Genetic analysis of shiga-toxigenic Escherichia coli isolates from cattle in a limited region

The ecology of shiga-toxigenic Escherichia coli (STEC) is important in the animal production environment. We investigated fecal shedding of STEC in one town in Miyagi, Japan by multiplex polymerase chain reaction (PCR) targeting shiga toxin gene 1 (Stx₁), gene 2 (stx₂) and malB promoter gene, and analyzed the PCR products of Stx₁ or Stx₂ (54 samples) by direct sequencing. Three of 46 (6.5%) beef cattle in the University Farm of Tohoku University (Kawatabi Farm) and 11 of 70 (15.7%) calves in neighboring dairy farms carried STEC. Rate of detecting genes of Stx ₁, stx₂ and Stx₇₊₂ was 3.4% (4/116), 8.6% (10/116) and 0.9% (1/116), respectively. Serotyping indicated that STEC contaminated farms at different times or through different routes. Isolates harbored no mutation among Stx₁, but six (Kawatabi Farm) and 38 (neighboring farms) base substitutions among stx₂, respectively. The diversity of substitutions of Stx₂ was observed among farms or even in a farm. Phylogenic analysis revealed that STEC detected in the area were classified into three clusters by the variety of stx₂. Sequence analysis of Stx₂ will be one of the tools for clarifying the source of outbreaks and the route of contamination of STEC.