Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 26, 2004
Publication Date: May 1, 2004
Citation: Shelton, D.R., Higgins, J.A., Van Kessel, J.S., Pachepsky, Y.A., Belt, K., Karns, J.S. 2004. Estimation of viable Escherichia coli 0157 in surface waters using enrichment in conjunction with immunological detection. Journal of Microbiological Methods. 58:223-231.
Interpretive Summary: There is increasing concern regarding the impact of water-borne pathogens on human health. Enterohemorrhagic E. coli O157:H7 is a serious health threat, particularly in children. It causes bloody diarrhea and, if not treated promptly, can result in kidney failure and death. There are an estimated 73,000 cases of E. coli O157 infections per year in the U.S., of which approximately 11,000 are water-borne. At present, the risk from water-borne transmission of E. coli O157 cannot be estimated because there are no reliable methods for the detection and enumeration of these organisms in water samples. Cultural methods are prohibitively laborious and expensive. We have previously developed a novel method for the quantitative detection of E. coli O157 in water samples using antibodies coupled to magnetic beads and a light producing catalyst (IM-ECL). The method has been optimized to detect as few as one living E. coli O157 cell in 100 mL of water by using a selective broth for growth of E. coli O157 cells in conjunction with IM-ECL for detection. The method is relatively fast (< 24 hours) and inexpensive, allowing for the analysis of multiple water samples daily. Preliminary studies with water samples from Baltimore County indicate that E. coli O157 cells are present, but at levels too low to present a public health threat.
The use of a minimal lactose enrichment both (MLB) in conjunction with immunomagnetic electrochemiluminescence detection (IM-ECL) was evaluated for the estimation of viable E. coli O157 populations in surface water samples. In principle, E. coli O157 populations (Cinitial E. coli O157) can be derived from enrichment data according to the equation:
Cinitial E. coli O157 = Cinitial coliforms x (Cfinal E. coli O157 / Cfinal coliforms), assuming that the growth rates and lag times of water-borne E. coli O157 and collective coliforms are sufficiently comparable, or at least consistent. We have previously described a protocol for determining Cfinal E. coli O157 in MLB-enriched water samples. In the present study, 80% of coliforms (red/pink colonies on MacConkey Agar) grew in MLB, indicating that this provides reasonably accurate estimates of Cinitial coliforms. Estimates of Cfinal coliforms were determined from turbidity data. Initial E. coli O157 populations (Cinitial E. coli O157) were calculated for 33 Baltimore watershed samples giving a positive IM-ECL response. The majority of samples contained E. coli O157 concentrations of <1 cell per 100 mL. These data indicate that E. coli O157 are present in surface water samples but at very low levels. Growth rates for MLB-enriched coliforms were highly variable (k = 0.47±0.13; n = 72). There was no correlation between growth rates and any measured water parameter, suggesting that water samples are spatially and temporally unique. Although variability in growth rates would be expected to yield some low values, the fact that most E. coli O157 concentrations were < 1 suggests that other factor(s) were also responsible. Studies with E. coli O157:H7 and wild-type E. coli suggest that increased lag times due to starvation were at least partially responsible for the observed data. Based on estimates of Cinitial coliforms and kcoliforms, MLB was evaluated for sensitivity and quantitativeness. Simulated populations of E. coli O157:H7 at stationary phase varied from ca. 103 to 108 cells mL-1 enrichment culture. Although not suitable for quantitation, MLB enrichment in conjunction with IM-ECL can detect as few as 1 viable water-borne E. coli O157 cell per 100 mL surface water. Experiments are in progress to evaluate alternative media for sensitivity and quantitative detection of enterohemorrhagic E. coli.