Single nucleotide polymorphisms that differentiate two subpopulations of Salmonella enteritidis within phage type

Abstract

BackgroundSalmonella Enteritidis is currently the world's leading cause of salmonellosis, in part because of its ability to contaminate the internal contents of eggs. Previous analyses have shown that it is an exceptionally clonal serotype, which nonetheless generates considerable phenotypic heterogeneity. Due to its clonality, whole genome analysis is required to find genetic determinants that contribute to strain heterogeneity of Salmonella Enteritidis. Comparative whole genome mutational mapping of two PT13a strains that varied in the ability to contaminate eggs and to form biofilm was achieved using a high-density tiling platform with primers designed from a PT4 reference genome. Confirmatory Sanger sequencing was used on each putative SNP identified by mutational mapping to confirm its presence and location as compared to the reference sequence. High coverage pyrosequencing was used as a supporting technology to review results.ResultsA total of 250 confirmed SNPs were detected that differentiated the PT13a strains. From these 250 SNPS, 247 were in the chromosome and 3 were in the large virulence plasmid. SNPs ranged from single base pair substitutions to a deletion of 215 bp. A total of 15 SNPs (3 in egg-contaminating PT13a 21046 and 12 in biofilm forming PT13a 21027) altered coding sequences of 16 genes. Pyrosequencing of the two PT13a subpopulations detected 8.9% fewer SNPs than were detected by high-density tiling. Deletions and ribosomal gene differences were classes of SNPs not efficiently detected by pyrosequencing.ConclusionsThese results increase knowledge of evolutionary trends within Salmonella enterica that impact the safety of the food supply. Results may also facilitate designing 2nd generation vaccines, because gene targets were identified that differentiate subpopulations with variant phenotypes. High-throughput genome sequencing platforms should be assessed for the ability to detect classes of SNPs equivalently, because each platform has different advantages and limits of detection.

Genetic Diversity and Evolution of Salmonella enterica Serovar Enteritidis Strains with Different Phage Types

“…Among the 160 loci investigated, only 23 loci contained informative SNPs, mostly from genes in COG category F (nucleotide transport and metabolism), K (transcription), Q (secondary metabolites, biosynthesis, transport, and catabolism), or S (unknown function). Among these 23 loci, 9 loci, including 6 coding regions (cpsG, iagB, marA, marC, narP, and wza) and three intergenic regions (SEN0084-SEN0085, SEN0999-SEN1000, and SEN3612-ivbL), were newly discovered in this study to contain polymorphic sites, while the other 14 were also found to contain variable sites that were able to differentiate two S. Enteritidis PT13a strains in a recent study (28). Examination of phage and plasmid profiles revealed several interesting findings.…”

“…Single nucleotide polymorphisms (SNPs) seem to be the most valuable molecular markers for studying the evolutionary relationships of isolates within homogeneous pathogenic clones (19,(25)(26)(27)(28)(29). During 2010, a large multistate outbreak of human S. Enteritidis infections associated with shell eggs occurred (http://www.cdc.gov /salmonella/enteritidis/).…”

Development of a New Molecular Subtyping Tool for Salmonella enterica Serovar Enteritidis Based on Single Nucleotide Polymorphism Genotyping Using PCR

“…Another source of variation in S. Enteritidis genomes is the frequent incorporation of bacteriophages (24). Future work is needed to explain how the observed genetic diversity in S. Enteritidis may mechanistically explain the phenotypic and behavioral differences seen when different isolates are used in studies such as animal infection trials, in vitro cell invasion assays, and studies of the ability of various isolates to contaminate internal contents of eggs and survive (5,34).…”

“…Detailed genetic studies of Salmonella Enteritidis have consistently shown the underlying causes of the poor discriminatory abilities of existing subtyping tools: isolates of Salmonella Enteritidis are extremely similar (i.e., are highly clonal), and this poses a difficulty in finding a definitive, distinguishing trait that could be used to track lineages (5,19). An ideal subtyping procedure should lead to a high level of discrimination among isolates, in contrast to existing methods, which tend to group apparently unrelated isolates together.…”

Pulsed-Field Gel Electrophoresis as a Molecular Tool for Characterizing Genomes of Certain Food-Borne Bacterial Isolates - A Review

“…It also represents one of the most genetically homogeneous serotypes of (Guard et al, 2011). This variation was observed both as SNP variation among 366 genes as well as the presence and absence of numerous phages and plasmids among these close relatives.…”