Diversity and virulence of the genus Cronobacter revealed by multilocus sequence typing (MLST) and comparative genomic analysis Susan Manju Joseph A thesis submitted in partial fulfilment of the requirements of Nottingham Trent University for the degree of Doctor of Philosophy July 2013 Experimental work contained in this thesis is original research carried out by the author, unless otherwise stated, in the School of Science and Technology at the Nottingham Trent University. No material contained herein has been submitted for any other degree, or at any other institution. This work is the intellectual property of the author. You may copy up to 5% of this work for private study, or personal, non-commercial research. Any re-use of the information contained within this document should be fully referenced, quoting the author, title, university, degree level and pagination. Queries or requests for any other use, or if a more substantial copy is required, should be directed in the owner(s) of the Intellectual Property Rights. Susan Manju Joseph ACKNOWLEDGEMENTS I would like to express my immense gratitude to my supervisor Prof. Stephen Forsythe for having offered me the opportunity to work on this very exciting project and for having been a motivating and inspiring mentor as well as friend through every stage of this PhD. His constant encouragement and availability for frequent meetings have played a very key role in the progress of this research project. I would also like to thank my co-supervisors, Dr. Alan McNally and Prof. Graham Ball for all the useful advice, guidance and participation they provided during the course of this PhD study. Special thanks are due to Esin Cetinkaya (Ankara University, Turkey), Arturo Levican and Prof. M. J. Figueras (Rovirai Virgili University, Spain) for all their help and inputs in the speciation studies. I would like to thank the team at Life Technologies (Foster City, CA) for having sequenced the Cronobacter spp. genomes used in the latter half of this project, and Prof. Michael McClelland and Dr. Prerak Desai (Vaccine Research Institute, San Diego) for their technical expertise and valuable guidance in the bioinformatic analysis of these genomes. The journey through this PhD, away from family, would not have been possible for me without the love, laughter and support of some very fine friends I made over the past few years. Many thanks to the Microbiology Prep Room staff, especially Pam and Mike, for their invaluable help at every stage. A huge round of acknowledgement and gratitude goes to all the colleagues and friends (some of whom have now moved on) in the Microbiology Research group as well as Office 104 for their constant support and camaraderie. Last but definitely not the least; I would like to thank my wonderful family for always showering me with love, blessings and encouragement, even though we have been miles apart. Special thanks are due for my parents for all their sacrifices and hard work that has made me what I am today. Lots of love and gratitude are also due to my wonderful husband, Vishakh, for being my pillar of strength and for patiently enduring the long-distance relationship while I pursued this PhD. I owe all of them a lot. I would like to dedicate this thesis to my parents and Vishakh, for having always believed in me and supported me unconditionally. TABLE OF CONTENTS PUBLICATIONS..........................................................................................................................v ABSTRACT.................................................................................................................................vi LIST OF FIGURES……………………………………………………...................................vii LIST OF TABLES........................................................................................................................x LIST OF APPENDICES…………………………………………………………….……..….xii LIST OF ABBREVIATIONS………………………………………………………………...xiii CHAPTER 1: General introduction and aims 1.1 The genus Cronobacter: An introduction.............................................................................2 1.1.1 History of Cronobacter taxonomy to 2008...........................................................2 1.1.2 Physiology..............................................................................................................7 1.1.3 Reservoirs...............................................................................................................8 1.1.4 Epidemiology........................................................................................................10 1.1.5 Pathogenicity and virulence factors....................................................................13 1.1.6 Culture and phenotypic identification methods................................................16 1.1.7 Molecular typing and identification methods....................................................19 1.1.8 Genome studies.....................................................................................................23 1.1.9 Public health significance and future directions...............................................24 1.2 Aims of the study……..........................................................................................................26 CHAPTER 2: Materials and Methods 2.1 Safety considerations............................................................................................................28 2.2 Bacterial strains....................................................................................................................28 2.3 Preparation of media............................................................................................................38 2.4 MLST and analysis...............................................................................................................39 2.4.1 Choice of bacterial strains for MLST.................................................................39 2.4.2 Genomic DNA extraction.....................................................................................40 2.4.3 PCR amplification of the MLST loci..................................................................40 2.4.4 PCR amplification of the 16S rDNA loci............................................................40 2.4.5 Agarose gel electrophoresis.................................................................................41 2.4.6 PCR product purification....................................................................................41 2.4.7 DNA sequencing...................................................................................................41 2.4.8 Allele and sequence type designation..................................................................43 2.4.9 Analysis of DNA sequences..................................................................................43 i 2.4.10 Evolutionary timescale estimations..................................................................43 2.4.11 Recombination analysis.....................................................................................43 2.4.12 PCR screening for serotype identification.......................................................44 2.4.13 Inter-species nucleotide divergence analysis....................................................44 2.4.14 Linkage and clonality.........................................................................................44 2.5 Whole genome sequencing and comparative genomic analysis.......................................45 2.5.1 Choice of bacterial strains for genome sequencing...........................................45 2.5.2 Genomic DNA extraction.....................................................................................45 2.5.3 Genome sequencing..............................................................................................47 2.5.4 Genome assembly.................................................................................................47 2.5.5 Core and pan-genome identification...................................................................48 2.5.6 Whole genome alignments and phylogenetic analysis.......................................48 2.5.7 Prophage region identification............................................................................49 2.5.8 Plasmid region identification...............................................................................49 2.5.9 Plasmid profiling..................................................................................................49 2.5.10 CRISPR region identification............................................................................50 2.5.11 Genome comparisons and presence/absence of key genes..............................50 2.5.12 Characterisation of O-LPS genes......................................................................51 2.5.13 Phylogenetic analysis of sialic acid gene cluster..............................................51 CHAPTER 3: MLST reveals recombination and evolutionary relationships of the Cronobacter genus 3.1 Introduction..........................................................................................................................53 3.1.1 Aims of this chapter.............................................................................................53 3.1.2 Multilocus sequence typing..................................................................................53 3.1.3 Recombination studies using MLSA...................................................................55 3.1.4