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Discriminating E. coli Isolated from Various Human and Nonhuman Sources Based on Analysis of Random Amplified Polymorphic DNA (RAPD) Patterns Evert Ting, Deborah Johnson, and Charles Tseng Department of Biological Sciences Purdue University Calumet Hammond, IN 2/11/03 1 INTRODUCTION High E. coli counts in water -- indicate fecal pollution Bacterial Source Tracking (BST) – a key step to control fecal pollution in water Random Amplified Polymorphic DNA (RAPD) – a DNA fingerprinting technique to characterize bacteria -- a useful tool for BST 2/11/03 2 OBJECTIVE 2/11/03 To determine the efficacy of using RAPD to differentiate E. coli from various human (HM) and nonhuman (NHM) sources. 3 MATERIALS & METHODS E. coli Collection (544 Isolates) Human -- 165 Feces 30 Urine 94 Sewage 30 Blood Nonhuman – 53 Horse 10 Pig 55 Goose 57 Cow 20 Chicken 30 Seagull * Except sewage, single isolate/ sample was used. 2/11/03 4 RAPD 2/11/03 DNA extraction DNA amplification w/ RCR (using 3 separate primers) Gel electrophoresis RAPD pattern documentation RAPD pattern analysis – BioNumericsTM 5 Gel Image of RAPD primer 1283 primer 1247 primer 2 100pb ladder HM175A HM175B HM175C HM175D HM175E HM176A 100pb ladder HM176B HM176C HM176D HM176E E. coli B None 100pb ladder 2/11/03 6 Fig. 1. MANOVA analysis of RAPD patterns of E. coli isolated from HM and NHM sources. HS: • - Feces, • - Urine, • - Blood, • - Sewage 2/11/03 NHS: • 7 Fig. 2. MANOVA analysis of RAPD patterns of E. coli isolated from human sources. • - Feces, • - Urine, • - Blood, • - Sewage 2/11/03 8 Fig. 3. MANOVA analysis of RAPD patterns of HM and NHM E. coli isolated from • HM sources, • birds, and • farm animals ( • - 10 Extra Sewage ) 51 2/11/03 9 Table 1. Identification of 10 sewage isolates outside the database against library units based on maximum similarity Isolate Best Matching Unit % Similarity Quality _______________________________________________________________ SW057 Human-Fecal/Urine-Sewage279 82.7% ■1 SW058 Human-Fecal/Urine-Sewage279 73.7% ■ SW059 Human-Fecal/Urine-Sewage279 86.6% ■ SW060 Human-Fecal/Urine-Sewage279 84.3% ■ SW061 Human-Fecal/Urine-Sewage279 77.6% ■ SW062 Bird-Sg/Gs/Ck105 77.1% ■ SW063 Human-Fecal/Urine-Sewage279 91.7% ■ SW064 Human-Fecal/Urine-Sewage279 80.8% ■ SW065 Human-Fecal/Urine-Sewage279 59.8% ■ SW066 Mammal-Pg/Cw/Hs120 77.4% ■ ________________________________________________________________ 1 Quality factors appear as color dots. They range from red (improbable identification), over orange, yellow (doubtful identification) to green (faithful identification). 2/11/03 10 Table 2. % of E. coli isolates correctly and incorrectly assigned to HM and NHM sources ___________________________________________________ Source # of % correctly * % incorrectly isolates Identified Identified ___________________________________________________ HM NHM __________________________________________________ * % values were determined by Jackknife Analysis 2/11/03 11 279 225 79.93 77.33 20.07 22.67 RESULTS 2/11/03 The Jackknife analysis indicated that 80% and 77% of HM and NHM isolates respectively can be correctly identified using RAPD analysis. The Discriminant analysis (MANOVA) showed that HM and NHM isolates can be separated from each other on the 2-D plot. 12 (Cont.) 2/11/03 E. coli isolated from different HM sources (sewage, feces, blood and urine) can be further separated into 3 clusters on the MANOVA plot. Ten sewage isolates not in data base were grouped in HM cluster on MONOVA plot. Seven of the 10 sewage isolates were identified as human isolates based on maximum similarity. 13 CONCLUSIONS 2/11/03 Both Jackknife and MANOVA discriminant analyses showed that RAPD is a useful tool for discriminating E. coli from HM and NHM sources. E. coli isolated from sewage are different from those isolated from human feces. To track the source of human contamination in natural water, it is important to include RAPD patterns of sewage isolates in the library. 14 ACKNOWLEDGEMENT This research project was supported by Illinois-Indiana Sea Grant College Program grants R/PS-05-98 and R/PS-01-99. 2/11/03 15