Limited genetic diversity in Salmonella enterica serovar Enteritidis PT13

Abstract

Background: Salmonella enterica serovar Enteritidis has emerged as a significant foodborne pathogen throughout the world and is commonly characterized by phage typing. In Canada phage types (PT) 4, 8 and 13 predominate and in 2005 a large foodborne PT13 outbreak occurred in the province of Ontario. The ability to link strains during this outbreak was difficult due to the apparent clonality of PT13 isolates in Canada, as there was a single dominant pulsed-field gel electrophoresis (PFGE) profile amongst epidemiologically linked human and food isolates as well as concurrent sporadic strains. The aim of this study was to perform comparative genomic hybridization (CGH), DNA sequence-based typing (SBT) genomic analyses, plasmid analyses, and automated repetitive sequence-based PCR (rep-PCR) to identify epidemiologically significant traits capable of subtyping S. Enteritidis PT13.

Results: CGH using an oligonucleotide array based upon chromosomal coding sequences of S. enterica serovar Typhimurium strain LT2 and the Salmonella genomic island 1 successfully determined major genetic differences between S. Typhimurium and S. Enteritidis PT13, but no significant strain-to-strain differences were observed between S. Enteritidis PT13 isolates. Individual loci (safA and fliC) that were identified as potentially divergent in the CGH data set were sequenced in a panel of S. Enteritidis strains, and no differences were detected between the PT13 strains. Additional sequence-based typing was performed at the fimA, mdh, manB, cyaA, citT, caiC, dmsA, ratA and STM0660 loci. Similarly, no diversity was observed amongst PT13 strains. Variation in plasmid content between PT13 strains was observed, but macrorestriction with BglII did not identify further differences. Automated rep-PCR patterns were variable between serovars, but S. Enteritidis PT13 strains could not be differentiated.

Conclusion: None of the methods identified any significant variation between PT13 strains. Greater than 11,300 base pairs of sequence for each of seven S. Enteritidis PT13 strains were analyzed without detecting a single polymorphic site, although diversity between different phage types of S. Enteritidis was observed. These data suggest that Canadian S. Enteritidis PT13 strains are highly related genetically.

Figure 1

Pulsed-field gel electrophoresis of S. Enteritidis PT13 using XbaI. Two macrorestriction patterns (SENXAI) were observed, and are presented with high to low molecular weight fragments from left to right.

Figure 2

DNA microarray-based comparative genomics of S. Enteritidis PT13. Array probes represent the linear order of S. Typhimurium LT2 coding sequences from left to right, with the custom Salmonella genomic island 1 (SGI1) at the far-left side. White denotes similarity to LT2, green denotes putative divergence and red represents putative duplication or copy number change. Clusters of bacteriophage-related determinants that are divergent in S. Enteritidis compared to S. Typhimurium: A, STM893–929 (Fels-1 prophage); B, STM1005–1024 (Gifsy-2 prophage); C, STM2230–2240 (putative phage); D, STM2589–2636 (Gifsy-1 prophage); E, STM2732–2772 (Fels-2 prophage); F, STM4198–4218 (putative phage).

Figure 3

Plasmid profiles for S. Enteritidis strains used in this study. Preparations were not digested with restriction endonuclease. Lanes 1–7: S. Enteritidis PT13 strains 04-6191, 04-6387, 04-7505, 05-6746, 05-0513, 05-1219 and 05-6733 respectively. Lanes 8 and 9: S. Enteritidis PT1 strains 06-1230 and 06-1751. Lanes 10 and 11: S. Enteritidis PT4 strains 06-1216 and 06-1231. Lane 12: plasmid extracted from S. Typhimurium LT2. Supercoiled DNA ladder molecular weights are to the left of lane 1. Arrow indicates a chromosomal DNA fragment.

Figure 4

Plasmid RFLP patterns for S. Enteritidis PT13, PT1 and PT4 strains. Restriction fragment patterns generated with BglII were analyzed in BioNumerics version 4 and a dendrogram was created using the UPGMA method with a coefficient of correlation, 2% optimization and 12% position tolerance. PP; RFLP plasmid pattern, PT; phage type.

Figure 5

Comparison of S. Enteritidis and select Salmonella serovars by automated rep-PCR. The dendrogram represents the relatedness of strains based upon analyses of the amplification products using DiversiLab software. The vertical grey threshold line represents 95% similarity; PT = phage type.