Detection of virulence‐associated genes characteristic of intestinal Escherichia coli pathotypes, including the enterohemorrhagic/enteroaggregative O104:H4, in bovines from Germany and Spain

Abstract

Cattle are reservoirs of enterohemorrhagic Escherichia coli; however, their role in the epidemiology of other pathogenic E. coli remains undefined. A new set of quantitative real-time PCR assays for the direct detection and quantification of nine virulence-associated genes (VAGs) characteristic of the most important human E. coli pathotypes and four serotype-related genes (wzx O104 ,fliC H4 , rbf O157 , fliC H7 ) that can be used as a surveillance tool for detection of pathogenic strains was developed. A total of 970 cattle fecal samples were collected in slaughterhouses in Germany and Spain, pooled into 134 samples and analyzed with this tool. stx1, eae and invA were more prevalent in Spanish samples whereas bfpA, stx2, ehxA, elt, est and the rbf O157 /fliC H7 combination were observed in similar proportions in both countries. Genes characteristic of the hybrid O104:H4 strain of the 2011 German outbreak (stx2/aggR/wzx O104 / fliC H4 ) were simultaneously detected in six fecal pools from one German abattoir located near the outbreak epicenter. Although no isolate harboring the full stx2/aggR/wzx O104 /fliC H4 combination was cultured, sequencing of the aggR positive PCR products revealed 100% homology to the aggR from the outbreak strain. Concomitant detection by this direct approach of VAGs from a novel human pathogenic E. coli strain in cattle samples implies that the E. coli gene pool in these animals can be implicated in de novo formation of such highly-virulent strains. The application of this set of qPCRs in surveillance studies could be an efficient early-warning tool for the emergence of zoonotic E. coli in livestock.

Experimental Infection of Calves with Escherichia coli O104:H4 outbreak strain

“…Although being fairly non-pathogenic for cattle similar to classical STEC strains, EHEC O104:H4 appear to be less well adapted to bovine intestine than classical STEC strains. EHEC O104:H4 was not yet found in cattle herds272844 but the present experimental infection study, as well as epidemiological evidence gathered in the outbreak region30, support the hypothesis that EHEC O104:H4 and probably other EAEC may be or become part of the E. coli microbiome in this livestock species and contribute to and participate in the genome plasticity through uptake and loss of virulence genes45. Future risk assessments must therefore take cattle as an animal reservoir for EHEC/EAEC hybrid strains and a potential source of transmission to humans into account.…”

“…The virulence plasmid of enteroaggregative E. coli (pAA) is a relatively unstable genetic element as demonstrated by its intra-host loss during course of human infection38. A role of the pAA for adherence of EHEC O104:H4 to the bovine intestine remains obscure, because agg R-positive EAEC have rarely been detected in cattle3039. pAA-negative derivatives isolated from humans lost their ability to efficiently colonize the intestinal epithelium, which results in lack of systemic absorption of Stx2 and diminished the ability of EHEC O104:H4 to cause HUS38.…”

“…It is tempting to speculate that the large 2011 outbreak in the human population affecting several thousand individuals and with an undisclosed number of asymptomatic shedders29 could have led to a spillover of the strain to wild-life or livestock populations including cattle. Indeed, we detected genes characteristic of the EHEC O104:H4 strain ( stx 2 , aggR , wzx O104 , fliC H4 ) in fecal pools sampled in a German abattoir processing animals from farms located near the outbreak epicenter two years after the outbreak onset, while contemporarily obtained samples from an unrelated geographical area (Spain) were negative for this combination of genes30. The epidemiological evidence to suggest that the outbreak strain has already conquered an animal reservoir is weak.…”

Human influence and biotic homogenization drive the distribution ofEscherichia colivirulence genes in natural habitats

“…Water samples and pooled fecal samples were processed and analyzed by using a previously described qPCR assay in order to detect a set of nine VGs (see Table S1) characteristic of different E. coli enteric pathotypes ( stx 1, stx 2, eae , Inv A, ehx A, est , elt , bfp A, agg R), four serotype‐related genes ( rfb O157 , fli C H7 , wzx O104 , fli C H4 ), and one genus‐specific gene ( uid A) (Cabal et al., ; Cabal et al., ). Pooled fecal samples were processed in a 1/3 proportion of phosphate‐buffered saline.…”

Fitness of Enterohemorrhagic Escherichia coli (EHEC)/Enteroaggregative E. coli O104:H4 in Comparison to That of EHEC O157: Survival Studies in Food and In Vitro

“…Several studies have also provided evidence of a reservoir for SF E. coli O157:NM in cattle (14)(15)(16)(17). Recently, genes characteristic of the O104:H4 German outbreak strain were found in cattle herd samples from one abattoir located near the outbreak epicenter (18). Consequently, like other STEC/EHEC serotypes, both E. coli O104:H4 and E. coli O157:NM might enter the food chain via cattle as the potential reservoir.…”