|VAN KESSEL, JO ANN|
|Wachtel, Marian - NIH, BETHESDA, MD|
|Belt, Kenneth - USDA/FS, BALTO., MD|
Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 29, 2003
Publication Date: December 1, 2003
Citation: Shelton, D.R., Van Kessel, J.S., Wachtel, M.R., Belt, K.T., Karns, J.S. 2003. Evaluation of parameters affecting detection of Escherichia Coli 0157 in enriched water samples using immunomagnetic electrochemiluminescence. Journal of Microbiological Methods. p. 717-725. 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 small numbers of these organisms in water samples. We have previously developed a novel method for the quantitative detection of E. coli O157 in water samples using commercial antibodies coupled to magnetic beads (IM) and a light producing catalyst (ECL). In order to detect E. coli O157 cells in water samples at levels which may pose a public health threat, it is first necessary to increase or enrich the number of E. coli O157 cells by growth in a semi-selective broth. However, bacteria other than E. coli O157 can also grow in the broth. We observed that non-E. coli O157 bacteria can interfere with the binding of E. coli O157 to IM beads, thereby causing an underestimate of E. coli O157 cell numbers in enriched water samples. However, since competitive binding can be determined for individual water samples by including a control sample spiked with a known amount of E. coli O157, a correction factor can be calculated which allows for quantification of E. coli O157 cell numbers in enriched water samples. Research is currently being conducted to utilize this information to estimate the initial number of E. coli O157 in raw water samples.
Technical Abstract: We report here on the use of immunomagnetic (IM) electrochemiluminescence (ECL) for quantitative detection of E. coli O157:H7 in water samples following enrichment using a minimal lactose broth (MLB). In-house IM beads, prepared using four commercial anti-O157 monoclonal antibodies, were compared for efficiency of cell capture. IM-ECL responses for E. coli O157:H7 Odwalla were similar for all 4 commercial anti-O157 LPS monoclonal antibodies. Twenty-two strains of E. coli O157:H7 or O157:HNM gave comparable ECL signals using in-house prepared IM beads. To assess the potential for interference from background bacteria in MLB-enriched water samples, 104 cells of E. coli O157:H7 Odwalla were added to enriched samples prior to analysis. There was considerable variability in recovery of Odwalla cells; net ECL signals ranged from 1 to 100% of expected values (i.e., percent inhibition from 0 to 99%). Cultures of Klebsiella pneumoniae, Klebsiella oxytoca, and Enterobacter cloacae, subsequently isolated from MLB-enriched water samples via IM separation, were observed to interfere with binding of Odwalla cells to IM beads. Recoveries of 104 Odwalla cells were 10% in the presence of ca.108 of K. pneumoniae, K. oxytoca, or E. cloacae cells. None of these strains gave a positive IM-ECL signal. These studies indicate that IM-ECL in conjunction with MLB enrichment is capable of quantitatively detecting as few as 103 to 105 E. coli O157:H7 cells mL-1 in enriched water samples that contain ca. 109 total bacteria mL-1, when percent binding is determined. Assuming comparable growth rates for E. coli O157:H7 and other bacteria in MLB, it may be possible to detect as few as one E. coli O157:H7 per 104 to 106 total coliforms 100 mL-1 of raw water.