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Title: Evolutionary trends in two strains of Salmonella enterica subsp. I serovar Enteritidis PT13a that vary in virulence potential.

item Guard, Jean

Submitted to: National Center for Biotechnology Information (NCBI)
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/8/2009
Publication Date: 8/10/2009
Citation: Guard, J.G. 2009. Evolutionary trends in two strains of Salmonella enterica subsp. I serovar Enteritidis PT13a that vary in virulence potential. National Center for Biotechnology Information (NCBI). Available:

Interpretive Summary: Researchers at the Egg Safety and Quality Research Unit (ESQRU) of the U. S. Department of Agriculture, Agricultural Research Service (USDA-ARS) have used microarray technology to identify a set of small scale polymorphisms that distinguish two PT13a strains of S. Enteritidis that are genetically related but phenotypically divergent. Using the S. Enteritidis PT4 genomic sequence from the Sanger Institute as a starting point, a set of overlapping primers were generated and used in conjunction with Nimblegen Systems technology to distinguish S. Enteritidis PT13a 21027, which forms biofilm but does not contaminate eggs, from S. Enteritidis PT13a 21046, which contaminates eggs but does not form biofilm. BLAST comparisons of the polymorphisms with other Salmonella enterica annotated databases potentially matched relevant phenotypic traits to genetic loci. Version 7 contains results from confirmation of polymorphisms by primer-based sequencing of PCR-amplified DNA (PCR sequencing) in forward and reverse orientations from the two target PT13a strains and from a PT4 strain that has high similarity to the Sanger reference strain. All polymorphisms were eventually confirmed by sequencing. Version 7 includes results to evaluate if a single nucleotide polymorphism (SNP) occurred within an open reading frame (ORF), if it altered amino acid sequence, and what class of amino acid substitution occurred if any. Version 7 also includes dbSNP accession numbers, which are planned to be released in dbSNP Build B131 during August 2009. Nimblegen states that combining mutational mapping with high-throughput resequencing detects within 5% of true SNP content. Mutational mapping alone previously detected 3 of 3 SNPs previously detected by ribotype analysis and PCR sequencing (rrlC, rrlA, cyaA); thus, mutational mapping was reliable for detection of SNPs. Some false positives occurred; for example, a SNP reported as present in only one of the two PT13a subpopulations in comparison to the PT4 genome was sometimes present in both PT13a strains. However, in a group of 48 regions that were recently analyzed by PCR-based sequencing, 47 were true SNPs that differentiated PT13a strains from each other. In contrast to mutational mapping, high-throughput resequencing methodology missed identifying all three control polymorphisms. The control polymorphisms and many true SNPs that differentiated PT13a strains were moved to a category referred to as "non-called ROI.” For this reason, high-throughput resequencing at first looked like it had a strong tendency towards reporting false negative results. However, sequencing of polymorphisms in the non-called ROI category indicated that high-throughput sequencing produced more false positives than did mutational mapping alone. For these reasons, the decision was made to re-sequence all putative SNPs in F/R directions in all 3 strains that had been detected by mutational mapping. Locations of SNPs and other polymorphisms are subject to change if the genomic database for S. Enteritidis PT4 is revised by the Sanger Institute after May 22, 2006.

Technical Abstract: Salmonella enterica subsp. I serovar Enteritidis (S. Enteritidis) is the world's leading cause of salmonellosis. Eggs contaminated by apparently healthy hens can result in illness in humans who consume them. Although the incidence of this pathogen within the United States has not been as high as it has been in Europe, current estimates from the Centers for Disease Control and from the USDA-Food Safety Inspection Service respectively suggest that this pathogen has the capacity to contaminate meat as well as eggs. It is considered a persistent threat to the safety of the food supply (1). It has prevented export of breeding stock to other countries. To investigate the genetic basis for the ability of the pathogen to contaminate eggs as well as to identify other evolutionary trends associated with growth potential and colonization of the avian host, the genomes of two monomorphic strains of S. Enteritidis PT13a with variant phenotypes were subjected to virtual subtraction hybridization against dimorphic S. Enteritidis PT4 (3). Direct comparison of the closely related genomes was possible by using the genome database for S. Enteritidis PT4 NCTC 13349 to generate overlapping sets of primers (Nimblegen, Inc.) that were capable of detecting single nucleotide polymorphisms (SNPs). The database of the S. Enteritidis PT4 reference genome is from the Pathogen Sequencing Group at the Sanger Institute and can be obtained from Version 7 lists 247 confirmed polymorphisms in the chromosome, one of which changes an amino acid and introduces a deletion (248 line entries). The large virulence plasmid had 3 additional SNPs. Version 7 is the endpoint of analysis. Future updates to this website will focus on editorial content rather than de novo discovery.