•Maryland Department of the Environment

•Delaware Department of Natural Resources and Environmental Control

•Salisbury University

Drs. Elichia Venso and Mark FranaBacterial 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 SorbitolE. coli O157:H7

CINYersinia

CAMPY CSMCampylobacter

XLT4: BG: Salmonella

SS AgarSalmonella/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 338DS 7 204 164GC 206 383 5594SP 82 90 101

Enterococci Mean Densities (MPN/100 ml or 100 g)Location Water Wet Dry

AI 6 77 251DS 6 15 69GC 35 170 820SP 34 72 235

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 DS 61% at GC 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