Quantification of Shiga Toxin-Converting Bacteriophages in Wastewater and in Fecal Samples by Real-Time Quantitative PCR

Shiga toxin-converting bacteriophages (Stx phages) are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7. Stx phages are released from their bacterial hosts after lytic induction and remain free in the environment. Samples were analyzed for the presence of free Stx phages by an experimental approach based on the use of real-time quantitative PCR (qPCR), which enables stx to be detected in the DNA from the viral fraction of each sample. A total of 150 samples, including urban raw sewage samples, wastewater samples with fecal contamination from cattle, pigs, and poultry, and fecal samples from humans and diverse animals, were used in this study. Stx phages were detected in 70.0% of urban sewage samples (10 to 10³ gene copies GC] per ml) and in 94.0% of animal wastewater samples of several origins (10 to 10¹⁰ GC per ml). Eighty-nine percent of cattle fecal samples were positive for Stx phages (10 to 10⁵ GC per g of sample), as were 31.8% of other fecal samples of various origins (10 to 10⁴ GC per g of sample). The stx₂ genes and stx₂ variants were detected in the viral fraction of some of the samples after sequencing of stx₂ fragments amplified by conventional PCR. The occurrence and abundance of Stx phages in the extraintestinal environment confirm the role of Stx phages as a reservoir of stx in the environment.Shiga toxin-producing Escherichia coli (STEC) is associated with diarrhea, hemorrhagic enterocolitis, and hemolytic-uremic syndrome in humans (46). Escherichia coli serotype O157:H7 is the main cause of these diseases, although other serotypes of E. coli and other enterobacteria species have been described (36). These E. coli serotypes produce at least two immunologically distinct Shiga toxins, called Stx1 and Stx2. In addition to these, several variations of these toxins have been reported in recent years, showing differences in virulence and distribution in the host populations examined (48, 51). Shiga toxin genes are carried by temperate bacteriophages (19, 35). Stx-encoding bacteriophages investigated to date consist of double-stranded DNA and have lambdoid genetic structures (19, 27, 32, 37, 47). The induction and regulation of these phages are directly involved in the production of toxin and, therefore, in the pathogenicity of the strains (8, 50). Stx phages are efficient vectors for the transfer of toxin genes, being able to convert nonpathogenic bacterial hosts into Stx-producing strains by transduction of stx, as has been demonstrated under various conditions (1, 4, 27, 28, 41, 49).Most of the reported outbreaks of STEC infections are associated with cattle products (10, 17), with the consumption of contaminated foods (10, 34), and with several waterborne infections (30). Stx phages are present within fecally contaminated aquatic environments (9, 28, 30, 32, 45). Moreover, a high percentage of STEC strains present in extraintestinal environments carry inducible Stx phages (14, 30).As individuals infected with STEC strains shed large quantities of Stx phages in feces, Stx phages should be prevalent in the environment, as are other viruses transmitted by the fecal-oral route (5, 11) or bacteriophages infecting bacteria present in the intestinal tract (16, 23). Moreover, those STEC strains isolated from food and animals carry inducible Stx phages (24, 27, 42). The virulence profiles of STEC strains isolated from food also suggest the presence of inducible Stx phages (10).Stx phages in sewage have been detected by nested PCR (28, 29, 31). However, to quantify them, the most probable number (MPN) method was applied, which allows only a rough estimate of the amount of Stx phages present in the sample. To assess the number of Stx phages accurately, real-time quantitative PCR (qPCR) technology is a useful tool. This technology is both sensitive and specific, and it gives accurate quantitative results (25). Comparison with a standard enables the number of copies of stx to be quantified, which can then be translated into the number of Stx phage particles.Little is known about the prevalence of phages carrying stx in fecal samples. The data available on the numbers of these phages in fecally contaminated water samples were only roughly estimated. The first step to evaluate the role of Stx phages in the environment as lateral gene transfer vectors is to know the extent of these viruses in the environment. The aim of this study is to report quantitative data on the abundance of Stx phages in urban sewage samples, in wastewater samples from cattle, pigs, and poultry, and in diverse fecal samples, calculated by means of a methodology based on qPCR.