Investigating the adherence factors of Escherichia coli at the bovine recto-anal junction.

Abstract

Shiga toxin-producing Escherichia coli (STEC) are major foodborne pathogens that result in thousands of hospitalizations each year in the United States. Cattle, the natural reservoir, harbor STEC asymptomatically at the recto-anal junction (RAJ). The molecular mechanisms that allow STEC and non-STEC E. coli to adhere to the RAJ are not fully understood, in part because most adherence studies utilize human cell culture models. To identify a set of bovine-specific E. coli adherence factors, we used the primary RAJ squamous epithelial (RSE) cell-adherence assay to coculture RSE cells from healthy Holstein cattle with diverse E. coli strains from bovine and nonbovine sources. We hypothesized that a comparative genomic analysis of the strains would reveal factors associated with RSE adherence. After performing adherence assays with historical strains from the E. coli Reference Center (n = 62) and strains newly isolated from the RAJ (n = 15), we used the bioinformatic tool Roary to create a pangenome of this collection. We classified strains as either low or high adherence and using the Scoary program compiled a list of accessory genes correlated with the "high adherence" strains. While none of the correlations were statistically significant, several gene clusters were associated with the high-adherence phenotype, including two that encode uncharacterized proteins. We also demonstrated that non-STEC E. coli strains from the RAJ are more adherent than other isolates and can outcompete STEC in coculture with RSEs. Further analysis of adherence-associated gene clusters may lead to an improved understanding of the molecular mechanisms of RSE adherence and may help develop probiotics targeting STEC in cattle.

Importance: E. coli strains that produce Shiga toxin cause foodborne illness in humans but colonize cattle asymptomatically. The molecular mechanisms that E. coli uses to adhere to cattle cells are largely unknown. Various strategies are used to control E. coli in livestock and limit the risk of outbreaks. These include vaccinating animals against common E. coli strains and supplementing their feed with probiotics to reduce the carriage of pathogens. No strategy is completely effective, and probiotics often fail to colonize the animals. We sought to clarify the genes required for E. coli adherence in cattle by quantifying the attachment to bovine cells in a diverse set of bacteria. We also isolated nonpathogenic E. coli from healthy cows and showed that a representative isolate could outcompete pathogenic strains in cocultures. We propose that the focused study of these strains and their adherence factors will better inform the design of probiotics and vaccines for livestock.