Prevalence of the stx 2 Gene in Coliform Populations from Aquatic Environments

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2004, p Vol. 70, No /04/$ DOI: /AEM Copyright 2004, American Society for Microbiology. All Rights Reserved. Prevalence of the stx 2 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, 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 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. Shiga toxin-producing Escherichia coli (STEC) strains are important human pathogens linked to the development of hemorrhagic colitis and hemolytic uremic syndrome worldwide. 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 responsible 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 are bacteriophage borne (24, 27, 36, 38, 40), which may be important for the spread of STEC strains. Recent studies reported significant numbers of free stx 2 -bearing bacteriophages in the environment (24, 25), which may maintain the gene and infect new bacteria. Some studies have already shown that the transduction of stx-bearing bacteriophages to new bacterial hosts occurs in vivo (1) and in vitro (34). E. coli O157:H7 was the first serotype associated with hemorrhagic colitis (33, 45), although more than 100 STEC serotypes have since been isolated from different sources, including contaminated food (22) and recreational (14) and drinking (21, 28, 39) water. Usually, the isolation of pathogens from the environment is difficult due to the low proportion of pathogens compared with the generally high microbial concentration. Consequently, the confirmation of a presumptive causative agent involved in an * Corresponding author. Mailing address: Departament de Microbiologia, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal, 645, 4 a Planta, Barcelona, Spain. Phone: Fax: ablanch@ub.edu. outbreak is not always achieved (5). Moreover, the detection of non-o157 STEC 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 -glucuronidase activity). As a result, they cannot be identified with selective culture media such as sorbitol-macconkey agar supplemented with cefixime and tellurite (46) or Rainbow agar (3), which are recommended for E. coli O157:H7 isolation. Some molecular methods 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. Furthermore, they are not able to detect strains carrying stx genes that do not express the toxin. Recently, a new molecular method suitable for the screening and isolation of potential Shiga toxin-producing strains has been developed based on the detection of the stx 2 gene (4). This method has allowed the detection of stx 2 gene-carrying bacteria among coliform bacteria grown on Chromocult coliform agar (Merck, Darmstadt, Germany) by using a specific probe to detect a fragment of 378 bp of the A subunit of the stx 2 gene. The advantage of this method is that individual colonies can be obtained, and then the clone can be isolated and further characterized, which is important to a better understanding of the ecology of the stx 2 gene in bacterial populations. This study reports the prevalence and the isolation of bacteria carrying the stx 2 gene in several samples of raw human sewage from different treatment plants and wastewater from different slaughterhouses by the most probable number (MPN) method combined with a nested PCR and the colony hybridization method (4). Additionally, the effects of secondary and 3535

2 3536 GARCÍA-ALJARO ET AL. APPL. ENVIRON. MICROBIOL. TABLE 1. Characteristics of the municipal sewage treatment plants and animal slaughterhouses and enumeration of stx 2 gene-carrying bacteria and bacterial indicators analyzed in this study a Plant Characteristics of the sewage of the treatment plant or slaughterhouse No. of samples stx 2 gene-carrying bacteria [log 10 (MPN 1)/ml] E. coli Organisms [log 10 (CFU 1)/ml] Mean no. (SD) of: FC [log 10 (CFU 1)/ml] TC [log 10 (CFU 1)/ml] 1 H; 400,000 inhabitants (0.3) 5.1 (0.2) 5.4 (0.3) 6.1 (0.2) 2 H; 5,000 inhabitants (0.4) 5.1 (0.3) 5.3 (0.2) 5.8 (0.3) 3 H; 1,400,000 inhabitants (0.4) 4.9 (0.1) 5.3 (0.2) 5.9 (0.1) 4 H; 1,500 inhabitants (0.5) 4.8 (0.2) 5.1 (0.1) 6.2 (0.1) 5 H; 1,500 inhabitants (0.2) 4.6 (0.1) 4.7 (0.1) 5.5 (0.2) 6 A; pig slaughterhouse (0.4) 6.0 (0.2) 6.0 (0.3) 6.3 (0.1) 7 A; cattle slaughterhouse (0.9) 4.5 (0.7) 4.6 (0.7) 4.6 (0.6) 8 M; mixed origin (0.4) 5.0 (0.1) 5.0 (0.1) 5.4 (0.2) a H, human origin; A, animal origin; M, mixed-animal origin (cattle, goat, lamb, and poultry). The percentage of samples that tested positive for stx 2 detection was 100%. tertiary treatments on the bacterial indicators (numbers of total coliforms [TC], fecal coliforms [FC], and E. coli organisms) in relation to the stx 2 gene-carrying bacteria were also determined for samples from two sewage treatment plants. The population of stx 2 gene-carrying bacteria was also monitored over a 1-year period to assess seasonal changes in STEC shedding, which has previously been described for E. coli O157:H7 (10). Finally, stx 2 gene-carrying strains were phenotypically characterized. MATERIALS AND METHODS Bacterial strains and media. E. coli O157:H7 strain ATCC 43889, which produces Stx2, was used as a positive control. E. coli O157:H7 strain ATCC and E. coli C600, which produce neither Stx1 nor Stx2 and do not possess the genes for these toxins, were used as negative controls. These bacteria were grown on tryptic soy agar (TSA) (Difco, Le Pont de Claix, France) overnight at 37 C and then grown without agitation in tryptic soy broth (Difco) for bacterial DNA extraction. Sample collection. Samples were collected aseptically and transferred into sterile containers according to standard procedures (6). The samples were then placed in coolers, transported to the laboratory, and kept at 4 C. Analysis was performed within 6 h of sampling. The characteristics of the sampling sites are summarized in Table 1. Raw wastewater samples were taken from the influents of five different urban wastewater treatment plants and three different slaughterhouses to compare the prevalences of stx 2 in waters of different origins. In addition, the effect on the elimination of these bacteria of a treatment based exclusively on lagooning (plant 4) and of a secondary (activated-sludge) and tertiary (lagooning) treatment (plant 5) was analyzed. Samples from treatment plant 4 were taken during the winter season, when a lower reduction of microbial populations has been described to occur (7). This treatment plant consists of four successive lagoons. Samples were taken from raw sewage, lagooning effluent 2 (between lagoons 2 and 3), and lagooning effluent 3 (between lagoons 3 and 4). Seasonal variations in the presence of the stx 2 gene in bacterial populations from sewage were studied by sampling raw sewage from treatment plant 1 every 2 weeks over a 1-year period. The enumeration of stx 2 gene-carrying bacteria was performed by the MPN nested-pcr method described previously (4). Enumeration of bacterial indicators. Numbers of FC, E. coli organisms, and TC were also analyzed for all the samples. The enumeration was performed using the membrane filtration method according to previously standardized methods (6). M-FC agar (Difco) was used for the enumeration of FC at 44.5 C, and Chromocult coliform agar (Merck) was used for the enumeration of E. coli organisms and TC at 37 C. DNA extraction. DNA extraction was performed according to a previously described protocol with minor modifications (12). Samples were centrifuged at 250 g and 4 C for 15 min to remove solid particles that could interfere with the DNA extraction and subsequent PCR. In order to quantify the stx 2 gene-carrying bacteria by the MPN nested-pcr method (4), bacterial cells from five aliquots of 10, 1, 0.1, and 0.01 ml of the supernatant were harvested by centrifugation at 16,000 g for 5 min. Supernatants were then removed, and 200 l of lysis buffer consisting of 10 mm Tris-HCl, 1 mm EDTA, and 0.5% Triton X-100 was added to each tube. The samples were resuspended, heat treated in a bath at 100 C for 10 min, and immediately transferred to ice-cold absolute ethanol. Samples were then centrifuged at 16,000 g for 5 min, and 2 l of the supernatant was used as a template for nested-pcr amplification. Detection and quantification of stx 2 gene-carrying bacteria. The detection and quantification of stx 2 gene-carrying bacteria were performed by a combination of the MPN technique and nested PCR and by colony hybridization. The MPN of bacteria carrying the stx 2 gene was determined after the nested- PCR amplification (performed as described below) of DNA extracted from 10-fold dilutions of each sample. All PCR assays were performed in 25- l volumes containing 2 mm MgCl 2,10 buffer provided by the manufacturer (Eppendorf, Hamburg, Germany), 200 M deoxynucleoside triphosphate,2uof Taq DNA polymerase (Eppendorf), a 0.3 M concentration of each primer, and 2 l of the extracted DNA, and the final volume was adjusted with sterile double-distilled water. External and internal reverse and forward primers used in this study were described previously (24). First, PCR and nested-amplification products of 378 and 169 bp, respectively, were resolved on a 2% agarose gel and stained with ethidium bromide. Nested-PCR amplification products were confirmed by dot blotting using an internal digoxigenin-labeled probe consisting of 26 bp of the A subunit of the stx 2 gene (24), according to previously established protocols (4). The MPN was calculated by counting the obtained bands corresponding to positive amplification. The sensitivity of this quantification method was calculated by analyzing 10-fold dilutions of a culture of E. coli O157:H7 ATCC grown in tryptic soy broth at 37 C for 6 h both by MPN-nested PCR and by plating replicate dilutions on TSA. The enumeration of bacteria carrying the stx 2 gene by colony hybridization was performed as follows. Tenfold dilutions of each sample were prepared with Ringer 1/4 Solution (Oxoid, Basingstoke, England). Aliquots of 250 l were spread onto 140-mm-diameter Chromocult coliform agar plates and incubated at 37 C for 24 h. The counting of blue and red colonies was performed for the enumeration of TC and E. coli organisms, respectively. Later, plates from dilutions presenting heavy but nonconfluent colony growth were selected for colony transfer, which was performed by carefully placing a nylon membrane onto the surface of the agar and quickly peeling it from the plate. Later, the bacterial cells on the membranes were lysed and fixed as described previously (26). The membranes were washed twice with a solution consisting of 0.1% sodium dodecyl sulfate and 3 SSC (20 SSC is 3 M NaCl plus 0.3 M sodium citrate, ph 7.0) at 68 Cfor1h30minandprehybridized with standard prehybridization solution at 68 C for 2 h. The membranes were then hybridized with the specific probe at 65 C. The external PCR primers for the stx 2 gene that were described previously (24) were used to prepare the stx 2 -specific probe used in the colony hybridization. The DNA extracted from temperate bacteriophages induced from stx 2 -positive E. coli strains was used as the template for the PCR as previously described (4). The 378-bp amplimer obtained was labeled with digoxigenin to be used later as a specific probe according to the manufacturer s instructions (Roche Diagnostics, Mannheim, Germany). Labeling was performed by the incorporation of digoxigenin-11-deoxyuridine-triphosphate during the PCR as described previously (23). The specificity of the probe was evaluated by using the collection strains E. coli ATCC 43889, E. coli C600, E. coli CN13, and E. coli DH5. The digoxigenin- DNA luminescent detection kit (Roche Diagnostics) was used to detect positive hybridization by placing membranes in contact with X-ray films according to the

3 VOL. 70, 2004 stx 2 GENE IN AQUATIC ENVIRONMENTS 3537 manufacturer s instructions. Colonies showing a positive hybridization signal were counted. Then, stx 2 -positive strains were isolated on TSA from the original Chromocult coliform agar plate, and the presence of the stx 2 gene was confirmed by specific PCR as described below. Sequencing. Some of the fragments obtained in the nested PCR were sequenced with the Big Dye terminator cycle sequencing ready reaction kit (Applied Biosystems, Madrid, Spain) and the ABI PRISM 3700 DNA sequencer (Applied Biosystems) according to the manufacturer s instructions. The samples were sequenced in both the reverse and forward orientations with the same primers used in the nested PCR. Phenotypic characterization. The strains carrying the stx 2 gene were biochemically phenotyped using the Phene-Plate system with 96-well PhP-RE microplates (Ph-Plate Microplate Technique AB, Stockholm, Sweden) developed for FC phenotyping analyses. They were used according to the manufacturer s instructions. Additionally, the presence of -D-glucuronidase activity and the ability to ferment sorbitol in 24 h were assessed. The sorbitol fermentation test was performed by inoculating purple bromocresol broth tubes containing 1% D- sorbitol (Sigma, St. Louis, Mo.) with the strain to be tested and incubating them at 37 C for 24 h. The -glucuronidase activity was assessed by inoculating the strain in 250 l of phosphate-buffered saline with a -glucuronidase tablet (Diatabs, Rosko, Denmark) and incubating it for 4 h or overnight at 37 C. The expression of the E. coli O157 serotype was tested by Western blot analysis of the lipopolysaccharide (LPS) with specific anti-o157 antibodies (Oxoid) as described below. Strains from different samples showing different PhP phenotypes and LPS patterns were identified with the commercial identification test API 20E (BioMérieux, La Balme, France). These strains were stored at 70 C in15% glycerol for further studies. Extraction of LPS and Western blotting. The extraction of LPS was conducted with the proteinase K method (11). Then, 10 l of the extracted LPS was subjected to electrophoresis in duplicate by using denaturing polyacrylamide gels (17). One gel was silver stained (41) to study LPS patterns, and the other was used for Western blotting with a Mini-Protean II dual-slab cell and a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad, Richmond, Calif.). Briefly, the gel was electrotransferred onto nitrocellulose membranes (Hybond ECL; Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, England) in a solution containing 25 mm Tris, 192 mm glycine, and 20% methanol. The gel was run at 150 V for 1 h according to the manufacturer s instructions. Later, membranes were processed by immunoblotting (31) using O157-specific antibodies (Oxoid) as specific primary antibodies. Shiga toxin protein production. The production of Shiga toxin proteins, both Stx1 and Stx2, was detected by methods described previously (29) using the commercial Duopath Verotoxin detection kit (Merck) according to the manufacturer s instructions. Briefly, five to six colonies from the TSA plates were inoculated in 1 ml of CAYE broth modified according to Evans (Merck) and incubated for 6hat37 C. After incubation, 180 l of the culture was mixed with 20 l of polymyxin B solution and further incubated for 10 min at 37 C. A volume of 160 l of the mixture was dispensed into the circular sample port on the test device. Results were observed after 20 min of room temperature incubation. RESULTS Presence of the stx 2 gene in raw sewage. The MPN nested- PCR method was efficient for the enumeration of stx 2 genecarrying bacteria (Fig. 1). Bacteria carrying the stx 2 gene were detected in all of the 67 sewage samples analyzed, independently of the human or animal origin of the samples. The values obtained with the MPN-nested PCR in the urban wastewater treatment plants ranged from 1.2 to 2.3 log 10 units (MPN 1) per ml. For all of the municipal sewage samples (human origin), the ratios of stx 2 gene-carrying bacteria to bacterial indicators were of the same order of magnitude. These proportions ranged from 1:1,000 to 1:10,000 (Table 1). However, a higher variability in the presence of stx 2 gene-carrying bacteria was observed in animal wastewater samples. In the cattle samples (the most numerous of our samples), the proportion of the numbers of E. coli organisms, FC, and TC that carried stx 2 was 1:100, 1 log unit higher than in human sewage samples. A similar ratio of stx 2 -carrying strains was found among FC and FIG. 1. Amplification products of the nested PCR used for MPN enumeration of stx 2 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. E. coli organisms in wastewater samples from other animal origins, although the number of samples is too low to consider these data to be any more than a mere indication. However, the ratio of stx 2 -carrying strains to TC for any wastewater sample was slightly lower than that for human sewage samples. The lowest proportion was observed in wastewater samples from the cattle slaughterhouse (Table 1). Seasonal distribution of the stx 2 gene. The values obtained for stx 2 gene-carrying bacteria, E. coli organisms, FC, and TC over a 1-year period of monitoring plant 1 were similar throughout the period, showing only minor variations. MPNs up to 100/ml were found for stx 2 gene-carrying bacteria, while TC, FC, and E. coli were present in quantities up to 10 6,10 5, and 10 5 CFU/ml, respectively (Fig. 2). The proportions of each of these bacterial populations that carried stx 2 were similar to those indicated above. No seasonal differences were observed for any of the measured parameters, as indicated by an analysis of variance test (P 0.05). Reduction of numbers of stx 2 gene-carrying bacteria in the effluents of sewage treatment plants. There was a reduction in the numbers of all of the studied bacterial indicators and stx 2 gene-carrying bacteria in the effluents of the two sewage treatment plants analyzed. There were some differences between values from the activated-sludge treatment plus lagooning system (plant 5) and the lagooning treatment (plant 4) (Table 2). At plant 4, there was a reduction of 2 log 10 units of stx 2 genecarrying bacteria and of around 1 log 10 unit of TC, FC, and E. coli organisms between raw sewage and the most distant lagooning effluent analyzed. At plant 5, the reduction of the numbers of TC, FC, and E. coli organisms in the tertiary effluent ranged from 3.5 to 4.5 log 10 units. However, the exact reduction in the number of stx 2 gene-carrying bacteria, which must be greater than 1 log 10 unit, could not be determined, since stx 2 gene-carrying bacteria were not detected in the effluents. Isolation and characterization of stx 2 gene-carrying bacteria. One hundred thirty-eight coliform strains were isolated from the different samples by using the colony hybridization method. The presence of the stx 2 gene was confirmed by spe-

4 3538 GARCÍA-ALJARO ET AL. APPL. ENVIRON. MICROBIOL. FIG. 2. Monitoring of stx 2 strains (}), E. coli ( ), FC ( ), and TC (Œ) over a 1-year period in the municipal sewage treatment plant. cific PCR for all of these isolates with the exception of five strains that apparently lost the gene upon subcultivation. Some of the 378-bp amplimers were sequenced, and they showed a high homology to the stx 2 gene sequence from the 933W phage. A total of 125 isolates showed the expected pattern of typical E. coli on Choromocult coliform agar (blue-colored colonies indicating -D-glucuronidase activity). According to the LPS pattern and the phenotype obtained with the PhP system, 59 representative strains were selected for further characterization. A total of 28 different LPS patterns and 44 different Phene system phenotypes with an identity level of were observed. Strains from different samples with different LPS patterns and phenotypes were considered representatives of each sample. Later, these strains were identified with the API 20E system. Fifty-five of these isolates were classified as E. coli (all of these, with one exception, were indole positive), while the others could not be identified with the API 20E system. The majority of the isolated strains was able to ferment sorbitol within 24 h and had -D-glucuronidase activity (46). Only five strains were unable to ferment sorbitol within 24 h, of which only one belonged to the E. coli O157:H serotype. This serotype was also negative for the -D-glucuronidase test. Production of Shiga toxins. Despite the fact that all of these strains produced the PCR products of the stx 2 gene, only 23 isolates (39%) produced the toxin protein Stx2, 4 isolates (7%) produced Stx1 but failed to produce Stx2, and only 1 isolate (2%) produced Stx1 and Stx2 (Table 3). The last isolate corresponded to the E. coli O157 isolate. There was a higher proportion of bacteria that carried the stx 2 gene and were not capable of toxin production among municipal sewage isolates than among animal wastewater isolates: 94% of the bacteria from municipal sewage versus 43% from animal wastewater samples. DISCUSSION Since E. coli O157:H7 was first isolated from water in 1989 (21), numerous waterborne-stec outbreaks have been reported (14, 28, 39). The low dose and the difficulty of isolating these microorganisms in aquatic environments increase the need for the development of sensitive and specific methods for STEC detection. A method suitable for the enumeration of stx 2 gene-carrying bacteria in sewage samples has been applied in this study to analyze their presence in environmental samples. In recent years, the use of quantitative PCR for the enumer- TABLE 2. Effect of diverse sewage treatments on the number of bacteria Sample source and type a TC [log (CFU 1)/ml] FC [log (CFU 1)/ml] Mean no. of: E. coli organisms [log (CFU 1)/ml] stx 2 gene-carrying bacteria [log (MPN 1)/ml] Plant 4 RS LE LE Plant 5 RS SE b TE b a RS, raw sewage; SE, secondary effluent; TE, tertiary effluent; LE2, lagooning effluent 2 (sewage effluent between lagoons 2 and 3); LE3, lagooning effluent 3 (sewage effluent between lagoons 3 and 4). b The number of bacteria was below the limit of detection.

5 VOL. 70, 2004 stx 2 GENE IN AQUATIC ENVIRONMENTS 3539 TABLE 3. Toxin production and distribution of stx 1 and stx 2 genes between the different representative strains isolated Origin No. of strains (n 59) with: Toxin production a stx 1 Stx1 only Stx2 only Stx1 and Stx2 No toxin gene stx 2 gene Animal Human ation of microorganisms, particularly for E. coli O157:H7 (37), has become widely extended. However, the combined use of nested PCR and the MPN method in this study allowed the MPN detection of 10 to 100 of the targeted bacteria per ml (4). The colony hybridization method allowed the detection of 1 stx 2 gene-carrying colony among approximately 10 3 total colonies on 140-mm-diameter agar plates. The results obtained with the MPN nested-pcr method were consistent with the quantification of strains isolated by using the colony hybridization method. The data revealed high levels of stx 2 gene-carrying bacteria in raw human sewage and wastewater containing fecal wastes from different animals, independently of the origins of the samples. The ratio of stx 2 gene-carrying organisms to FC was approximately 1:1,000 in all the samples with the exception of cattle wastewater, which showed a higher proportion of stx 2 gene-carrying organisms (1:100), probably because cattle are the major reservoir of STEC (9). We observed some variations in the concentrations of different microbial indicators and stx 2 gene-carrying bacteria among the different human sewage treatment plants, which could be related to the different kinds of wastewater that they receive. Although some results were obtained from a small number of samples, significant values were obtained from plants which supplied high numbers of samples. These data are representative, since the incoming raw sewage of those treatment plants has been widely studied, showing very constant values for the analyzed bacterial indicators (7, 20). Unlike what happens with cattle shedding of E. coli O157:H7 (9), there were no significant seasonal differences in the levels of shedding of stx 2 gene-carrying bacteria in the monitored human wastewater treatment plant. These results are in agreement with those from studies performed with contaminated river waters in Japan, which found stx 2 gene-carrying bacteria at densities between 10 2 and 10 4 cells per ml independently of the season (16). The high diversity of E. coli serotypes and other enterobacteria which may carry the stx 2 gene may explain such a difference, because the MPN nested-pcr method analyzes all these bacterial groups at the same time. There was only a minor decrease in the number of stx 2 genecarrying bacteria for two samples obtained during the winter. Data from this study and the fact that E. coli O157:H7 cells have been demonstrated to survive for long periods in manure (15, 43) and in water (5, 32, 42) suggest that human sewage and animal wastewater, in addition to cattle and other, wild animals, should be regarded as reservoirs of STEC. There was a reduction in the number of stx 2 gene-carrying strains after treatment in both treatment plants analyzed. In treatment plant 4, there was a larger reduction in the number of stx 2 gene-carrying strains than in the number of bacterial indicators, suggesting that STEC strains are not specifically resistant to the lagooning treatment. It was not possible to compare this treatment with the activated-sludge treatment plus lagooning system (plant 5), because after secondary treatment, the numbers of stx 2 gene-carrying bacteria were already under the limit of detection. Values of reduction could not be calculated; it can be stated only that the log 10 reduction was greater than 1.2 log 10 units. However, the activated-sludge treatment plus lagooning system was also observed to be efficient for the reduction in the number of stx 2 gene-carrying bacteria. The values obtained in treatment plant 4 confirm previous results showing a small reduction in the number of FC during the winter season, which is the period in which the results reported here were obtained (7). The specific colony hybridization method used in this study enabled the isolation of 138 coliform strains carrying the stx 2 gene. The expected PCR band of 378 bp corresponding to the stx 2 gene amplimer was produced by all strains, with the exception of five strains that appeared to lose the gene after subcultivation. This phenomenon is consistent with the results of previous studies of clinical samples that showed the loss of stx genes upon subcultivation (13; J. C. Paton and A. W. Paton, Letter, J. Clin. Microbiol. 35:1917, 1997). Only one strain belonged to the serotype O157:H, which suggests a low prevalence of this serotype in stx 2 -carrying strains in the analyzed samples. This study shows that the stx 2 gene is commonly present in municipal sewage and animal wastewaters of different origins, with a wide distribution range among coliform bacteria. Some of these bacteria not only harbor the gene but also are able to produce the toxins and consequently may represent a potential health risk, which must be taken into account. Most of the analyzed strains that expressed the Stx2 toxin arose from animal samples. Stx1 toxin production was also detected in some strains (4 of 59), usually arising from samples of human origin. It should be noted that in isolates from human wastewater and wastewater of animal origin, the gene is not always expressed. The toxin is produced in approximately 50% of the strains from animal wastewater, while of the strains from human sewage, although all of them carried the gene, less than 10% showed expression of the protein toxin. Hence, the animal strains seemed to maintain the characteristic, while the human strains have acquired the gene in a nonfunctional way, since they do not produce Stx2. The presence of the stx 1 gene evaluated in bacterial populations in this study has also been observed in the genomes of free bacteriophages infectious for E. coli or Shigella spp. present in sewage (8, 24). It is well known that the stx 2 gene is carried in the genomes of temperate bacteriophages, which may be the origin of free phages detected in sewage. Although the presence of inducible phages in the strains analyzed in this study has not been evaluated (this topic will be the subject of future studies), the potential contribution of phages to the mobility of the stx 2 gene should not be underestimated. The presence of the stx 2 gene in populations detected in sewage (phages or bacteria) indicates an exchange of this gene between these populations. More information is needed to understand the ecology of the stx 2 gene in water environments and its involvement in the pathogenicity of STEC strains.

6 3540 GARCÍA-ALJARO ET AL. APPL. ENVIRON. MICROBIOL. ACKNOWLEDGMENTS This study was supported by the research project BMC from the Ministerio de Ciencia y Tecnología, the project 2001SGR00099, and the Centre de Referència en Biotecnologia from the Generalitat de Catalunya. REFERENCES 1. Acheson, D. W. K., J. Reidl, X. Zhang, G. T. Keusch, J. J. Mekalanos, and M. K. Waldor In vivo transduction with Shiga toxin 1-encoding phage. Infect. Immun. 66: Bertin, Y., K. Boukhors, N. Pradel, V. Livrelli, and C. Martin Stx2 subtyping of Shiga toxin-producing Escherichia coli isolated from cattle in France: detection of a new Stx2 subtype and correlation with additional virulence factors. J. Clin. Microbiol. 39: Bettelheim, K. A Studies of Escherichia coli cultured on Rainbow Agar O157 with particular reference to enterohaemorrhagic Escherichia coli (EHEC). Microbiol. Immunol. 42: Blanch, A. R., C. Garcia-Aljaro, M. Muniesa, and J. Jofre Detection, enumeration and isolation of strains carrying the stx 2 gene from urban sewage. Water Sci. Technol. 47: Chalmers, R. M., H. Aird, and F. J. Bolton Waterborne Escherichia coli O157. Symp. Ser. Soc. Appl. Microbiol. 2000:124S 132S. 6. Clesceri, L. S., A. E. Greenberg, and A. D. Eaton, (ed.) Standard methods for the examination of water and wastewater, 20th ed. American Public Health Association, Washington, D.C. 7. Durán, A. E Comportamiento de los bacteriófagos propuestos como microorganismos modelo frente a diferentes procesos naturales y artificiales en aguas. Ph.D. thesis. Universisty of Barcelona, Barcelona, Spain. 8. Faruque, S. M., N. Chowdhury, R. Khan, M. R. Hasan, J. Nahar, M. J. Islam, S. Yamasaki, A. N. Ghosh, G. B. Nair, and D. A. Sack Shigella dysenteriae type 1-specific bacteriophage from environmental waters in Bangladesh. Appl. Environ. Microbiol. 69: Hancock, D., T. Besser, J. Lejeune, M. Davis, and D. Rice The control of VTEC in the animal reservoir. Int. J. Food Microbiol. 66: Heuvelink, A. E., F. L. A. M. van den Biggelaar, J. T. M. Zwartkruis-Nahuis, R. G. Herbes, R. Huyben, N. Nagelkerke, W. J. G. Melchers, L. A. H. Monnens, and E. de Boer Occurrence of verocytotoxin-producing Escherichia coli O157 on Dutch dairy farms. J. Clin. Microbiol. 36: Hitchcock, P. J., and T. M. Brown Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J. Bacteriol. 154: Holland, J. L., L. Louie, A. E. Simor, and M. Louie PCR detection of Escherichia coli O157:H7 directly from stools: evaluation of commercial extraction methods for purifying fecal DNA. J. Clin. Microbiol. 38: Karch, H., T. Meyer, H. Rüssmann, and J. Heesemann Frequent loss of Shiga-like toxin genes in clinical isolates of Escherichia coli upon subcultivation. Infect. Immun. 60: Keene, W. E., J. M. McAnulty, F. C. Hoesly, L. P. Williams, Jr., K. Hedberg, G. L. Oxman, T. J. Barrett, M. A. Pfaller, and D. W. Fleming A swimming-associated outbreak of hemorrhagic colitis caused by Escherichia coli O157:H7 and Shigella sonnei. N. Engl. J. Med. 331: Kudva, I. T., K. Blanch, and C. J. Hovde Analysis of Escherichia coli O157:H7 survival in ovine or bovine manure and manure slurry. Appl. Environ. Microbiol. 64: Kurokawa, K., K. Tani, M. Ogawa, and M. Nasu Abundance and distribution of bacteria carrying sltii gene in natural river water. Lett. Appl. Microbiol. 28: Laemmli, U. K Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: Law, D A review: virulence factors of Escherichia coli O157 and other Shiga toxin-producing E. coli. J. Appl. Microbiol. 88: Lindgren, S. W., J. E. Samuel, C. K. Schmitt, and A. D. O Brien The specific activities of Shiga-like toxin type II (SLT-II) and SLT-II-related toxins of enterohemorrhagic Escherichia coli differ when measured by Vero cell cytotoxicity but not by mouse lethality. Infect. Immun. 62: Lucena, F., X. Mendez, A. Moron, E. Calderon, C. Campos, A. Guerrero, M. Cardenas, C. Gantzer, L. Shwartzbrood, S. Skraber, and J. Jofre Occurrence and densities of bacteriophages proposed as indicators and bacterial indicators in river waters from Europe and South America. J. Appl. Microbiol. 94: McGowan, K. L., E. Wickersham, and N. A. Strockbine Escherichia coli O157:H7 from water. Lancet i: Mead, P. S., and P. M. Griffin Escherichia coli O157:H7. Lancet 352: Muniesa, M., J. Recktenwald, M. Bielaszewska, H. Karch, and H. Schmidt Characterization of a Shiga toxin 2e-converting bacteriophage from an Escherichia coli strain of human origin. Infect. Immun. 68: Muniesa, M., and J. Jofre Abundance in sewage of bacteriophages that infect Escherichia coli O157:H7 and that carry the Shiga toxin 2 gene. Appl. Environ. Microbiol. 64: Muniesa, M., F. Lucena, and J. Jofre Comparative survival of free Shiga toxin 2-encoding phages and Escherichia coli strains outside the gut. Appl. Environ. Microbiol. 65: Nizetic, D., R. Drmanac, and H. Lehrach An improved bacterial colony lysis procedure enables direct DNA hybridisation using short (10, 11 bases) oligonucleotides to cosmids. Nucleic Acids Res. 19: O Brien, A. D., J. W. Newland, S. F. Miller, R. K. Holmes, H. W. Smith, and S. B. Formal Shiga-like toxin-converting phages from Escherichia coli strains that cause hemorrhagic colitis or infantile diarrhea. Science 226: Olsen, S. J., G. Milller, T. Breuer, M. Kennedy, C. Higgins, J. Waldorf, G. McKee, K. Fox, W. Bibb, and P. Mead A waterborne outbreak of Escherichia coli O157:H7 infections and hemolytic uremic syndrome: implications for rural water systems. Emerg. Infect. Dis. 8: Park, C. H., H. J. Kim, D. L. Hixon, and A. Bubert Evaluation of the Duopath Verotoxin test for detection of Shiga toxins in cultures of human stools. J. Clin. Microbiol. 41: Paton, J. C., and A. W. Paton Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections. Clin. Microbiol. Rev. 11: Puig, A. R., R. Araujo, J. Jofre, and J. Frias-Lopez Identification of cell wall proteins of Bacteroides fragilis to which bacteriophage B40 8 binds specifically. Microbiology 147: Rajkowski, K. T., and E. W. Rice Recovery and survival of Escherichia coli O157:H7 in reconditioned pork-processing wastewater. J. Food Prot. 62: Riley, L. W., R. S. Remis, S. D. Helgerson, H. B. McGee, J. G. Wells, B. R. Davis, R. J. Hebert, E. S. Olcott, L. M. Johnson, N. T. Hargrett, P. A. Blake, and M. L. Cohen Hemorrhagic colitis associated with a rare Escherichia coli serotype. N. Engl. J. Med. 308: Schmidt, H., M. Bielaszewska, and H. Karch Transduction of enteric Escherichia coli isolates with a derivative of Shiga toxin 2-encoding bacteriophage 3538 isolated from Escherichia coli O157:H7. Appl. Environ. Microbiol. 65: Schmidt, H., J. Scheef, S. Morabito, A. Caprioli, L. H. Wieler, and H. Karch A new Shiga toxin 2 variant (Stx2f) from Escherichia coli isolated from pigeons. Appl. Environ. Microbiol. 66: Scotland, S. M., H. R. Smith, G. A. Willshaw, and B. Rowe Vero cytotoxin production in a strain of Escherichia coli is determined by genes carried on bacteriophage. Lancet ii: Sharma, V. K., and E. A. Dean-Nystrom Detection of enterohemorrhagic Escherichia coli O157:H7 by using a multiplex real-time PCR assay for genes encoding intimin and Shiga toxins. Vet. Microbiol. 93: Strockbine, N. A., L. R. M. Marques, J. W. Newland, H. W. Smith, R. K. Holmes, and A. D. O Brien Two toxin-converting phages from Escherichia coli O157:H7 strain 933 encode antigenically distinct toxins with similar biologic activities. Infect. Immun. 53: Swerdlow, D. L., B. A. Woodruff, R. C. Brady, P. M. Griffin, S. Tippen, H. D. Donnell, Jr., E. Geldreich, B. J. Payne, A. Meyher, and J. G. Wells A waterborne outbreak in Missouri of Escherichia coli O157:H7 associated with bloody diarrhea and death. Ann. Intern. Med. 117: Teel, L. D., A. R. Melton-Celsa, C. K. Schmitt, and A. D. O Brien One of two copies of the gene for the activatable Shiga toxin type 2d in Escherichia coli O91:H21 strain B2F1 is associated with an inducible bacteriophage. Infect. Immun. 70: Tsai, C. M., and C. E. Frasch A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119: Wang, G., and M. P. Doyle Survival of enterohemorrhagic Escherichia coli O157:H7 in water. J. Food Prot. 61: Wang, G., T. Zhao, and M. P. Doyle Fate of enterohemorrhagic Escherichia coli O157:H7 in bovine feces. Appl. Environ. Microbiol. 62: Weinstein, D. L., M. P. Jackson, J. E. Samuel, R. K. Holmes, and A. D. O Brien Cloning and sequencing of a Shiga-like toxin type II variant from an Escherichia coli strain responsible for edema disease of swine. J. Bacteriol. 170: Wells, J. G., B. R. Davis, I. K. Wachsmuth, L. W. Riley, R. S. Remis, R. Sokolow, and G. K. Morris Laboratory investigation of hemorrhagic colitis outbreaks associated with a rare Escherichia coli serotype. J. Clin. Microbiol. 18: Zadik, P. M., P. A. Chapman, and C. A. Siddons Use of tellurite for the selection of verocytotoxigenic Escherichia coli O157. J. Med. Microbiol. 39: