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University of Limerick Genotypic and Phenotypic Analysis of Ralstonia

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University of Limerick Genotypic and Phenotypic Analysis of Ralstonia University of Limerick Ollscoil Luimnigh Genotypic and Phenotypic Analysis of Ralstonia pickettii High Purity Water Isolates Michael P Ryan B.Sc. Chemical and Environmental Sciences Dept., University of Limerick A thesis submitted to the University of Limerick in candidature for the degree of Doctor of Philosophy Supervisors: Dr. Catherine C Adley and Prof. J Tony Pembroke Submitted to the University of Limerick: June 2009 Declaration I hereby declare that the work detailed in this thesis is the result of my own investigations. No part of this work has been or is being submitted in candidature for any other degree. ______________________________________________ Michael Ryan Date ______________________________________________ i Abstract Ralstonia is a newly characterised genus that includes former members of the Burkholderia species (Ralstonia pickettii and Ralstonia solanacearum). The type species of the genus-Ralstonia pickettii (type strain, ATCC27511) is a clinical isolate which has been isolated from a wide variety of clinical specimens. Recently it has been isolated mainly as a contaminant of industrial high purity water circulation systems, in space ship water systems and in laboratory high purity water systems including the Millipore systems. To generate a strain collection of R. pickettii for phenotypic and genotypic analysis strains were initially isolated from Millipore laboratory purified water; these were supplemented with culture collection strains, clinical and industrial isolates until a culture collection of fifty-eight strains from different geographic locations and environmental origins was generated. All were initially identified as R. pickettii. A review of the literature demonstrated that this collection represents one of the largest collections of R. pickettii in the world. These fifty-eight strains gave a wide range of isolates from several different environmental niches allowing a phylogenetic analysis of R. pickettii to be undertaken. Characterisation of the fifty-eight R. pickettii isolates of our strain collection using phenotypic and genotypic techniques was undertaken. To determine if any genomic variations were present in the strains, species-specific PCR was preformed. Fourteen strains were found to be in fact the closely related species Ralstonia insidiosa. Following this it was decided to investigate phylogenetic markers such as the 16S rDNA gene. Given that the analysis on the published 16S rDNA gene of R. pickettii from a wide range of environments and geographic locations showed almost no variation, two other sequences were selected for typing and were analysed, the 16S-23S rDNA ISR and the fliC gene. Like the 16S rDNA gene analysis, the 16S-23S rDNA ISR analysis (through RFLP and sequencing) showed very little variation between the R. pickettii isolates. The 16S-23S rDNA ISR analysis also supported the species-specific PCR and showed fourteen strains to be R. insidiosa. The fliC gene sequencing analysis indicated that there are four different types of fliC gene in R. pickettii but these types do not group together based on the origin of the source strain. ii Further genotypic characterisation was undertaken using RAPD (Random Amplification of Polymorphic DNA)-PCR and BOX-PCR and the results of these experiments were analysed by dendrogram analysis. Four different primers were used for the RAPD analysis P3, P15, M13 and OPA3OU which gave twenty-five, twenty-one, twenty-one and fifteen groups respectively. BOX primer BOX-A1R was used for the BOX-PCR and resulted in eighteen groups. No relationship was discovered between or within the different groupings based on their origin. Phenotypic analysis was carried out using both biochemical identification kits (Remel RapID NF Plus, BioMérieux API 20NE) and traditional biochemical tests such as nitrate usage tests, virulence factor determination (elastase, protease, haemolysin) and motility testing to establish if their was any variation due to different environments of isolation. No major variations were found based on the isolate source or between the R. pickettii isolates and those found to be R. insidiosa. Antibiogram profiles for the R. insidiosa subset of strains showed that the majority for were multi-resistant to a range of antibiotics similar to previously published reports for R. pickettii. Mobile Genetic Elements (MGE) can allow bacteria to survive in different environments. To determine if these could be aiding the adaptability of R. pickettii to different environments a study was undertaken to determine if any MGE’s were present in the R. pickettii culture collection. Three ICE-like elements related to Tn4371 were discovered in R. pickettii strains ULM001, ULM003 and ULM006 form our collection and were partially characterised using a PCR approach. Bioinformatic analysis led to the discovery of ten previously uncharacterised Tn4371-like ICE’s in the genomes of several different bacteria. These elements were bioinformatically characterised. Several different genes putatively involved in metabolism and antibiotic and heavy metal resistance were identified. No known virulence determinates were discovered. A common genetic scaffold for all Tn4371-like ICE’s was found and a new nomenclature for all Tn4371-ICE’S was proposed. R. pickettii is a persistant contaminant of HPW and this study provides both phenotypic and genotypic analysis of a collection of R. pickettii and its close neighbour R. insidiosa. iii Acknowledgements I would like to express my sincere thanks to Dr. Catherine Adley and Prof. Tony Pembroke for their invaluable supervision, advice and encouragement throughout the course of my research. I would like to thank all the lads from LG007 for all good times, laughs, debates, cups of tea and free chocolate. Thanks to the guys from the lab Farag, Camelia, Karen, Kamila and Colm for putting up with me at times I probably didn’t really deserve it. I would like to thank my Mum, Dad and sister for all their encouragement and support throughout the years of the PhD. I would like to thanks all my friends Roibeard, Kev, Ivor, Maurice, Mary, Finola, Nicola, Conor, Damien, Rob, Vincent, Sarah and the undoubted many names that I’m forgetting for their friendship and all their support through both good and not so good times. iv Table of Contents DECLARATION ...................................................................................................................... I ABSTRACT ............................................................................................................................ II ACKNOWLEDGEMENTS .................................................................................................. IV TABLE OF CONTENTS ....................................................................................................... V LIST OF TABLES ............................................................................................................. VIII LIST OF FIGURES ............................................................................................................... X LIST OF ABBREVIATIONS ............................................................................................ XII UNITS OF MEASUREMENT .......................................................................................... XIII CHAPTER 1: INTRODUCTION .......................................................................................... 1 1.1.TAXONOMY OF THE RALSTONIA GENUS ............................................................................. 2 1.2.PHENOTYPIC CHARACTERISTICS OF THE GENUS RALSTONIA ............................................. 7 1.3.NATURAL HABITAT OF RALSTONIA SPP ............................................................................. 8 1.4. RALSTONIA PICKETTII ....................................................................................................... 9 1.4.1. Taxonomy of Ralstonia pickettii ............................................................................... 9 1.4.2. Genotypic and Phenotypic Characteristics of Ralstonia pickettii ........................... 10 1.4.3. Clinical significance of Ralstonia pickettii ............................................................. 11 1.4.4. Antibiotic Resistance Patterns of Ralstonia pickettii .............................................. 16 1.4.5. Industrial significance of Ralstonia pickettii .......................................................... 19 1.5. RALSTONIA INSIDIOSA ..................................................................................................... 21 1.6. CLINICAL INSTANCES OF R. MANNITOLILYTICA ............................................................... 23 1.7. BACTERIA IN HIGH-PURITY WATER ............................................................................... 23 1.7.1. Oligotrophic Bacteria .............................................................................................. 24 1.7.2. Water Quality .......................................................................................................... 25 1.8. GENOTYPIC TYPING OF BACTERIAL ISOLATES .............................................................. 31 1.8.1. The 16S rRNA Gene ............................................................................................... 32 1.8.2. The 16S-23S rRNA spacer region ........................................................................... 34 1.8.3. fliC gene Analysis ..................................................................................................
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