Detection of Shiga toxin-encoding genes in small community water supplies

Shiga toxin (Stx), one of the most potent bacterial toxins known, can cause bloody diarrhea, hemolytic uremic syndrome, kidney failure and death. The aim of this pilot was to investigate the occurrence of Shiga toxin-encoding genes, stx ( stx 1 and stx 2) from total coliform (TC) and E. coli positive samples from small community water systems. After aliquots for TC and E. coli analyses were removed, the remnant volume of the samples was enriched, following a protocol developed for this study. Fifty-two per cent of the samples tested by multiplex PCR were positive for the presence of the stx genes; this percentage was higher in raw water samples. The stx2 gene was more abundant. Testing larger volumes of the samples increase the sensitivity of our assay, providing an alternative protocol for the detection of Shiga toxin-producing E. coli (STEC) that might be missed by the TC assay. This study con ﬁ rms the presence of Stx encoding genes in source and distributed water for all systems sampled and suggests STEC as a potential health risk in small systems.

about the occurrence and prevalence of STEC in water.
Most research on STEC has focused on clinical samples, environmental samples following an outbreak or river and drinking water samples impacted by either sewage, slaughterhouses or agricultural processes (Ram et  The increase in the number of infections associated with STEC in drinking water has exposed the need to improve methods for its detection in environmental samples. Moreover, available methods detect only one serotype of E. coli, which can produce false negative results (Hamner et al. ; Yang et al. ). Recent developments for the molecular identification of diarrheagenic E. coli are expensive or time consuming and include quantitative PCR or multiplex PCR (mPCR) methods (Ram et al. ; Omar & Barnard ).
This pilot study attempts to adapt methods currently used in the monitoring of E. coli in potable water to develop a protocol for the detection of Shiga toxin-encoding genes in drinking water focusing on small potable water supply systems serving fewer than 3,300 people. Even though small systems comprise the largest number of potable water supplies around the world, they are often excluded from comprehensive sampling campaigns to evaluate the prevalence and occurrence of emerging pathogens and other contaminants. Small systems around the world, as in the USA, typically serve remote, underserved populations, those most at risk from waterborne illness due to the lack of capacity to operate, improper treatment and disinfection (Minnigh &  This study investigates the occurrence of Shiga toxinencoding genes in raw and distributed water from seven small potable water supplies operated by communities using an mPCR assay and a protocol for rapid and efficient detection of STEC in water.

Sample collection
Water samples were collected from seven small potable water systems located in the municipalities of Patillas and Guayama, in the southeast of the island of Puerto Rico ( Figure 1). Because of the absence or inconsistency of treatment, for the purpose of this paper, the samples collected within the distribution systems are referred to as 'distributed' water samples. Following convention, source water samples are called 'raw'. From each site, a 1-L water sample was collected for detection and enumeration of coliforms and E. coli. Physicochemical parameters (pH and turbidity) were also determined. Total and free chlorine was measured from distributed water samples. Samples were kept at 4-8 C until processing and were analyzed within 30 h of collection.
Total coliform and E. coli  Nalgene© centrifuge bottles and centrifuged for 7 min at 3360 × g in a Hermle© Z513 K. The supernatant was discarded, and the pellet was transferred using a sterile 10 mL serological pipette to a 50 mL sterile centrifuge tube. The pellet was then washed twice using DNA-grade 1× PBS and stored at À20 C until processing. Pellets were split into 2 mL portions to ease examination.

DNA extraction
DNA from all samples (E. coli positive and enriched samples) was extracted using a PureLink™ Genomic DNA Mini Kit (Invitrogen™) following manufacturer's protocols.
Initial digestion was done for 2 h at 55 C in a dry bath.
DNA from all samples was stored at À20 C.

Multiplex PCR
All mPCR reactions were performed in an Eppendorf unit Mastercycler ep Realplex 4S in a total reaction volume of 20 μL. The mPCR protocol for the mPCR kit (QIAGEN©) was used for all PCR reactions. The genes targeted by our mPCR assay included stx1 and stx2 genes which encode the two main forms of the toxin; the lt gene which encoded the heat-labile toxin produced by the enterotoxigenic E. coli (ETEC); the eaeA gene which encodes for the adhesion protein intimin; and an E. coli marker gene mdh.

RESULTS
In this study, two distinct sampling programs were per-  with densities between 10 0 and 10 2 per 100 mL, and E. coli between 0 and 10 2 . Three samples (3 of 31) were in the 10 3 log for TC and in the 10 2 log for E. coli. Additionally, two distributed samples (2 of 31) were in the 10 5 and 10 4 log for TC and were in the 10 3 and the 10 0 log, respectively, for E. coli (Table S1). The two distributed samples with the highest TC log had no chlorine and the turbidity for the sample with higher log counts was 4.6 NTUs (Table S1).
Raw water samples had TC log ranging from 10 to 10 5 and 0 to 10 4 E. coli per 100 mL (Table S1).
Even though all systems under this study showed stx positive samples in either or both raw and distributed water (Table 1) (Table 1).
Only one system showed a sample positive to the gene encoding the Lt toxin (data not shown). The mPCR method used included an internal control, the mdh gene.
However, the amplification of this gene was very difficult to screen in the 2.5% agarose gel used; the bands were small and faint and their visualization in the gel was not constant, thus mPCR results for this gene were not considered accurate and were not included in the analysis (data not shown).
The gene eaeA was detected in 63 of the 98 samples (Table 2). The two samples that contained only stx1 were also positive for eaeA. A total of 21 of the 27 samples that were positive to just stx2 were also positive to eaeA (78%).
A total of 19 of the 22 samples that had a combination of stx1 and stx2 were also positive to eaeA (86%) ( Table 2).
The only sample that was positive to the gene encoding  the Lt toxin was also eaeA positive (data not shown).
Twenty-one samples had only eaeA, with no stx1 or stx2 associated (Table 2).
In an effort to detect serotypes that could be missed by the standard MUG assay, such as E. coli O157:H7, we Of the 19 distributed samples with chlorine measurements (Table S1), stx genes were not found in waters with total chlorine >0.5 mg/L (Fishers exact test P ¼ 0.004) and not found after enrichment in samples with total chlorine >0.05 mg/L. Turbidity was not significantly related to stx occurrence in either raw or distributed samples.

DISCUSSION
Small (  This study also shows the need to include small systems in surveillance studies and efforts to gather information to assess the risk to the total population. The presence of the genes encoding Shiga toxin represents a health risk for the people drinking water from these systems, especially since most of these systems provide little or no disinfection. The presence of eaeA gene without Shiga toxin-encoding genes might represent an additional health risk, suggesting that other virulent factors might be present at these sites.
Additional studies are needed for the development of methods to detect E. coli serotype O157:H7 as it represents a health risk and its presence is easily missed by conventional methods.