Application of Shiga toxin-specific digoxigenin-labeled DNA probes for identification of Shigatoxigenic Escherichia coli strains (STEC) in bovine feces

The aim of the present study was to develop and validate a method for specific identification of Shiga toxin-producing E. coli (STEC) in bovine feces by the use of DIG-labeled DNA probes. The stx1- and stx2-specific probes were prepared by means of PCR with stx1- and stx2-positive reference E. coli strains, respectively. The nylon membranes were utilized for making replicas from the LB or MacConkey´s master plates that had been inoculated with bovine feces artificially infected with stx-positive or stx-negative E. coli bacteria. The membranes were then incubated with stx-specific probes, anti-DIG conjugates, and developed with enzyme substrates (BCIP and NBT). Dark spots visible on the replicas were observed which corresponded to the stx-positive E. coli colonies on the master agar plates. Those bacteria were then isolated and characterized using multiplex PCR for the amplification of the rfbO157 (LPS O157), eaeA (intimin), and stx1/stx2 (Shiga toxins) genes. The analysis of the 69 bovine fecal samples derived from animals naturally infected with STEC demonstrated that 41 of them were either stx1-, stx2- or stx1/stx2-positive. The present method may be used for the identification and characterization of bacteria belonging to the STEC group in feces of bovine origin, which is the natural reservoir of these microorganisms.