Subtyping Salmonella enterica serovar enteritidis isolates from different sources by using sequence typing based on virulence genes and clustered regularly interspaced short palindromic repeats (CRISPRs)

Abstract

Salmonella enterica subsp. enterica serovar Enteritidis is a major cause of food-borne salmonellosis in the United States. Two major food vehicles for S. Enteritidis are contaminated eggs and chicken meat. Improved subtyping methods are needed to accurately track specific strains of S. Enteritidis related to human salmonellosis throughout the chicken and egg food system. A sequence typing scheme based on virulence genes (fimH and sseL) and clustered regularly interspaced short palindromic repeats (CRISPRs)-CRISPR-including multi-virulence-locus sequence typing (designated CRISPR-MVLST)-was used to characterize 35 human clinical isolates, 46 chicken isolates, 24 egg isolates, and 63 hen house environment isolates of S. Enteritidis. A total of 27 sequence types (STs) were identified among the 167 isolates. CRISPR-MVLST identified three persistent and predominate STs circulating among U.S. human clinical isolates and chicken, egg, and hen house environmental isolates in Pennsylvania, and an ST that was found only in eggs and humans. It also identified a potential environment-specific sequence type. Moreover, cluster analysis based on fimH and sseL identified a number of clusters, of which several were found in more than one outbreak, as well as 11 singletons. Further research is needed to determine if CRISPR-MVLST might help identify the ecological origins of S. Enteritidis strains that contaminate chickens and eggs.

Fig. 1.

Graphic representation of spacer arrangements in CRISPR1 and CRISPR2 of the 27 S. Enteritidis sequence types. Human, chicken, egg, and environmental isolates are designated h, c, e, and env, respectively. The number of isolates from each source is indicated after the source designation. Repeats are not included; only spacers are listed. The direction of the spacer arrangements is from 5′ to 3′. Each unique spacer is represented by a unique combination of the background color and the color of a particular character. Spacers were aligned, and gaps represent the absence of a particular spacer. Singletons had very distinct arrays of spacers and thus could not be aligned. P125109 is the strain of S. Enteritidis deposited in GenBank (accession number AM933172).

Fig. 2.

Frequency of the five predominant sequence types (E ST1, -3, -4, -8, and -10) in human, chicken, egg, and environmental isolates. The five predominant sequence types accounted for 76% of all isolates analyzed in the present study. All unlabeled pie slices in panels b, c, d, and e are STs unique to that given source, except the pie slice of E ST10, which contained only isolates from environmental samples.

Fig. 3.

Cluster diagram based only on fimH and sseL for all 27 sequence types. MLST identified three clusters (CI, CII, and CIII). Human, chicken, egg, and environmental isolates are designated h, c, e, and env, respectively. The number of isolates from each source is indicated before the source designation.

Fig. 4.

Cluster diagram based on virulence genes and CRISPRs for all 27 sequence types. Human, chicken, egg, and environmental isolates are designated h, c, e, and env, respectively. The number of isolates from each source is indicated before the source designation.