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•Maryland Department of the Environment •Delaware Department of Natural Resources and Environmental Control •Salisbury University Drs. Elichia Venso and Mark Frana Bacterial Source Tracking Laboratory To determine levels of fecal indicator organisms (E. coli & Enterococcus) in beach sediment and water samples at 1 Delaware and 3 Maryland sampling sites To investigate survival and/or regrowth of fecal indicator organisms found in sediment and water samples To determine if an association was present between bacterial density in beach sand and in beach water To select for and identify potential bacterial pathogens in these same water and sediment samples 4 sampling locations: Maryland Assateague Island (bay side) Granary Creek (Wye River tributary) Sandy Point Delaware Delaware Shore 12 month project: Jan–Dec 2008 Sample collections: Monthly (Jan-Apr; Oct – Dec) Bimonthly (May – Sep) Samples collected at each study site: Water at knee depth Sediment at water sampling location (“wet”) Sediment in foreshore between high tide line and water’s edge (“dry”) Collection data sheets included information on: Water Temperature Conductivity Dissolved Oxygen pH Salinity Sediment Temperature Note: Analysis was conducted on one set of samples for nitrogen, phosphorus, pH, etc. Other Air Temperature and Direction Weather Tide Unusual observations Water: Analysis of triplicate water samples to enumerate the fecal indicators using Colilert ®-18 Most Probable Number analysis for the detection of E. coli and Enterolert™ for the detection of Enterococci Membrane filtration onto selective media for the isolation of potential pathogens Sediment: Determination of moisture content Agitation of 10 grams in 50 ml of extraction buffer for 30 minutes using a wrist-action shaker , then allowed to settle for 30 min Membrane filtration of supernatant for enumeration of E. coli and Enterococci and identification of potential pathogens. Regrowth: Incubation of select sediment and water samples at 4°C, 21°C, and 37 °C, followed by enumeration of E. coli and Enterococcus MacConkey Sorbitol E. coli O157:H7 CIN Yersinia CAMPY CSM Campylobacter XLT4: BG: Salmonella SS Agar Salmonella/Shigella Carbon Source Utitilization using Biolog© Polymerase Chain Reaction (PCR)-Midi Labs© DNA amplification Sequence matching E. coli Mean Densities (MPN/100 ml or 100 g) Location Water Wet Dry AI 181 56 338 DS 7 204 164 GC 206 383 5594 SP 82 90 101 Enterococci Mean Densities (MPN/100 ml or 100 g) Location Water Wet Dry AI 6 77 251 DS 6 15 69 GC 35 170 820 SP 34 72 235 Assateague: E. coli Bacteria/100mls or grams 2500 12000 2000 5180 1500 1000 500 0 Water Wet Sed Dry Sed Bacteria/100mls or grams Assateague: Enterococci 2500 2000 170000 24000 1500 1000 500 0 Water Wet Sed Dry Sed Bacteria/100mls or grams Delaware Shore: E. coli 1600 1400 1200 1000 800 600 400 200 0 Water Wet Sed Dry Sed Bacteria/100mls or grams Delaware Shore: Enterococci 1600 1400 1200 1000 800 600 400 200 0 Water Wet Sed Dry Sed Granary Creek: E. coli Bacteria/100mls or grams 1600 5140 1400 7130 14000 1200 23000 1000 800 600 400 200 0 Water Wet Sed Dry Sed Granary Creek: Enterococci Bacteria/100ml or grams 1600 1400 5840 9290 1200 3100 2130 1000 3500 2100 800 600 400 200 0 Water Wet Sed Dry Sed Bacteria/100mls or grams Sandy Point: E. coli 1600 1400 1200 1000 800 600 400 200 0 5440 2490 5770 Water Wet Sed Dry Sed Sandy Point: Enterococci Bacteria/100mls or grams 1600 1400 110000 1200 1000 800 600 400 200 0 Water Wet Sed Dry Sed Sandy Point Water: E. coli 250 MPN/100 mls 200 150 4oC 100 21oC 37oC 50 0 Granary Creek Water: Enterococci 250 MPN/100mls 200 150 4oC 21oC 100 37oC 50 0 0 hr 24hr 48hr 72hr 96hr 120 hr 144 hr 168 hr 192 hr Granary Creek dry sediment cfu/100 dry grams 2500 2000 1500 GC dry 4oC GC dry 21oC 1000 GC dry 37oC 500 0 0 hr 24 hr 48 hr 72 hr 96 hr 120 hr 144 hr 168 hr 192 day 9 day day day hr 10 11 12 Assateague wet sediment 300 cfu/100 dry grams 250 200 AI wet 4oC 150 AI wet 21oC 100 AI wet 37oC 50 0 0 hr 24 hr 48 hr 21°C 4°C 30000 30000 25000 25000 20000 20000 15000 GC dry 10000 GC wet 5000 15000 10000 GC dry 5000 GC wet 0 30000 0 hr 24 hr 48 hr 72 hr 96 hr 120 hr 144 hr 168 hr 192 hr day 9 day 10 day 11 day 12 day 13 day 13 day 12 day 11 day 10 day 9 192 hr 168 hr 144 hr 120 hr 96 hr 72 hr 48 hr 24 hr 0 hr 0 37°C 25000 20000 15000 GC dry 10000 GC wet 5000 0 0 hr 24 48 72 96 120 144 168 192 day day day day day hr hr hr hr hr hr hr hr 9 10 11 12 13 Density in water versus in wet and dry sediments: E. coli: Enterococci 49% at SP 29% at GC Density in water versus in wet and dry sediments and % moisture: E. coli: Enterococci: 72% at AI 58% at AI 82% at AI 92% at DS 99% at GC 61% at GC 100% at SP 50% at SP Density in water versus in dry sediment and % moisture: Enterococci: 92% DS 35% at GC 25% at SP Percentage of experiments with regrowth in water: E. coli: 47% at AI 27% at DS 73% at GC 67% at SP Enterococci: 33% at GC 27% at SP Percentage of experiments with regrowth in wet sediment: E. coli: 67% at GC 20% at SP Enterococci: 33% at GC 20% at SP Percentage of experiments with regrowth in dry sediment: E. coli: 33% at GC 20% at SP Enterococci: 27% at GC 20% at AI and SP One “potential” pathogen with a definitive ID: Sandy Point Wet Sediment Vibrio furnissii Six additional potential pathogens, but with low confidence ID: Granary Creek Water E. coli O157:H7 (2) Shigella dysenteriae (2) Sandy Point Wet Sediment E. coli O157:H7 Shigella flexneri Other Genera identified: Aeromonas Citrobacter Enterobacter Klebsiella Proteus Pseudomonas Serratia Shewanella Detectable indicator bacterial densities were found in 47% of 408 assays. The highest fecal indicator counts were found in sediment samples from Granary Creek. The lowest fecal indicator counts were found at the Delaware Shore site. Regrowth/survivability data was measurable at all four sites, although survival was relatively short-term for samples held at 21 °C and 37 °C as compared to samples held at 4 °C. Only one potential pathogen was definitively identified. MDE Kathy Brohawn William Beatty John “Rusty” McKay Heather Morehead Kathy Bassett Sarah Harvey Ann McManus DNREC John Pingree Debbie Rouse Glenn King Salisbury University Lesley Frana Annie Adkins Chris Labe Isha Choudhary Mary Vendetti, Leo Cabrera Cloe Manarinjara Megan Robison