Prevalence of the stx2 gene in coliform populations from aquatic environments

Abstract

Shiga toxin-producing Escherichia coli strains are human pathogens linked to hemorrhagic colitis and hemolytic uremic syndrome. The major virulence factors of these strains are Shiga toxins Stx1 and Stx2. The majority of the genes coding for these toxins are borne by bacteriophages. Free Stx2-encoding bacteriophages have been found in aquatic environments, but there is limited information about the lysogenic strains and bacteria present in the environment that are susceptible to phage infection. The aim of this work was to study the prevalence and the distribution of the stx(2) gene in coliform bacteria in sewage samples of different origins. The presence of the stx(2) gene was monitored every 2 weeks over a 1-year period in a municipal sewage treatment plant. A mean value of 10(2) genes/ml was observed without significant variation during the study period. This concentration was of the same order of magnitude in raw municipal sewage of various origins and in animal wastewater from several slaughterhouses. A total of 138 strains carrying the stx(2) gene were isolated by colony hybridization. This procedure detected approximately 1 gene-carrying colony per 1,000 fecal coliform colonies in municipal sewage and around 1 gene-carrying colony per 100 fecal coliform colonies in animal wastewaters. Most of the isolates belonged to E. coli serotypes other than E. coli O157, suggesting a low prevalence of strains of this serotype carrying the stx(2) gene in the wastewater studied.

FIG. 1.

Amplification products of the nested PCR used for MPN enumeration of stx₂ gene-carrying bacteria in sewage samples. Lanes: 1ml, replicates of 1-ml sample; 0.1ml, replicates of 0.1-ml sample; 0.01ml, replicates of 0.01-ml sample; M, 100-bp DNA marker; H, I, and J, negative DNA extraction controls; K, negative PCR control; L, positive PCR control (strain ATCC 43889); N, negative nested-PCR control.

FIG. 2.

Monitoring of stx₂ strains (⧫), E. coli (×), FC (▪), and TC (▴) over a 1-year period in the municipal sewage treatment plant.