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Comparative Analysis of Cronobacter Sp. with Respect to Genomic Diversity and Physiology

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Comparative Analysis of Cronobacter Sp. with Respect to Genomic Diversity and Physiology COMPARATIVE ANALYSIS OF CRONOBACTER SP. WITH RESPECT TO GENOMIC DIVERSITY AND PHYSIOLOGY EVA KUCEROVA A thesis submitted in partial fulfilment of the requirements Nottingham Trent University for the degree of Doctor of Philosophy. This research programme was carried out in collaboration with Nottingham University, UK and Vaccine Research Institute of San Diego, California, US November 2010 i 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. ii i ABSTRACT In recent years, some outbreaks of bacterial infection in neonatal intensive care units have been traced to powdered infant formula contaminated with Cronobacter species, causing life-threatening diseases such as necrotizing enterocolitis and meningitis. This study for the first time presents growth data and whole genome comparisons for five different species of Cronobacter after the taxonomic revision by Iversen et al. (2008). Growth data for 28 bacterial strains across 21 bacterial species in casein- and whey-dominant infant formula at temperatures 21, 27, 37, 41 and 44oC were determined, covering category A and B organisms as defined by WHO 2006. The data revealed potential of some Cronobacter species to grow at 44oC, posing a significant risk of infection by the bacterium when following the current formula preparation guidelines. The results were presented to the Food Standards Agency with the aim to update the current risk assessment model and improve infant formula preparation guidelines. The first sequenced Cronobacter genome (C. sakazakii BAA-894) was used to construct a 384,030 probe oligonucleotide tiling DNA microarray covering its 4 Mb chromosome and plasmids pESA2 (31 kb) and pESA3 (131 kb). Comparative genomic hybridization (CGH) was undertaken on five C. sakazakii strains, and representatives of four other Cronobacter species. CGH highlighted 15 clusters of genes that were divergent or absent in more than half of the tested strains. Six of these were of probable prophage origin; other regions included type VI secretion systems, the O-antigen gene locus, a tellurite resistance cluster, a fimbriae cluster, and a copper resistance operon cop. The CGH analysis highlighted the role of horizontal gene transfer, as a significant part of the variable gene pool was due to acquisition of mobile DNA. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance operon cus linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. By comparing the neonatal intensive care unit outbreak strains with less virulent strains and by analysis of the variable regions, we identified a list of putative virulence factors that may improve our limited understanding of Cronobacter’ s pathogenesis. ii ACKNOWLEDGEMENTS I would like to express my gratitude to my supervisor Prof. Steve Forsythe for his support and guidance throughout the whole course of my PhD, inspiring comments on my work and supernaturally quick corrections of my thesis. It has been a great journey for me and I thoroughly enjoyed (almost) every part of it. I would also like to thank to my external supervisor Prof. Mike McClelland for his invaluable help with CGH data analysis and with the publication of the manuscript. Another thank you belongs to Dr. Gina Manning for being a great inspiration for me, both professionally and personally. I would also like to express my gratitude to all my colleagues and staff in the Microbiology department for creating such a lovely working atmosphere. A very special thank you belongs to my fellow PhD students Abiyad Baig, Gemma Croxall, Naomi Dunning, Muriel Funck, Jenny Ince, Susan Joseph and Sandra Reuter for being my true friends and giving me inspiration and support. I have enjoyed working with you so much. An even more special thank you belongs to Dr. Tim Baker, who supported me in every possible way during the course of my PhD. Having you by my side made the writing-up of this thesis so much easier. I apologise for the lack of cooked meals and meaningful conversations that you patiently endured for several months. It is indeed reversible. Finally, I would like to dedicate this thesis to a very special lady, my mum Dr. Jitka Kucerova. I will never be able to thank you enough for all the support you have been giving me throughout my entire life. Thank you for all the opportunities that you have given to me, it is you who made this possible. iii CONTENTS ABSTRACT…………………………………………………….i ACKNOWLEDGEMENTS…………………………………….ii CONTENTS…………………………………………………...iii GENERAL INDEX……………………………………...iii INDEX OF TABLES……………………………………vii INDEX OF FIGURES………………………………….viii ABBREVIATIONS…………………………………………...xii GENERAL INDEX CHAPTER 1. INTRODUCTION…………………………………………………..1 1.1. Literature review ............................................................................................ 2 1.1.1 History ........................................................................................................ 3 1.1.2 Sources of the organism ............................................................................. 4 1.1.2.1 Environmental sources and food contamination ................................ 4 1.1.2.2 Breast milk substitutes ....................................................................... 6 1.1.2.3 Clinical sources .................................................................................. 8 1.1.3 Taxonomy .................................................................................................. 9 1.1.4 Molecular typing methods ....................................................................... 10 1.1.5 Pathogenesis and virulence factors .......................................................... 12 1.1.5.1 Infant meningitis .............................................................................. 12 1.1.5.2 Necrotizing enterocolitis .................................................................. 13 1.1.5.3 Virulence studies .............................................................................. 14 1.1.6 Antibiotic susceptibility ........................................................................... 18 1.1.7 Contamination of powdered infant formula ............................................. 19 1.1.7.1 Thermal resistance ........................................................................... 20 1.1.7.2 Desiccation tolerance ....................................................................... 21 1.1.7.3 Biofilm formation and capsule production ...................................... 23 1.1.8 Safety of breast milk substitutes .............................................................. 25 1.1.9 Control methods ....................................................................................... 27 1.2. General aims ................................................................................................ 28 1.3. General materials and methods .................................................................... 30 iv 1.3.1 Media and reagents .................................................................................. 30 1.3.2 Molecular biology kits and reagents, biochemical kits ............................ 31 1.3.3 Preparation of media ................................................................................ 32 1.3.4 Preparation of media with antibiotics ...................................................... 33 CHAPTER 2. FOOD STANDARDS AGENCY PROJECT: BACTERIOCIDAL PREPARATION OF POWDERED INFANT FORMULA………………………49 2.1. Introduction .................................................................................................. 35 2.1.1 Aims ......................................................................................................... 35 2.1.2 Growth rates of Cronobacter and other Enterobacteriaceae in powdered infant formula ........................................................................................... 38 2.2. Materials & Methods ................................................................................... 40 2.2.1 Bacterial strains and their maintenance ................................................... 40 2.2.2 Introduction to impediometry .................................................................. 42 2.2.3 Calculation of doubling time ................................................................. 43 2.2.4 Direct impedance method ........................................................................ 46 2.2.5 Indirect impedance method ...................................................................... 48 2.2.6 Quality control of impedance curves and TTDs generated by the automated RABIT system ........................................................................ 48 2.2.7 Determination of generation times and lag times by viable count ........... 49 2.3. Results .......................................................................................................... 51 2.3.1 Laboratory determination of generation times ......................................... 51 2.3.2 Growth of Category A organisms: Cronobacter
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