The Pennsylvania State University The Graduate School Department of Veterinary and Biomedical Sciences Pathobiology Program PATHOGENOMICS AND SOURCE DYNAMICS OF SALMONELLA ENTERICA SEROVAR ENTERITIDIS A Dissertation in Pathobiology by Matthew Raymond Moreau 2015 Matthew R. Moreau Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2015 The Dissertation of Matthew R. Moreau was reviewed and approved* by the following: Subhashinie Kariyawasam Associate Professor, Veterinary and Biomedical Sciences Dissertation Adviser Co-Chair of Committee Bhushan M. Jayarao Professor, Veterinary and Biomedical Sciences Dissertation Adviser Co-Chair of Committee Mary J. Kennett Professor, Veterinary and Biomedical Sciences Vijay Kumar Assistant Professor, Department of Nutritional Sciences Anthony Schmitt Associate Professor, Veterinary and Biomedical Sciences Head of the Pathobiology Graduate Program *Signatures are on file in the Graduate School iii ABSTRACT Salmonella enterica serovar Enteritidis (SE) is one of the most frequent common causes of morbidity and mortality in humans due to consumption of contaminated eggs and egg products. The association between egg contamination and foodborne outbreaks of SE suggests egg derived SE might be more adept to cause human illness than SE from other sources. Therefore, there is a need to understand the molecular mechanisms underlying the ability of egg- derived SE to colonize the chicken intestinal and reproductive tracts and cause disease in the human host. To this end, the present study was carried out in three objectives. The first objective was to sequence two egg-derived SE isolates belonging to the PFGE type JEGX01.0004 to identify the genes that might be involved in SE colonization and/or pathogenesis. Both genomes were almost identical (99% identity) being approximately 4.67Mb in size and the GC content was 52%. Both genomes contained about 4,600 open reading frames, of which 600 (or 12.5% of the genome) were related to virulence. Nine genes contained single nucleotide polymorphisms (SNPs) when these virulence-associated genes of egg isolated SE were compared with human isolated SE providing evidence of host-adapted microevolution of SE. Among these SNPs, two resulted in non-conservative changes in a fimbrial usher and a lipopolysaccharide biosynthesis gene, whereas four SNPs were located in promoter regions. The second objective of the study was to identify the host-specific genes (poultry vs. human) by pan-genomic analysis of the newly sequenced genomes and the genomes of Salmonella serovars published in the NCBI database. There were approximately 2, 800 cluster of orthologous genes (COGs) conserved among all serovars tested of which 247 were associated with Salmonella virulence. These core virulence genes may be associated with colonization and/or subsequent infection in all host species whereas the ‘host-specific’ genes most likely determine the host-specific mechanisms. This analysis identified 10 poultry-specific potential iv virulence genes, including the genes of two fimbrial operons, lpf- and sti- and four genes with hypothetical functions. Twelve other genes (e. g. iroD, ttrB/C, and sthA) were present only in Salmonella serovars that can infect the human host. The third objective of the study was to elucidate if the source attributes have an effect on SE virulence. Here, the colonization ability and virulence potential of SE grown in Luria broth (LB) medium and egg yolk in the laboratory and SE recovered from feces of mice experimentally infected with SE) were compared using a mouse colitis model of SE infection . The results demonstrated that SE grown in the egg yolk possesses enhanced colonization, shedding, and virulence capabilities as compared to the SE grown in the LB medium or SE recovered from mouse feces as determined by clinical signs, gross pathology, histological lesion scoring, and bacterial enumeration of feces, small and large intestines, and internal organs of the infected mice. These data suggest that egg yolk may condition SE to be better ‘primed’ for transmission to and infection of a second host by upregulating the expression of certain genes. Future studies should be directed towards understanding the mechanisms involved in microevolution of SE virulence in egg yolk using approaches, such as microarray and RNA sequencing. In conclusion, this study provides new insights into the current understanding of SE virulence and identifies potential targets for rational development of vaccines and antimicrobials to minimize human foodborne illness due to SE. v TABLE OF CONTENTS List of Figures .......................................................................................................................... viii List of Tables ........................................................................................................................... ix Abbreviations ........................................................................................................................... xi Acknowledgements .................................................................................................................. xii Chapter 1 Overview of the Pathobiological Perspectives of Salmonella enterica Serovar Enteritidis ......................................................................................................................... 1 1.1 Classification and General Background of Salmonella and Salmonella enterica Subspecies enterica .................................................................................................. 2 1.2 Salmonella Enteritidis Colonization, Infection and Contamination of Poultry and Shell Eggs ................................................................................................................. 4 1.3 Salmonella Enteritidis Colonization and Pathogenesis in Humans............................ 9 1.4 References .................................................................................................................. 26 Chapter 2 Evolution of Pathogenicity Revealed by Whole Genome Sequencing and Comparative Genomics of Two Egg Isolates of Salmonella Enteritidis .......................... 33 2.1 Abstract ...................................................................................................................... 34 2.2 Background ................................................................................................................ 35 2.3 Methods ...................................................................................................................... 36 2.3.1 Bacterial Strains. ............................................................................................. 36 2.3.2 PFGE Profiling. ............................................................................................... 36 2.3.3 Genomic DNA Purification. ............................................................................ 36 2.3.4 Sequencing and Assembly............................................................................... 37 2.3.5 Construction of the whole genome optical map (OpMap). ............................. 37 2.3.6 Genome Annotation. ....................................................................................... 37 2.3.7 Comparative Genomics and SNP Analysis. .................................................... 38 2.4 Results ........................................................................................................................ 38 2.4.1 General Overview of SEE1 and SEE2 Genomes ............................................ 38 2.4.2 Phage Regions in SEE1 and SEE2 .................................................................. 39 2.4.3 Genes That Exhibit Frameshifts ...................................................................... 40 2.4.4 Pathogenomics-Based Virulence Gene Profiling of SEE1 and SEE2 ............. 41 2.4.5 Bioinformatic Evidence of Genomic Microevolution during Human Infection............................................................................................................ 46 2.5 Discussion and Conclusions ....................................................................................... 47 2.6 Author Contributions ................................................................................................. 53 2.7 References .................................................................................................................. 98 vi Chapter 3 Patho-Pan-Genomics of Salmonella enterica Serovars Reveals Host-Specific Factors and Potential Vaccine Targets of Salmonella Enteritidis .................................... 101 3.1 Abstract ...................................................................................................................... 102 3.2 Background ................................................................................................................ 104 3.3 Materials and Methods ............................................................................................... 107 3.3.1 Orthologous gene clusters and pan-genome matrices. .................................... 107 3.3.2 Pan-Genome Analysis. .................................................................................... 107 3.4 Results ........................................................................................................................ 108 3.4.1 Genetic Relatedness of the Various Salmonella enterica Serovars. ................ 108 3.4.2 Virulence-Associated