Escheria Colli

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2004, p. 3535–3540 Vol. 70, No. 6 0099-2240/04/$08.00+0 DOI: 10.1128/AEM.70.6.3535–3540.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Prevalence of the stx2 Gene in Coliform Populations from Aquatic Environments

Cristina García-Aljaro, Maite Muniesa, Juan Jofre, and Anicet R. Blanch*Departament de Microbiologia, Facultat de Biologia, Universitat de Barcelona, 08028 Barcelona, Spain

Received 26 November 2003/Accepted 3 February 2004

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 forthese 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 stx2 gene in coliform bacteria in sewage samples of differentorigins. The presence of the stx2 gene was monitored every 2 weeks over a 1-year period in a municipal sewage treatment plant. A mean value of 102 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 strainscarrying the stx2 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 serotypecarrying the stx2 gene in the wastewater studied.

Shiga toxin-producing Escherichia coli (STEC) strains are important human pathogens linked to the development of hemorrhagic colitis and hemolytic uremic syndrome world- wide. The major virulence characteristics of STEC strains are the production of Shiga toxins and the ability to colonize the bowel through the adhesin protein intimin,which is responsi- ble for attaching and effacing lesions in the intestinal mucosa (18). However, not all pathogenic STEC strains have been shown to produce intimin, suggesting that other factors could contribute to human disease (30). There are two main groups of Shiga toxins (Stx1 and Stx2) and several Stx2 variants (c, d, e, and f) (2, 19, 35, 44). The majority of stx genes arebacte- riophage borne (24, 27, 36, 38, 40), which may be important for the spread of STEC strains. Recent studies reported significant numbers of free stx2-bearing bacteriophages in the environ- ment (24, 25), which may maintain the gene and infect new bacteria. Some studies have already shown that the transduc-

outbreak is not always achieved (5). Moreover, the detection of non-O157STEC strains is particularly difficult due to the lack of common phenotypic characteristics such as those presented by typical E. coli O157:H7 strains (delayed fermentation of sorbitol and the absence of þ-glucuronidase activity). As a result, they cannot be identified with selective culture media such as sorbitol-MacConkey agar supplemented with cefixime and tellurite (46) orRainbow agar (3), which are recom- mended for E. coli O157:H7 isolation. Some molecular meth- ods based on the detection of Shiga toxins have been described because these are the unique common features among non- O157 strains (46). However, most of them are expensive and time-consuming and do not allow the isolation of clones. Fur- thermore, they are not able to detect...