiology of CF: General considerations 29 1.2.3 Staphylococcus aureus 30 L2. 4 P s eu dom onas aeru gi nosa 3l 7 .2.5 Haemophilus influenzae JJ 1 .2.6 Stenotrophomonas maltophilia 33 1.2.7 Other bacterial, fungal and viral agents 34 1.3 ß. cepacía in cystic fïbrosis 35 I.3.L B. cepacia respiratory disease 36 1.3.2 Virulence factors & immune evasion 38 l.4Isolation and identification of B. cepacía 43 1.4.1 Isolation of-B. cepacia 43 I.4.2Identification 45 1.4.3 Typing 48 L.5 Epidemiology of B. cepøciø in CF 53 1.5. I Person to person transmission 53 2 1.5.2 Non-CF infections 56 1.6 B. cepacía in the environment 58 1.6.1 B. cepacia as a ph¡opathogen 58 1.6.2 Production of antibiotics and biocontrol by B. cepacia 59 1.6.3 B. cepacia biodegradation and bioremediation 62 1.7 Antibiotics and antimicrobial chemotherapy and their 65 use in B. cepacía infections 1.7.1 Action of antimicrobial drugs 65 L.7.2Drugs that inhibit cell wall synthesis 65 1.7.3 Drugs that inhibit protein synthesis 67 I.7.4Drugs that inhibit nucleic acid synthesis 69 1.7.5 Drugs that inhibit membrane integrity 7T 1.7.6 Antibiotic uptake 72 1.7 .7 Antibiotic therapy in B. cepacia infection 72 1.8 Antibiotic resistance in ß. cepacía 76 1.8.1 Mechanisms of resistance 76 1.8.2 Intrinsic and acquired resistance in B. cepacia 77 L.9. Plasmids in B. cepacía 82 1.9.1 Antibiotic resistance plasmids in.B. cepacia 84 1.9.2 Degradative plasmids in B. cepaciø 86 1.10 Comparative properties and relationships of clinical 88 and environmental B. cepacía isolates 1. 10. 1 Epidemiology of environmental and clinical isolates 90 l,.lL Aims of study 92 CIIAPTER 2 Isolation and typing of Burkholdería cepacía 93 2.1 Introduction 93 2.2 Materials and methods 94 2.3 Results 105 2.3 .I Environmental survey # I 105 3 2.3.2 Growth of,B. cepacia and other species 106 on selective media 2.3 .3 Environmental survey #2 lll 2.3.4Recovery rates of B. cepacia in Environmental 113 Surveys #l&#2 2.4 Discussion tt4 2.4.1 Assessment of selective media tt4 2.4.2Recovery rate of B. cepacia from the environment t17 2.4.3Efficacy of selective media in environmental and 119 clinical isolation of B. cepacia 2.4.4 Isolation and identification ofB. cepøcia'. t2t General considerations CHAPTER 3 Phytopathogenicity of ßurkholdería cepacia 122 3.L lntroduction 122 3.2 Mnterials and methods t23 3.3 Results 127 3 .3.I In vitro maceration of onion tissue 127 3.3.2 Quantitative test of onion maceration t32 3.4 Discussion 135 3.4.1 Invilro maceration of onion tissue- 135 3 .4 .2 Development of a quantitative assay of onion 136 maceration CHAPTER 4 Antibiotic resistance and susceptibility of 138 Burkholdería cepacía 4.1 Introduction 138 4. 1.1 Kirby Bauer disc diffi.rsion test 138 4. 1 .2 D etermination of minimum inhibitory concentration t40 (MIC) by agar dilution 4.2 Materials and methods 142 4 4.3 Results 1,45 4.3.1 Kirby Bauer disc diffirsion test for determination of t45 antibiotic reisitance/susceptibility 4.3.?MIC determination by agar dilution 153 4.4 Discussion t57 4.4. I Antibiotic resistance/susceptibilþ r57 4 . 4 .2 lÙr/.IC determination 160 4.4.3 Resistance in environmental and clinical isolates 162 4 . 4 . 4 Antimicrobi al re si stance/susceptibility : 163 General considerations CHAPTER 5 Isolation and purification of plasmid DNA from 165 ßurkholdería cepacía 5.1 fntroduction 165 5. 1. I Principles of plasmid DNA isolation and purification 165 5.1.2 Plasmid isolation and detection in B. cepacia t69 5.1.3 Methods for detection and purification of plasmid DNA r69 used in this study 5.2 Materials and methods t70 5.3 Results 178 5.3.1 Isolation of plasmid DNA 178 5.3.2 Estimation of plasmid size and distribution of plasmids l8l 5,3.3 Isolation of plasmid DNA for transformation and 188 plasmid mapping studies 5.3.4 Amplification of plasmid DNA by growth in Terrific Broth 188 and by chloramphenicol amplification 5.4 Discussion 189 5.4.1 Detection of plasmid DNA 189 5.4.2 Sizing of B. cepacia plasmids 191 5.4.3 Distribution and sizes of plasmids 193 5 5.4.4 Isolation of8. cepacia plasmid DNA for restriction t94 mapping and transformation studies 5.4.5 Improvements, alternatives and future work 195 CHAPTER 6 Plasmid curing and its effect on phenotype 197 6.1 Introduction t97 6.2 Materials and methods 198 6.3 Results 201 6.3.1 Loss of antibiotic resistance by curing 201 6.3.2 Investigation of plasmid loss 202 6.4 Discussion 2to CIIAPTER 7 Conjugal transfer of plasmid DNA in Burkholdería cepacía 2L3 7.1 fntroduction 2t3 7. l. I Investigation of conjugal transfer of plasmid DNA 213 7 .l.2 Coryugative DNA transfer in the epilithon 214 7 .2 lÑf.nterials and methods 2t6 7.3 Results 226 7.3. I Transfer of broad host range plasmids 226 7 .3 .2 Transfer of antibiotic resistance between B. cepacia strains 232 7 .3.3 Exogenous isolation of mercury resistance plasmids 233 from river epilithon with aB. cepacia recipient 7.4 Discussion 234 7.4. t Conjugal transfer of broad host range plasmids 234 7.4.2Transfer of piperacillin resistance between B. cepacia 238 strains 7 .4.3 Exogenous isolation of mercury resistance plasmids 239 from the environment 7.4.4 General considerations and future work 239 6 CHAPTER 8 Typing of Burkholdería cepøcía isolates 240 8.1 fntroduction 240 8.1.1 PCRRibotyping 240 8.1.2 Typing ofB. cepacia by macrorestriction analysis 241 8.2 Materials and methods 244 8.3 Results 253 8.3.1 PCR ribotyping 253 8. 3.2 Macrorestriction analysis 256 8.4 Discussion 260 8.4.1 Typing ofB. cepacia by PCR ribotyping and 260 macrorestriction analysis 8.4,2 Signifïcance of typing results 261 8.4.3 Failure of specific PCR primers 263 8.4.4 Problems with, and improvements to methods 263 CHAPTER 9 Investigation of multiple replicons in Burkholderia cepacía 266 9.1 Introduction 266 9. 1.1 Multiple bacterial replicons 266 g.l.ZDetection of rRNA genes on multiple replicons 267 9.2 Materials and methods 269 9.3 Results 272 9.3.1 Detection of multiple replicons 272 9.3.2 Size estimation of large replicons inB. cepacia 282 9.3.3 Detection of rRNA genes on multiple replicons by 289 PCR ribotyping 9.4 Discussion 290 9.4.1 Multiple replicons in B. cepacia 290 9.4.2Multtple replicons in other species 294 9.4.3 Identification of rRNA genes on replicons by 295 PCR ribotyping 7 9.4.4Identification and sizing of multiple replicons: 297 improvements and alternatives CHAPTER 10 General discussion and conclusions 299 10.1,B. cepacía in the environment 299 10.2 Relationship between clinical and environmental isolates of 304 B.
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