Genetic Characterization of Escherichia coli O157:H7/– Strains Carrying the stx₂ Gene but Not Producing Shiga Toxin 2

Nine Escherichia coli O157:H7/– strains isolated primarily from non-clinical sources in Thailand and Japan carried the stx₂ gene but did not produce Stx2 toxin in a reversed passive latex agglutination (RPLA) assay. A strain (EDL933) bearing a stx₂ phage (933W) was compared to a strain (Thai-12) that was Stx2-negative but contained the stx₂ gene. To study the lack of Stx2 production, the Thai-12 stx₂ gene and its upstream nucleotide sequence were analyzed. The Thai-12 stx₂ coding region was intact and Stx2 was expressed from a cloned stx₂ gene using a plasmid vector and detected using RPLA. A lacZ fusion analysis found the Thai-12 stx₂ promoter non-functional. Because the stx₂ gene is downstream of the late promoter in the stx₂ phage genome, the antitermination activity of Q protein is essential for strong stx₂ transcription. Thai-12 had the q gene highly homologous to that of Φ21 phage but not to the 933W phage. High-level expression of exogenous q genes demonstrated Q antitermination activity was weak in Thai-12. Replication of stx₂ phage was not observed in Stx2-negative strains. The q-stx₂ gene sequence of Thai-12 was well conserved in all Stx2-negative strains. A PCR assay to detect the Thai-12 q-stx₂ sequence demonstrated that 30% of O157 strains from marketed Malaysian beef carried this sequence and they produced little or no Stx2. These results suggest that stx₂-positive O157 strains that produce little or no Stx2 may be widely distributed in the Asian environment.