Differential phenotypic diversity among epidemic-spanning Salmonella enterica serovar enteritidis isolates from humans or animals

Abstract

Nontyphoidal salmonellae are major causes of food-borne disease worldwide. In Uruguay, Salmonella enterica serovar Enteritidis was the most commonly isolated serovar throughout the last decade, with a marked epidemic period between 1995 and 2004. In a previous study, we conducted comparative genomics of 29 epidemic-spanning S. Enteritidis field isolates, and here we evaluated the pathogenic potential of the same set of isolates using several phenotypic assays. The sample included 15 isolates from human gastroenteritis, 5 from invasive disease, and 9 from nonhuman sources. Contrary to the genetic homogeneity previously observed, we found great phenotypic variability among these isolates. One-third of them were defective in at least one assay, namely, 10 isolates were defective in motility, 8 in invasion of Caco-2 cells, and 10 in survival in egg albumen. Twelve isolates were tested for invasiveness in 3-day-old chickens, and five of these were significantly less invasive than the reference strain. The two oldest preepidemic isolates were reduced in fitness in all assays, providing a plausible explanation for the previous negligible incidence of S. Enteritidis in Uruguay and supporting the view that the introduction or emergence of a more virulent strain was responsible for the marked rise of this serovar. Further, we found differences in fitness among the isolates which depended on the source of isolation. A total of 1 out of 14 isolates from human gastroenteritis, but 6 out of 13 isolates from other sources, was impaired in at least two assays, suggesting enhanced fitness among strains able to cause intestinal disease in humans.

FIG. 1.

(A) Motility of S. Enteritidis isolates. The dashed line indicates the 20% cutoff below which an isolate is assigned a motility-impaired phenotype. S. Gal, S. Gallinarum strain. A representative experiment is shown. (B) Caco-2 adhesion and invasion assays. Total associated bacteria (adhesion) or intracellular bacteria (invasion) were quantified. The dashed line indicates the 0.11% cutoff used as a threshold to differentiate invasion-negative from invasion-positive isolates. Data shown are means ± standard errors. (C) Frequency of invasion-positive S. Enteritidis isolated from human gastroenteritis compared to the frequency of isolates obtained from other sources, i.e., isolates obtained from human systemic disease and nonhuman sources. The P value obtained using Fisher's exact test is shown.

FIG. 2.

Quantitative real-time PCR of CCL20, IL-8, and TNF-α genes from Caco-2 cells infected with S. Enteritidis isolates. Averages ± standard errors are shown. n/i, not infected; fliC−, cells infected with the fliC::aphT PT4-derived strain; *, significant difference related to cells infected with PT4 shown by one-way ANOVA (P < 0.05).

FIG. 3.

(A) S. Enteritidis survival in egg albumen at 2 days postinoculation. Values are expressed as average percentages of initial inocula ± standard errors. The dashed line indicates the 30% cutoff used as a threshold to differentiate survival-negative from survival-positive isolates. (B) Frequency of survival-positive S. Enteritidis isolates obtained from human sources compared to that of isolates obtained from nonhuman sources. The P value obtained using Fisher's exact test is shown.

FIG. 4.

S. Enteritidis colonization of chicken spleens at 7 days postinoculation. Each symbol represents an individual animal from two independent experiments. The corresponding median for each group is represented with a solid line. *, significant difference compared to that of PT4 shown by one-way ANOVA (P < 0.05).