GEORGE MICHAEL HUMPHREY BIRCHENOUGH Analysis of intestinal factors contributing to the age- dependency of systemic neuropathogenic Escherichia coli K1 infection in the neonatal rat Thesis submitted in accordance with the requirements of the UCL School of Pharmacy for the degree of Doctor of Philosophy Microbiology Group, Department of Pharmaceutics, UCL School of Pharmacy July 2012 PLAGIARISM STATEMENT This thesis describes research conducted in the UCL School of Pharmacy between October 2008 and July 2012 under the supervision of Professor Peter W. Taylor. I certify that the research described is original and that any parts of the work that have been conducted by collaboration are clearly indicated. I also certify that I have written all the text herein and have clearly indicated by suitable citation any part of the dissertation that has already appeared in publication. Signature: Date: Acknowledgements Firstly I wish to thank my supervisor, Professor Peter Taylor, for giving me the opportunity to work on such an interesting and rewarding project. Your continued support and enthusiasm has been a constant source of encouragement and I greatly appreciate all the advice and help (both scientific and general!) that you have provided over the last four years. I owe you a lot of beer. I also wish to thank my amazing parents for all their love and support over the eight years of my higher education. Without your enthusiasm and belief I would not have been able to follow this path. I sincerely promise I will now get a job! Furthermore, I wish to thank colleagues at the London School of Hygiene & Tropical Medicine, Dr. Richard Stabler for all the help with the SSU arrays and Dr. Ozan Gundogdu and Melissa Martin for assistance with the gene expression arrays. Finally, I am also very grateful to all members, past and present, of the Microbiology Group who have made the last four years such an enjoyable experience. Thank you Patri, Helena, Joao, Dave, Sarah, Christina, Lucia and Fatosh for all the great times, for all the advice, and for being guinea pigs for my JCR experiments! I would also like to extend my thanks to all the staff and students at the UCL School of Pharmacy who have made the institution such a friendly working environment. I wish you all the best for the future. Dedicated to the memory of Charlie Abstract Systemic infections by encapsulated bacteria are a major aetiological agent of neonatal mortality. Neonatal meningitic Escherichia coli (NMEC) are isolated in a significant proportion of these infections. 80-85% of NMEC isolates express the K1 polysaccharide capsular antigen, a homopolymer of α-2,8-linked polysialic acid (PSA) which mimics the PSA modulator of neuronal plasticity in mammalian hosts and enables these strains to evade components of the innate and adaptive neonatal immune system. Systemic E. coli K1 infection is age-dependent. The basis of age-dependency is the capacity of the pathogen to translocate from the gastrointestinal (GI) tract into the systemic circulation. This initial step in pathogenesis is poorly characterized and the mechanistic basis of age-dependency is unknown. Post-partum development of the GI microbial population (microbiota) and host tissue may modulate susceptibility to E. coli K1. Age-dependency was characterized in the neonatal rat model of infection. Two- day old (P2) neonates were highly susceptible to infection after oral dosing with E. coli K1 strain A192PP, whereas P9 neonates were highly refractive. This variation was not caused by the capacity of the pathogen to colonize the GI tract. The P2-P9 GI microbiota was assessed using culture-independent methods. Quantitative and qualitative analysis of the microbiota revealed that the P2-P9 microbiota was significantly different to that of the adult, but that very little variation occurred between the neonatal groups examined. Suppression of the P9 microbiota using combined antibiotic treatment did not increase the susceptibility of this group to E. coli K1. The P2-P9 development of the GI tissues and the response of P2 and P9 tissues to E. coli K1 colonization were assessed at the transcriptional level. A substantial degree of developmental expression was observed over P2-P9, including the up-regulation of putative components of the small intestinal (α-defensin peptides Defa24 and Defa-rs1) and colonic (trefoil factor peptide Tff2) mucus barrier. Colonization with E. coli K1 modulated expression of these peptides: The developmental expression of Tff2 was dysregulated in P2 tissues, likely due to IL-1β and NFκB signalling, and was accompanied by a decrease in the gel-forming mucin Muc2. Conversely, α-defensin expression was up-regulated in P9 tissues. These results indicated that the intestinal barrier function of the P9 GI tract is more developed than the P2 equivalent. Furthermore, E. coli K1 colonization may compromise the development of the colonic mucus barrier in P2 neonates. This supports the hypothesis that the developmental state of the GI tissue, but not the microbiota, modulates susceptibility to systemic E. coli K1 infection. In addition, these results imply that supplementation of the neonatal GI with recombinant α-defensins or Tff2 represent potential strategies for the prophylaxis of neonatal E. coli K1 infection. 1 Table of Contents ABSTRACT……………………………………………………………………… 1 Table of Contents………………………………………………………………... 2 Figures & Tables………………………………………………………………... 7 Abbreviations………………………………………………………………….... 11 CHAPTER 1 – GENERAL INTRODUCTION…………….…………………. 15 1.1 Infant mortality in the 21st Century………………………………. 16 1.1.1 Overview…………………………………………………. 16 1.1.2 The Increasing Importance of Neonatal Mortality………. 18 1.1.3 Aetiology of Neonatal Mortality…………………………. 20 1.1.3.1 Non-infectious disease…………………………………... 21 1.1.3.2 Infectious Disease……………………………………….. 22 1.1.3.2.1 Sepsis……………………………………………………. 23 1.1.3.2.2 Bacterial Meningitis……………………………………... 28 1.1.4 Reducing Neonatal Mortality……………………………. 35 1.2 Escherichia coli………………………………………………….. 38 1.2.1 Natural History…………………………………………... 38 1.2.2 One Species, Multiple Pathovars………………………… 45 1.2.2.1 Intestinal Pathovars………………………………………. 45 1.2.2.2 Extra-Intestinal Pathovars………………………………... 48 1.3 NMEC…………………………………………………………….. 50 1.3.1 The molecular epidemiology of NMEC………………….. 50 1.3.2 Pathogenesis of E. coli K1 infection……………………… 52 1.4 The age-dependency of E. coli K1 infection……………………... 61 2 1.4.1 The basis of age-dependency……………………………… 61 1.4.2 The intestinal microbiota………………………………….. 63 1.4.3 The intestinal tissues………………………………………. 66 1.5 Aims & Objectives………………………………………………… 70 CHAPTER 2 – MODEL & METHOD DEVELOPMENT……………………. 72 2.1 Introduction……………………………………………………….. 73 2.2 Materials & Methods……………………………………………… 77 2.2.1 Bacteria: strains, growth conditions and stock maintenance 77 2.2.2 Animals……………………………………………………. 78 2.2.3 Bacteriophage K1E propagation, purification and titration. 78 2.2.4 Oral inoculation of neonates and adults…………………… 79 2.2.5 Processing of tissue & stool samples……………………… 80 2.2.6 Detection of E. coli K1 colonization and bacteraemia……. 80 2.2.7 E. coli K1 quantification…………………………………… 81 2.2.8 DNA extraction……………………………………………. 81 2.2.9 DNA extraction of GI tissues and stool samples………….. 83 2.2.10 neuS PCR and amplicon agarose gel electrophoresis……... 84 2.2.11 Amplicon cleanup and DNA sequencing………………….. 84 2.2.12 E. coli K1 quantification by neuS qPCR…………………... 85 2.3 Results……………………………………………………………… 87 2.3.1 Characterization of the neonatal rat model of E. coli K1 infection……………………………………………………. 87 2.3.1.1 Age-dependency…………………………………………… 87 2.3.1.2 Relationship between colonization, bacteraemia and mortality…………………………………………………… 88 2.3.1.3 Onset of systemic infection……………………………….. 90 2.3.2 The maternal-neonatal route of infection…………………. 92 3 2.3.2.1 Colonization of adults rats with E. coli K1………………. 92 2.3.2.2 Colonization of pregnant rats with E. coli K1…………… 93 2.3.3 Quantification of E. coliK1 by neuS qPCR……………… 94 2.3.3.1 Specificity of the primers………………………………… 95 2.3.3.2 Validation of the qPCR assay……………………………. 96 2.3.3.3 Comparison of culture/phage and qPCR methods in vivo 100 2.4 Discussion……………………………………………………….. 102 CHAPTER 3 – THE INTESTINAL MICROBIOTA………………………… 105 3.1 Introduction………………………………………………………. 106 3.2 Methods & Materials…………………………………………….. 111 3.2.1 SSU rDNA PCR primers………………………………… 111 3.2.2 SSU rDNA qPCR………………………………………... 112 3.2.3 Whole SSU rDNA amplification and cleanup…………... 113 3.2.4 Microarray reference pool………………………………. 113 3.2.5 SSU rDNA amplicon labelling and purification………... 114 3.2.6 Microarray hybridization and washing…………………. 114 3.2.7 Microarray scanning and data normalization…………… 115 3.2.8 Preparation of competent A192PP cells………………… 115 3.2.9 Transformation of competent A192PP with pUC19……. 116 3.2.10 Minimum inhibitory concentration……………………… 117 3.2.11 Antibiotic treatment of neonatal rats……………………. 117 3.3 Results…………………………………………………………… 119 3.3.1 E. coli K1 intestinal colonization………………………... 119 3.3.2 P2-P9 neonatal intestinal microbiota……………………. 120 3.3.2.1 Quantitative analysis of the microbiota…………………. 121 3.3.2.2 Qualitative analysis of the microbiota…………………… 123 4 3.3.2.2.1 Relative intestinal population overview…………………... 124 3.3.2.2.2 Comparison of P2, P5 and P9 intestinal microbiota……… 128 3.3.3 Antibiotic-mediated suppression of the microbiota and susceptibility to E. coli K1 infection……………………… 130 3.3.3.1 Antibiotic-mediated suppression of the neonatal microbiota 130 3.3.3.2 Colonization of microbiota-suppressed