Intimin Gene ( eae ) Subtype-Based Real-Time PCR Strategy for Specific Detection of Shiga Toxin-Producing Escherichia coli Serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 in Cattle Feces

Abstract

g Shiga toxin-producing Escherichia coli (STEC) strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are known to be associated with particular subtypes of the intimin gene (eae), namely, ␥1, ␤1, , , and ␥1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targeted eae subtypes. The simultaneous presence of stx, eae, and one of the five O group markers was found in 58.0% of the samples, and the five targeted stx plus eae plus O genetic combinations were detected 143 times. However, taking into consideration the association between eae subtypes and O group markers, the resulting stx plus eae subtype plus O combinations were detected only 46 times. The 46 isolation assays performed allowed recovery of 22 E. coli strains belonging to one of the five targeted STEC serogroups. In contrast, only 2 of 39 isolation assays performed on samples that were positive for stx, eae and an O group marker, but that were negative for the corresponding eae subtype, were successful. Characterization of the 24 E. coli isolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11, and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenic E. coli (aEPEC). Finally, the more discriminating eae subtype-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces.

Prevalence of Carriage of Shiga Toxin-Producing Escherichia coli Serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 among Slaughtered Adult Cattle in France

“…The proportion of isolation assays leading to the isolation of one of the top five STEC strains was relatively low, as previously observed in various studies (14,15,17). The reasons for this discrepancy, including PCR-based strategy and isolation assays limitations, have been already discussed elsewhere (26,(40)(41)(42). In this study, three isolation procedures were used in parallel and allowed us to improve the isolation fraction of the top five STEC strains.…”

“…The PCR-based strategy used to detect suspect samples was that described in the ISO 13136:2012 technical specification, to which we added a screening of the eae subtypes associated with the five major EHEC (11,26). Indeed, we previously showed that this additional screening step helped to be more discriminating for the specific detection of suspect samples likely to contain the five major STEC in cattle feces.…”

“…DNA extracts were subjected to a sequential real-time PCR-based approach for the detection of EHEC-associated genetic markers. This PCR-based strategy used to detect suspect samples was the same as the one described in the ISO 13136:2012 technical specification, to which a screening of the eae subtypes associated with the five major EHEC was added (11,26). An initial screening step was performed for the detection of stx, eae genes, and eae subtypes ␥1, ␤1, ε, and .…”

“…Upon arrival, each sample (10 g) was diluted 10-fold (wt/vol) in 90 ml of modified tryptone soya broth (Oxoid, Dardilly, France) supplemented with novobiocin (Oxoid, Dardilly, France) at 16 mg · liter Ϫ1 and incubated overnight at 37°C. Bacterial DNA was extracted from 1 ml of each enriched broth using lysis tubes (Pall GeneDisc Technologies, Bruz, France), as already described (26).…”

Molecular Profiling of Shiga Toxin-Producing Escherichia coli and Enteropathogenic E. coli Strains Isolated from French Coastal Environments

“…Eighteen types and nine subtypes of the eae gene, namely, ␣, ␣2, ␤1 to ␤3, ␥1, ␥2, ␦, ε, ε2 to ε4, , , 2, , , 2, , , , , , , , , and , have been deposited in the GenBank database (14). The eae subtypes are responsible for some host tissue cell tropisms (15) and are related to human infections; eae subtypes ␤, ␥, ε, and are those most frequently associated with human infections (16,17).…”