Molecular characterization of biofilm production and whole genome sequencing of selected Campylobacter concisus oral and clinical strains A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Mohsina Huq BSc; MSc (Microbiology); MS (Biotech) School of Applied Sciences College of Science Engineering and Health RMIT University September, 2016 DECLARATION I certify that except where due acknowledgement has been made, the work is that of the author alone; the work has not been submitted previously, in whole or in part, to qualify for any other academic award; the content of the thesis is the result of work which has been carried out since the official commencement date of the approved research program; any editorial work, paid or unpaid, carried out by a third party is acknowledged; and, ethics procedures and guidelines have been followed. Mohsina Huq 30/09/2016 I ACKNOWLEDGEMENTS I would like to express my sincere appreciation and gratitude to my senior supervisor Dr. Taghrid Istivan for her invaluable comments and comprehensive feedback on my work. I am grateful to her for her patient support, kind assistance, consistent encouragement and great guidance throughout the entire period of my research. My sincere gratitude goes to my associate supervisors Professor Peter Smooker and Dr. Thi Thu Hao Van for their continuous encouragement and guidance throughout this research program. I would like to thank the Department of Education and Training, Australian Government for awarding me with the Endeavour Postgraduate Scholarship. Without their funding, this work would not have been possible. My sincere gratitude goes to the Dean of Research and Innovation, Professor Andrew Smith for granting me the top-up scholarship which was a great help to complete this research study. I am grateful tor Dr. Anna Walduck for her scientific editing and proofreading of my thesis. Thanks for Dr.Lisa Dias; I really appreciate your guidance, and the time you spent with me to make my PhD study go well. I would like to thank the past and current students in Dr. Istivan’s laboratory, especially Dr. Eltaher Elshagmani and Dr. Khaled Allemailem for their continuous assistance and encouragement. I would like to express my sincere thanks to all members in the School of Science (Bundoora) for their friendship and assistance, especially Layla Alhasan, Abdouslam Alsharif, Alaa Karkashan, Mohamed Said, Natalie Kikidopoulos, Thomas Mclean, Alekhya Penumarthi, Vishal Mistry, Danka Jovetic, Kim Stevenson Huan, Yong Yap, Gor Mian Chee and Ben Vazina and to all staff members of Biotechnology and Environmental Biology for their friendship, assistance, and company. II Special thanks to Dr. Eric Adetutu and Vijay Bhaskarla for helping me with the next generation sequencing. Sincere gratitude to my associate supervisor Dr. Van for sequencing two strains in the Illumina Miseq. Special thanks to Professor Rob Moore for performing the de novo assembly of one genome and guidance in my research. I would like to acknowledge the help of Dr. Chaitali Dekiwadia and all members of RMIT Microscopy and Microanalysis Facility for letting me use the electron microscope. I would like to acknowledge my parents, Mozammel Hoque and Ferdous Akhter, my three brothers, and my two sisters for their love, guidance and support. Their inspiration and encouragement helped me to get through the hardest times. I would like to express my sincere appreciation to my husband Monir and especially my son Mahrus, for their love, support and patience over many years throughout my current and previous studies. I hope that this thesis will assure them that one of their dreams in addition to mine has finally come true. Lastly, I owe everything to the Almighty God (Allah) without whose grace and guidance, any of this would be possible. III Table of contents DECLARATION .................................................................................................................. I ACKNOWLEDGEMENTS ................................................................................................. II Table of contents .................................................................................................................IV List of Figures ...................................................................................................................... X List of Tables .................................................................................................................... XIV Summary ........................................................................................................................... XV Publications ...................................................................................................................... XIX Abbreviations .................................................................................................................... XX Chapter One : General Introduction ...................................................................................... 1 1.1 The genus Campylobacter ...................................................................................... 1 1.1.1 History and association with diseases ............................................................. 3 1.2 Campylobacter concisus: the emerging pathogen .................................................. 5 1.2.1 C. concisus in the human oral cavity .............................................................. 5 1.2.2 C. concisus in acute gastroenteritis ................................................................. 8 1.2.3 C. concisus in chronic gastrointestinal diseases ............................................ 11 1.2.4 C. concisus in immunocompromised patients ............................................... 15 1.2.5 C. concisus in other parts of the human body ............................................... 15 1.3 Identification of C. concisus ................................................................................. 17 1.3.1 Identification of C. concisus by cultural and biochemical properties ........... 17 1.3.2 Identification of C. concisus by molecular methods ..................................... 17 1.4 Virulence factors in C. concisus ........................................................................... 18 1.4.1 Motility .......................................................................................................... 19 1.4.2 Toxins ............................................................................................................ 20 1.4.3 Adhesion and invasion .................................................................................. 25 1.5 Antibiotic Resistance ............................................................................................ 29 1.6 Biofilm formation by Campylobacter spp. ........................................................... 30 1.6.1 Mechanism of biofilm formation .................................................................. 33 1.6.2 Cell to cell signalling (Quorum-sensing) ...................................................... 35 1.7 Role of luxS in QS ................................................................................................ 39 1.7.1 The structure and activity of LuxS ................................................................ 39 IV 1.7.2 Metabolic role of LuxS ................................................................................. 42 1.7.3 LuxS and growth ........................................................................................... 44 1.7.4 LuxS and biofilm formation .......................................................................... 45 1.7.5 LuxS and chemotaxis .................................................................................... 45 1.7.6 LuxS and motility .......................................................................................... 46 1.7.7 LuxS and cellular adherence and invasion .................................................... 48 1.7.8 LuxS and virulence ....................................................................................... 49 1.7.9 LuxS and haemolysis .................................................................................... 50 1.8 Molecular typing of C. concisus ........................................................................... 50 1.8.1 Genetic diversity of C. concisus .................................................................... 50 1.8.2 Typing of C. concisus on the basis of phenotypic characteristics ................. 53 1.9 Genome analysis of Campylobacter spp. ............................................................. 54 1.9.1 Whole genome sequence of C. concisus ....................................................... 55 1.9.2 Comparative genomic analyses of C. concisus and C. jejuni........................ 56 1.9.3 The ‘pan and core’ genome of C. concisus ................................................... 61 1.9.4 Plasmids in C. concisus ................................................................................. 62 1.9.5 Phylogenetic analysis based on ribosomal RNA (rRNA) genes ................... 63 1.10 Aims of the study .................................................................................................. 63 Chapter Two : General Materials and Methods .................................................................. 64 2.1 General Procedures ............................................................................................... 64 2.2 Bacteriological Methods ......................................................................................