Exposure to rumen protozoa leads to enhancement of pathogenicity of and invasion by multiple-antibiotic-resistant Salmonella enterica bearing SGI1

Abstract

Multiple-antibiotic-resistant Salmonella enterica serotype Typhimurium is a food-borne pathogen that has been purported to be more virulent than antibiotic-sensitive counterparts. The paradigm for this multiresistant/hyperpathogenic phenotype is Salmonella enterica serotype Typhimurium phage type DT104 (DT104). The basis for the multiresistance in DT104 is related to an integron structure designated SGI1, but factors underlying hyperpathogenicity have not been completely identified. Since protozoa have been implicated in the alteration of virulence in Legionella and Mycobacterium spp., we attempted to assess the possibility that protozoa may contribute to the putative hypervirulence of DT104. Our study reveals that DT104 can be more invasive, as determined by a tissue culture invasion assay, after surviving within protozoa originating from the bovine rumen. The enhancement of invasion was correlated with hypervirulence in a bovine infection model in which we observed a more rapid progression of disease and a greater recovery rate for the pathogen. Fewer DT104 cells were recovered from tissues of infected animals when protozoa were lysed by preinfection chemical defaunation of the bovine or ovine rumen. The protozoan-mediated hypervirulence phenotype was observed only in DT104 and other Salmonella strains, including serovars Agona and Infantis, possessing SGI1.

FIG. 1.

Assessment of 1-h HEp-2 cell invasion for Salmonella recovered from RPz. Strain designations are summarized in Table 1. Black bars correspond to Salmonella recovered from RPz. Controls included Salmonella exposed to RPz culture medium (open bars), Salmonella exposed to RPz lysates (gray bars), Salmonella recovered from RPz that were exposed to cytochalasin D (horizontally hatched bars), Salmonella recovered from bovine macrophages (diagonally hatched bars), and Salmonella recovered from HEp-2 cells (checkered bars). Percent invasion equals 100(CFU recovered/CFU added).

FIG. 2.

Evaluation of the temporal nature of RPz-mediated DT104 hyperinvasion of HEp-2 cells. Time points correspond to length of DT104-HEp-2 cell coincubation. Squares represent the invasion of DT104 recovered from RPz, while the circles correspond to DT104 exposed to RPz culture medium. Percent invasion equals 100(CFU recovered/CFU added).

FIG. 3.

Transient hyperinvasion of HEp-2 cells (1 h) for DT104 exposed to RPz. Open bars represent bacteria isolated from RPz, i.e., the first invasion assay. Black bars represent the second invasion assay, i.e., bacteria recovered from the first assay. Gray bars represent the third invasion assay, i.e., bacteria recovered from the second assay. Percent invasion equals 100(CFU recovered/CFU added).

FIG. 4.

Evaluation of the relationship between RPz-mediated hyperinvasion of HEp-2 cells (1 h) and SGI1. Strain designations are summarized in Table 1, while the SGI1 status is indicated on the left. Black bars represent the controls (ctrl), which correspond to exposure to RPz culture medium, while open bars represent Salmonella recovered from RPz. Percent invasion equals 100(CFU recovered/CFU added).

FIG. 5.

Quantitation of DT104 recovered from calves experimentally challenged with the pathogen. (Bottom, graph) Enumeration of Salmonella bacteria recovered from tissues (spleen, lymph nodes [lnn], or ileum) removed from calves challenged with DT104. For spleen and lymph node studies, DT104 was either recovered from RPz (open bars, n = 3), present in RPz (open bars, n = 6), exposed to RPz culture medium (filled bars, n = 3), or present in HEp-2 cells (filled bars, n = 4). The data represent most-probable numbers of DT104 recovered from calves at 36 h following an oral infection. For ileal studies, ligated loops were employed and the data represent CFU recovered at 25 min following ileal inoculation. Ligated loops were inoculated with DT104 recovered from either RPz (open bars, n = 3) or HEp-2 cells (filled bars, n = 3). P values, comparing RPz-dependent and RPz-independent data, are given above the RPz-dependent data. (Top, gel photo) Agarose gel electrophoresis of 250-bp sipB-C amplicons (4) obtained from PCR amplification of infected tissues corresponding to that present in the graph. For each tissue, a standard amount of DNA was used for amplification as described in Materials and Methods.

FIG. 6.

Assessment of the onset of pyrexia in calves infected with DT104 exposed to RPz. Rectal temperatures were determined every 8 to 12 h for calves infected with DT104 exposed to RPz (squares, n = 6) or DT104 not exposed to RPz (circles, n = 4). P values, comparing RPz-dependent and RPz-independent data, are given above the RPz-dependent data. The upper limit for normal bovine rectal temperature (∼102.4°F) is indicated by the horizontal line.