Characterization of Salmonella enterica serotype Enteritidis subpopulations using Comparative Genome Sequencing and mutational analysis
Morales, Cesar Agusto
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Salmonella enterica serotype Enteritidis continues to be the leading cause of salmonellosis both worldwide and in the United States, in part due to its unique ability to contaminate the internal contents of table eggs. Subpopulations that express varying phenotypes within the same phage type of S. Enteritidis are successful in different ecological niches but complement each other to complete the overall egg contamination pathway. Comparative Genomic Sequencing was used to determine the nucleotide sequence identity of all polymorphisms between two subpopulations of S. Enteritidis that differ in their lipopolysaccharide structure, ability produce biofilm, ability to achieve high-cell density growth, Phenotype Microarray results, and in their ability to contaminate eggs. A total of 247 regions of polymorphisms were found to differentiate the two subpopulations, ranging in size from one single nucleotide polymorphism to a 215 base pair deletion. Twelve polymorphisms were predicted to disrupt open reading frames (ORFs), 99 were predicted to result in amino acid substitutions, 82 were predicted to occur within an ORF but were synonymous, 38 polymorphisms were found in non-coding intergenic regions, and 12 were found in RNA genes. Within these results, it is believed that the genetic variation reported is responsible for the phenotype variation observed between the two subpopulations. Three of the ORF-disrupting polymorphism were selected for further characterization based on their predicted genotype-phenotype relationships. Using site-directed mutagenesis, the genes sen4316 and dsdA were confirmed to be necessary for biofilm formation and D-serine catabolism, respectively. Phenotype Microarray results further suggested a role of osmotic resistance for dsdA. The gene sefD, encoding a minor fimbrial subunit that is unique to S. Enteritidis, was analyzed in an egg production hen experiment for its role in chicken reproductive tract tropism. No significant difference in egg production was observed between the sefD mutant and wild type strains, but overexpression of sefD resulted in attenuation, suggesting a possible role for modulating illness in the chicken. The research herein represents the first steps towards determining genetic determinants for subpopulation heterogeneity, including the ability to contaminate the egg.