Detection of virulence-associated genes characteristic of intestinal Escherichia coli pathotypes, including the Enterohemorrhagic / Enteroaggregative O104:H4 in bovines from Germany and Spain

Cattle are reservoirs of Enterohemorrhagic Escherichia coli but their role in the epidemiology of other pathogenic E. coli remains undefined. We describe 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 (wzxO104, fliCH4, rbfO157, fliCH7) that can be used as a surveillance tool for detection of pathogenic strains. A total of 970 cattle fecal samples were collected in slaughterhouses from Germany and Spain and 134 pooled samples and analyzed with this tool. Results showed that stx1, eae, and invA were more prevalent in Spanish samples while bfpA, stx2, ehxA, elt, est, and the rbfO157/fliCH7 combination were observed in similar proportions in both countries. Genes characteristic of the hybrid O104:H4 strain of the 2011 German outbreak (stx2/aggR/wzxO104/fliCH4) were simultaneously detected in six fecal pools from one German abattoir, near the outbreak epicenter. Although no isolate harboring the full stx2/aggR/wzxO104/fliCH4 combination was cultured, sequencing of the aggR positive PCR products revealed 100% homology to the aggR from the outbreak strain. Concomitant detection of VAGs from a novel human pathogenic E. coli strain in cattle samples by this direct approach 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 can be an efficient early-warning tool for the emergence of zoonotic E. coli in livestock.