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Comparative Genomic Approaches to Understanding Achromobacter Xylosoxidans

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Comparative Genomic Approaches to Understanding Achromobacter Xylosoxidans Comparative genomic approaches to understanding Achromobacter xylosoxidans Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy By Pisut Pongchaikul September 2015 Acknowledgements Acknowledgements First of all, I am very grateful to immeasurable supports and encouragement from my supervisors – Dr Alistair Darby, Professor Craig Winstanley, and Dr Svetlana Antonyuk. Sincerely thanks all members of Darby‟s lab, in particular, Dr Ian Goodhead, for all of your kindness and lively environment in the office. Thanks all from Winstanley‟s lab. Thanks to Dr Pitak and Clinical Microbiology unit for clinical isolates and microbiological skill training. And thanks all worms died for my project. Massive thanks all people in B231- all informaticians, Dr Richard Gregory, in particular. To, Jen (Dr Jennifer Kelly), thanks for your advise on pan-genome. To, Ian Wilson, many thanks for being nice to me, and for proofreading this thesis. To Sarah, thanks for being such a really nice friend. Thanks all places where allow me to sit and write up this thesis. Thanks Chaba Chaba Thai restaurant for being my second home in Liverpool. Special thanks Medical Scholar Programme for the opportunity to do intercalated degree. Thanks Mahidol-Chamlong Harinasuta PhD scholarship for an invaluable opportunity to come to Liverpool and to undertake such a great project. Thanks Dr Pansakorn, without your encouragement I would not have had a chance to do my PhD in Liverpool. Thanks PR601‟s members, especially Dr Ponpan, for my first experience in scientific research and for your mental support. Last but not least, this pride is for my family, of course, my Mum and my Dad, who always boost me up. Thanks for your everlasting support. To a special person, Jitpisuth, you are everything to me – good friend, nice chef, travelling companion, French teacher, thesis proofreader, and wonderful girlfriend. i Declaration Declaration I, myself, declare that all experiments described in this thesis have been carried out by me without any collaboration except for those indicated in acknowledgement and in the text. The works were produced in the Institute of Integrative Biology and the Institute of Infection and Global Health, the University of Liverpool. Pisut Pongchaikul ii Abstract Abstract The investigation into the genome of the emerging pathogen reveals the genetic basis of the pathogen. In this study, Achromobacter xylosoxidans was used as an example for the genomic study of emerging pathogens. A. xylosoxidans is an emerging and opportunistic pathogen in patients with various underlying diseases, such as Cystic Fibrosis and cancers. In chapter 3, the objectives of the study were to compare and evaluate the approach for A. xylosoxidans identification. A wide range of methods, including phenotypic test, 16S rDNA gene sequencing, MALDI-TOF, RAPD and MLST, were utilised to demonstrate the species identification. MALDI-TOF was considered as the most appropriate method due to the least time consuming. The application of multiple approaches to identify A. xylosoxidans was suggested. In chapter 4, comprehensive genomic feature of A. xylosoxidans has not been elucidated. The objective of this study was to use comparative genomic tool to investigate genomic feature of A. xylosoxidans. The analysis revealed the opened pan-genome of the species. The core genome accounted for approximately 50% of the size of the genome. Furthermore, the analysis revealed recombination events in the core genome of the species. Interestingly, phylogenetic relationships demonstrated global distribution of the species without geographical structure. This study provided pan-genome structure of the species, allowing for studies of genetic exchange mechanism in the species. In chapter 5, the main objective of the chapter was to investigate the antibiotic resistance and genes associated with the resistance in the species. The whole genome sequence and bioinformatics were used to search for genes associated with antibiotic resistance phenotypes. Single-molecule real time (SMRT) sequencing was also used to investigate the integron. The analysis revealed conserved RND-type efflux transporters across the species. The complete genome sequence revealed class 1 integron carrying IMP-14 on the chromosome of multidrug resistant isolates. This study demonstrated the identification of antibiotic resistance genes using bioinformatics and SMRT sequencing. In chapter 6, the objective was to investigate the virulence of A. xylosoxidans in organism model. The great moth larva Galleria mellonella was used to test for the virulence of A. xylosoxidans. Bioinformatics analyses were conducted to predict genes associated with virulence determinants. The analysis resulted in hydrolase-containing protein and PscD type III secretion system predicted as virulence determinants based on G. mellonella infection model, paving the way for further studies. This study demonstrated the application of whole genome sequencing and bioinformatics to understand the biology of the emerging pathogen. The identification of the genome of the emerging pathogen is required for the facilitation of prevention and therapeutic in the future. iii Glossary Glossary °C Degree celcius DNA Deoxyribonucleic acid FEV1 Forced expiratory volume in one second g Gram h Hour L Litre MgCl2 Magnesium Chloride m Metre μ Micro mM Millimolarity min Minute M Molarity mol Mole n Nano OD Optical density pmole Picomole RNA Ribonucleic acid rDNA Ribosomal DNA U.K. United Kingdom U.S. United States of America iv Table of Contents Table of Contents Acknowledgements ................................................................................................ i Declaration ........................................................................................................... ii Abstract ............................................................................................................... iii Glossary ................................................................................................................ iv Table of Contents ................................................................................................. v List of Figures ....................................................................................................... x List of Tables ..................................................................................................... xiv Chapter 1 General Introduction ........................................................................................... 1 1.1. Emerging infectious diseases and hospital-acquired infections ................... 3 1.1.1. Emerging pathogens ................................................................................... 3 1.1.2. Hospital-acquired infection problem .......................................................... 4 1.1.2.1. Hospital-acquired respiratory infections .......................................................... 5 1.1.2.2. Hospital-acquired surgical site infections ........................................................ 6 1.1.2.3. Hospital-acquired gastrointestinal tract infections ........................................... 7 1.1.2.4. Hospital-acquired urinary tract infections ........................................................ 8 1.1.3. The burden of hospital-acquired infection and prevention strategies ......... 9 1.2. Antibiotic resistance ...................................................................................... 10 1.2.1. Antibiotic resistance: a multi-dimensional problem ................................. 10 1.2.1.1. Causes of antibiotic resistance ....................................................................... 10 1.2.1.2. Economic burden of antibiotic resistance ...................................................... 11 1.2.2. Antibiotic resistance in hospital-acquired emerging infection ................. 12 1.3. Situation of antibiotic resistance in countries where are associated with this study: The U.K. and Thailand .......................................................................... 14 1.3.1. Antibiotic resistance problem in the U.K. ................................................ 14 1.3.2. Antibiotic resistance problem in Thailand ................................................ 15 1.4. Whole genome sequencing in clinical microbiology ................................... 16 1.4.1. Next-generation whole genome sequencing ............................................. 16 1.4.2. The application of whole genome sequencing in clinical microbiology....... .................................................................................................................. 19 1.4.3. The application of whole genome sequencing to emerging pathogens ........ .................................................................................................................. 21 1.5. Achromobacter xylosoxidans as an emerging pathogen .............................. 23 1.6. A. xylosoxidans as a Cystic Fibrosis pathogen ............................................. 24 1.7. Complete genome sequence of the A. xylosoxidans genome ....................... 25 1.8. Aims and objectives ....................................................................................... 25 1.8.1. The identification and strain typing of A. xylosoxidans (Chapter 3) ........ 26 1.8.2. General feature of pan-genomic analysis of A. xylosoxidans (Chapter 4) .... .................................................................................................................
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