Other mPCR assays have enabled detection of certain E. coli serogroups (O157, O111, and O113) in water. Here, researchers applied two previously developed mPCR methods (China et al., 1996; Paton and Paton, 1999) to detect these organisms in water sampled from dairy farms in Brazil (Vicente et al., 2005). They first targeted the virulence genes stx1 , stx2 , and eae (China et al., 1996); then for positive samples, a second mPCR assay was utilized targeting rfb O157, rfb O113, and rfb O111 (Paton and Paton, 1999). Others have used mPCR to identify potentially pathogenic E. coli from a beach environment (Lake Superior) frequently closed because of high E. coli counts (Ishii et al., 2007). In this study, four virulence genes [stx1 , stx2 , eaeA, and ehxA (Paton and Paton, 1998a)] were targeted. The researchers found that from 3557 E. coli strains obtained from lake water, sediment, and sand, only one could be classified as a potential human pathogen, an enteropathogenic E. coli (EPEC).
Microarrays have been especially valuable for investigating E. coli pathotypes in water samples (Hamelin et al., 2006, 2007). In one study, researchers found that a sig- nificant percentage (29%) of beach isolates carried a pathotype set of virulence-related genes, and a smaller percentage (14%) carried antimicrobial resistance genes (Hamelin et al., 2006). Another microarray-based study found that the distribution of E. coli pathotypes differed significantly between sampling sites (surface water at six locations), with ExPEC being the most commonly encountered pathotypes (Hamelin et al., 2007). A recent article illustrates the impressive potential of microarray technology for detecting a large number of pathogens (Miller et al., 2008). These researchers devel- oped and validated an In situ –synthesized biochip for the detection of 12 microbial pathogens relevant to clinical diagnostics as well as water and food safety (Miller et al., 2008). The method involved probes designed for multiple virulence and marker genes (VMGs) for each pathogen, with each VMG being targeted by an average of 17 probes. A split multiplex PCR design was used to amplify target genes simultane- ously, providing a detection limit of 0.1 to 0.01% relative abundance, depending on the VMG and the pathogen. In addition, the biochip was validated using DNA obtained from three different types of water samples spiked with pathogen genomic DNA. The ability to detect multiple pathogens in parallel and in complex matrixes indicates that microarrays will probably play a growing role for monitoring water quality.
Molecular methods offer both advantages and disadvantages over traditional meth- ods for detecting and identifying E. coli populations in water. Both sides are nicely illustrated by a recent investigation of ETEC in household and environmental water samples from Bangladesh, involving both a quantitative real-time PCR method and the toxin GM1 ganglioside-enzyme-linked immunosorbent assay (GM1-ELISA) (Lothigius et al., 2008). The real-time PCR method, which quantifies the ETEC enterotoxin genes for the production of heat-labile (LT) and heat-stable (ST) enterotoxins (estA, estB , and eltB ), found that 26 of the 39 samples (67%) were positive for ETEC, but only six samples (15%) were positive in the GM1-ELISA. The study highlights the common advantage of molecular-based methods, increased sensitivity, and thus reduced risk for false negatives. However, this advantage also comes with the concern of overes- timating the number of infectious cells, as the method does not discriminate between live and dead cells. In addition, the real-time method does not facilitate differenti- ation between different strains in the same sample. For example, the method does discriminate between samples with a double positive strain (ST/LT) or with separate ST-positive and LT-positive strains. In contrast, in GM1-ELISA, separate colonies can be tested for ST and LT enterotoxin production.
METHODS FOR ADDRESSING EMERGING PROBLEMS FOR WATER 67
Although molecular methods provide a powerful platform for pathogen detection, it appears that in some cases, more refinement is needed for the detection of specific pathogenic E. coli in water samples. For example, researchers involved in a 30-month surface water monitoring study concluded that current immunological and PCR assays (real-time PCR targeting stx1, stx2, and eae genes (Higgins et al., 2005) could not
reliably identify waterborne enterohemorrhagic E. coli (EHEC) (Shelton et al., 2006). Others have reported the inadequacies of using the gene (ehlyA) encoding for the puta- tive virulence factor enterohemolysin for detecting environmental EHEC (Boczek et al., 2006). They found that although enterohemolysin production among environmental E. coli isolates is common, the enterohemolysin positives isolates did not exhibit the necessary virulence factors to be classified as EHEC.
The most useful methods for detecting E. coli in water are those that provide information on cell viability. The importance of such methods was illustrated in a study investigating viable but nonculturable cells (VBNCs) in nondisinfected drinking water (municipal tap and private well water) (Bjergbaek and Roslev, 2005). These researchers examined the ability of four different E. coli strains to enter a VBNC state using both traditional and molecular-based methods [cultivation, gfp-tagged E. coli , fluorescent in situ hybridization (FISH) and direct viable counts]. In addition, various resuscitation procedures were used to investigate the recovery of stressed E. coli from drinking water samples. Interestingly, they reported that the E. coli strains could enter a state where they were undetectable with standard media but able to maintain the potential for metabolic activity and sometimes, cell division. They reported that apparently nonculturable cells were fully resuscitated to a culturable state. The authors conclude that potentially viable E. coli may not be detected with standard procedures during the routine analysis of drinking water.
More recently, a highly sensitive method of detecting viable E. coli O157:H7 cells in water samples was developed (Liu et al., 2006, 2008). In fact, according to the authors, the procedure resulted in the lowest limit of detection reported to date for viable but not culturable cells in environmental water samples. The technique involved efficient cell capture, RNA extraction and purification, followed by detection via reverse transcrip- tion PCR and electronic microarray detection of the E. coli O157 lipopolysaccharide gene (rfbE ) and the H7 flagellin gene (fliC ) gene of E. coli O157:H7. The electron microarray facilitated DNA concentration on the microchip and contributed signifi- cantly to assay sensitivity. The assay enabled detection of 3 to 4 CFU/L tap water, 7 CFU/L river water, and 50 VBNCs/L river water. Clearly, this method has great potential for monitoring viable E. coli in environmental and drinking water samples.
3.3 METHODS FOR ADDRESSING EMERGING PROBLEMS FOR WATER
3.3.1 Genetic Characterization of Toxic Algae in Water
Harmful algal blooms (HABs) are considered emerging waterborne pathogens because of the toxins produced by these organisms (cyanobacteria or “blue-green algae”). Thus, monitoring the toxin-producing cyanobacteria is critical for the continued protection of our drinking water sources. In fact, cyanotoxins are on the U.S. Environmental Protection Agency’s (EPA’s) current (version 3) contaminant candidate list. However, because each cyanotoxin can be produced by more than one cyanobacterial species and the same species can produce more than one toxin (Funari and Testai, 2008), this is a