Cell invasion of poultry-associated Salmonella enterica serovar Enteritidis isolates is associated with pathogenicity, motility and proteins secreted by the type III secretion system

Abstract

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne gastroenteritis in humans worldwide. Poultry and poultry products are considered the major vehicles of transmission to humans. Using cell invasiveness as a surrogate marker for pathogenicity, we tested the invasiveness of 53 poultry-associated isolates of S. Enteritidis in a well-differentiated intestinal epithelial cell model (Caco-2). The method allowed classification of the isolates into low (n = 7), medium (n = 18) and high (n = 30) invasiveness categories. Cell invasiveness of the isolates did not correlate with the presence of the virulence-associated gene spvB or the ability of the isolates to form biofilms. Testing of representative isolates with high and low invasiveness in a mouse model revealed that the former were more invasive in vivo and caused more and earlier mortalities, whereas the latter were significantly less invasive in vivo, causing few or no mortalities. Further characterization of representative isolates with low and high invasiveness showed that most of the isolates with low invasiveness had impaired motility and impaired secretion of either flagella-associated proteins (FlgK, FljB and FlgL) or type III secretion system (TTSS)-secreted proteins (SipA and SipD) encoded on Salmonella pathogenicity island-1. In addition, isolates with low invasiveness had impaired ability to invade and/or survive within chicken macrophages. These data suggest that not all isolates of S. Enteritidis recovered from poultry may be equally pathogenic, and that the pathogenicity of S. Enteritidis isolates is associated, in part, with both motility and secretion of TTSS effector proteins.

Fig. 1.

Cluster analysis of 53 S. Enteritidis isolates recovered from diverse poultry sources. Eight major clusters (4, 5 and 8–13) were defined as groups of closely related strains sharing on average ≥85 % allelic congruence, as resolved by MLVA genotypes.

Fig. 2.

A k-means cluster plot of the variable Caco-2 cell invasiveness of 53 S. Enteritidis isolates recovered from diverse poultry sources. Three groups of isolates with low (n = 7), medium (n = 18) and high (n = 30) invasiveness were identified based on their cell invasion potential.

Fig. 3.

Kaplan–Meier survival curve showing percentage survival rates in mice challenged with representative S. Enteritidis isolates with high and low invasiveness in cultured Caco-2 cells.

Fig. 4.

Numbers of Salmonella recovered from small intestine, caecum, liver and spleen of mice challenged with S. Enteritidis isolates with high/medium and low invasiveness in cultured Caco-2 cells.

Fig. 5.

Secretory protein profile of representative S. Enteritidis isolates with low and high invasiveness in cultured Caco-2 cells. Lane M, protein molecular mass standard. The deduced amino acid sequences from the five major protein bands (indicated by arrows) corresponded to SipA (p1, 74 kDa), FlgK (p2, 59 kDa), FljB (p3, 52 kDa), SipD (p4, 36 kDa) and FlgL (p5, 34 kDa).

Fig. 6.

Transmission electron micrographs of S. Enteritidis strains UK (a), BC8 (b), P21 (c), G1 (d), C19 (e), C45 (f), G45 (g) and A11 (h). Samples were taken from the edge of a motile colony. Bars, 0.2 µm.