Role of StdA in adhesion of Salmonella enterica serovar Enteritidis phage type 8 to host intestinal epithelial cells

Abstract

Background: Salmonella is often implicated in foodborne outbreaks, and is a major public health concern in the United States and throughout the world. Salmonella enterica serovar Enteritidis (SE) infection in humans is often associated with the consumption of contaminated poultry products. Adhesion to epithelial cells in the intestinal mucosa is a major pathogenic mechanism of Salmonella in poultry. Transposon mutagenesis identified stdA as a potential adhesion mutant of SE. Therefore, we hypothesize StdA plays a significant role in adhesion of SE to the intestinal mucosa of poultry.

Methods and results: To test our hypothesis, we created a mutant of SE in which stdA was deleted. Growth and motility were assayed along with the in vitro and in vivo adhesion ability of the ∆stdA when compared to the wild-type SE strain. Our data showed a significant decrease in motility in ∆stdA when compared to the wild-type and complemented strain. A decrease in adhesion to intestinal epithelial cells as well as in the small intestine and cecum of poultry was observed in ∆stdA. Furthermore, the lack of adhesion correlated to a defect in invasion as shown by a cell culture model using intestinal epithelial cells and bacterial recovery from the livers and spleens of chickens.

Conclusions: These studies suggest StdA is a major contributor to the adhesion of Salmonella to the intestinal mucosa of poultry.

Figure 1

Growth curves of the WT, ∆stdA, and complemented strains. The strains were grown in LB and the optical densities at 600 nm were measured each hour. The graph is representative of two independent experiments.

Figure 2

Motility assay of the WT, ∆stdA, and complemented strains. Bacteria were spotted onto soft agar, and migration of the bacteria was measured from the inoculation point to the periphery of the plate. (A) Images showing the migration of each SE strain. (B) Graph displaying the migration of each SE strain. The actual P values are given displaying a statistically significant difference between ∆stdA and the WT strain.

Figure 3

Cell culture assays using T84 intestinal epithelial cells. (A) Adhesion assay and (B) invasion assay with the WT, ∆stdA, and complemented strains. The actual P values are given displaying a statistically significant difference between ∆stdA and the WT strain.

Figure 4

Determination of adhesion ability in poultry. Bacterial counts in (A) small intestine and (B) cecum of chickens inoculated by oral crop gavage with 1 × 10⁷ CFU of WT and ∆stdA. The actual P values are given displaying a statistically significant difference between ∆stdA and the WT strain.

Figure 5

Systemic infection ability of the WT and ∆stdA SE strains. Bacterial counts in (A) liver and (B) spleen of chickens inoculated by oral crop gavage with 1 × 10⁷ CFU of WT and ∆stdA. There was no bacterial recovery at the 16 hour time point from either the liver or spleen of chickens inoculated with the WT or ∆stdA strain, so only the day 7 data is shown. The actual P values are given displaying a statistically significant difference between ∆stdA and the WT strain.