Author
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Shelton, Daniel |
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Karns, Jeffrey |
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Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 4/1/2001 Publication Date: 7/1/2001 Citation: SHELTON, D.R., KARNS, J.S. QUANTITATIVE DETECTION OF ESCHERICHIA COLI O157 IN SURFACE WATERS USING IMMUNOMAGNETIC-ELECTROCHEMILUMINESCENCE (IM-ECL). APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 2001. Interpretive Summary: There is increasing concern regarding the impact of water-borne pathogens on human health. In particular, 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. For example, the current USDA/Food Safety and Inspection Service (FSIS) method entails four enrichment/culturing steps for preliminary identification and ten biochemical tests for confirmation of E. coli O157. We have previously developed a novel method for the quantitative detection of E. coli O157 in water samples using antibodies coupled to magnetic beads in conjunction with a light detection system. This method has been modified to detect E. coli O157 cells in water samples concentrated 100-fold using vortex filtration. The modified method allows for detection of approximately 25 to 100,000 E. coli O157 viable or non-viable cells in 100 milliliters of concentrated water. If water samples are enriched using special growth media selective for E. coli O157, approximately 2 viable E. coli O157 cells can be detected in one liter of concentrated water. Technical Abstract: A protocol for the quantitative detection of E. coli O157 in concentrated surface waters using immunomagnetic-electrochemiluminescence (IM-ECL) was developed and optimized. Studies were conducted using an antibody "sandwich" format utilizing self-prepared IM beads conjugated with a monoclonal IgG specific to O157 LPS coupled with a polyclonal anti-O157:H7 IgG to which an electrochemiluminescent label (TAG) was attached. Background ECL signals from concentrated (100x) water samples were substantially higher and more variable than raw water samples or phosphate buffered saline blanks. The background signal was partially eliminated by the addition of polyvinylpolypyrrolidone (PVPP). Successive cell capture incubations, termed sequential bead capture (SBC), were evaluated as a means of establishing baseline ECL values for individual water samples. Optimal results were obtained with (i) a slow speed centrifugation (100xg) prior to the assay, (ii) a three hour cell capture incubation, and (iii) a three mL sample volume. The linear dynamic range was ca. 100 to 100,000 E. coli O157 cells per mL concentrated water (100x). Ten liter surface water samples were spiked with ca. 5,000 E. coli O157 cells, concentrated by vortex filtration, and aliquots analyzed by IM-ECL. Cell recoveries were essentially complete. ECL signals were generally consistent with standard curves for comparable cell concentrations. Based on Student's t values, the sensitivity of the IM-ECL appears to be ca. 25 E. coli O157 cells per 100 mL concentrated water. Enrichments were conducted using EC broth (ECB) and minimal lactose broth (MLB). All tubes with concentrated water became turbid and gave a positive ECL response. These data suggest that viable 2 cells per L concentrated water can be detected with MLB. |
