Rainbow O157 Agar-Black colonies are E. coli O157 Poster Q-422 Evaluation of Organisms Isolated Using an Antibody-Based Detection Method for Waterborne Escherichia coli O157:H7 M. J. Collins, R. M. Hoffman--WI State Laboratory of Hygiene, Madison, WI Marty Collins WI State Lab of Hygiene 2601 Agriculture Drive Madison, WI 53718 (608) 224-6260 Phone collinmj@mail.slh.wisc.edu Abstract Introduction: E. coli O157:H7 is a highly virulent pathogen that causes nearly 73,000 illnesses in the United States each year. From 1982-2002, the CDC reported 350 E. coli O157 outbreaks, 9% of which were waterborne. Existing methods for detection of E. coli O157:H7 in water are non-standardized, have poor sensitivity, and are time intensive. These factors have prompted the development of several antibody-based detection methods for detecting E. coli O157:H7 in water. However, methods based on antigenicity may not provide sufficient information of an organism’s virulence and public health risk. This study summarizes data obtained from water samples tested by an antibody-based method. Methods: During the 2005 sampling season, 121 surface water samples from lakes near Madison, WI and riverine systems and inshore areas of Lake Michigan near Milwaukee, WI were tested for the presence of E. coli O157:H7. Each sample was filtered through a 142 mm, 0.4 micron PCTE membrane filter, enriched in 25 mL modified buffered peptone water broth, and incubated at 42°C for 6 hours. Samples were concentrated by IMS using anti-E. coli O157:H7 Dynabeads®, stained with a FITC-conjugated anti-E. coli O157:H7 antibody and screened using flow cytometry. Samples exhibiting fluorescence were considered presumptive positive and plated onto CHROMagar™ O157 and Rainbow Agar O157 plates then incubated at 42°C for 18 hours. Isolated colonies were confirmed by O-antisera agglutination. Positive isolates were analyzed by PCR for detection of rfbE, eae, stx1, & stx2 genes and H-typed by RFLP-PCR.  Isolates were also tested for enterohemolysin production by plating onto EBA agar and glucoronidase activity as determined by the MUG reaction. Results: Of the121 samples collected, 30 samples tested presumptively positive for E. coli O157. Thirteen isolates were obtained from 11 water samples that tested positive by O157 antisera agglutination testing.  Three additional isolates collected in 2004 that also tested positive were also included for analysis.  All 16 isolates were PCR positive for the rfbE gene. Eight isolates lacked all toxin genes tested, 5 isolates contained eae only, and 1 isolate contained eae and stx2 genes. Overall, the 13 isolates were found to belong to one of six serotypes detected and 9 phenotypes detected. Conclusions: These data demonstrate that methods relying primarily on antigenicity may not provide sufficient information to adequately assess an organism’s virulence and public health risk. Detection methods should include detection of toxin-production genes. Introduction E. coli O157:H7 is a highly virulent pathogen that causes nearly 73,000 illnesses in the United States each year. From 1982-2002, the CDC reported 350 E. coli O157 outbreaks, 9% of which were waterborne. Existing methods for detection of E. coli O157:H7 in water are non-standardized, have poor sensitivity, and are time intensive. These factors have prompted the development of several antibody-based detection methods for detecting E. coli O157:H7 in water. However, methods based on antigenicity may not provide sufficient information of an organism’s virulence and public health risk. This study summarizes data obtained from water samples tested by an antibody-based method.  Results Of 121 samples collected, 30 samples tested presumptively positive for E. coli O157. Thirteen isolates were obtained from 11 water samples that tested positive by O157 antisera agglutination testing.  Three additional isolates collected in 2004 that also tested positive for E. coli O157 were also included in the analysis.  All 16 isolates tested positive for the rfbE gene. Eight isolates lacked all toxin genes tested, 5 isolates contained eae only, and 1 isolate contained eae and stx2 genes. From the 16 identified isolates, 6 serotypes and 9 phenotypes were observed.  Only one isolate (E. coli O157:H7) tested positive for enterohemolysin. Isolate Shiga Toxin Intimin Entero-hemolysin Glucoronidase Isolate ID 1 (2004) - + E. coli O157:H12 2 (2004) E. coli O157:H16 3 (2004) 4 E. coli O157:H- 5 6 7 8 9 10 11 12 13 14 E. coli O157:H32 15 E. coli O157:H6/41 16 Stx1 - Stx2 + E. coli O157:H7 Control strain Stx1 + Rainbow O157 Agar-Black colonies are E. coli O157 CT-SMAC-E.coli O157 CHROMagar O157-E.coli O157 Methods In 2005, 121 surface water samples were collected from lakes near Madison, WI and riverine systems and inshore areas of Lake Michigan near Milwaukee, WI. These samples were tested for the presence of E. coli O157:H7 using an antibody-based method developed in our laboratory. Samples were filtered through 142 mm, 0.4 µM PCTE membrane filters and enriched in modified buffered peptone water broth at 42°C for 6 hours. Samples were concentrated by IMS using anti-E. coli O157:H7 Dynabeads®, stained with a FITC-conjugated anti-E. coli O157:H7 antibody and screened using flow cytometry. Samples exhibiting fluorescence were considered presumptive positive and plated onto CHROMagar™ O157 and Rainbow Agar O157 plates then incubated at 42°C for 18-24 hours.   Isolated colonies were confirmed by O-antisera agglutination. Positive isolates were analyzed by PCR for detection of rfbE, eae, stx1, & stx2 genes and H-typed by RFLP-PCR. Isolates were also tested for enterohemolysin production by plating onto EBA agar and glucoronidase activity as determined by the MUG reaction. Conclusions The presumptive screening method used in this study detected several E. coli O157 strains in addition to E. coli O157:H7. These data demonstrate that E. coli O157:H7 detection methods relying primarily on O157 antigenicity may not provide adequate information to assess an organism’s virulence and public health risk. Furthermore, the data show that while antibody screening may prove a useful screening tool, these methods cannot detect other enterohaemorrhagic and enterotoxigenic strains that also pose a public health concern.  Therefore, detection methods should incorporate testing for toxin-production genes.