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

 Density in water versus in wet and dry sediments:  E. coli : 72% at AI 49% at SP  Enterococci 58% at AI 29% at GC  Density in water versus in wet and dry sediments and % moisture:  E. coli: 82% at AI 99% at GC 100% at SP  Enterococci: 92% at DS61% at GC 50% at SP  Density in water versus in dry sediment and % moisture:  Enterococci: 92% DS35% at GC 25% at SP

 Percentage of experiments with regrowth in water:  E. coli : 47% at AI27% 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