ted coolers, while water samples for enterococci culture were stored in an insulated container on ice upon collection, returned to the laboratory within four hours and processed immediately upon arrival. Physical and chemical water quality measurements Water-column PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19632594 depth and surface-water 118414-82-7 web salinity, temperature, dissolved oxygen, conductivity, and pH were measured on every sampling date and at each location with a YSI 556 Multi-probe system in accordance with the manufacturer’s instructions. Fecal indicator measurements Fecal indicator measurements were conducted following the standard methods as described for enterococci in Standard Methods for the Examination of Water and Wastewater. Briefly, surface-water samples were filtered in triplicate onto sterile 0.45 mm pore size, 47 mm diameter, nitrocellulose Fisherbrand water-testing membrane filters, and plated onto Difco m Enterococcus agar. According to manufacturer’s instructions, plates were incubated for 48 hours at 35uC. All light to dark red colonies were recorded as presumptive enterococci. Vibrio isolation Surface water samples were spread plated in triplicate onto Chromagar Vibrio media and incubated for 24 hours at 37uC. After incubation, each plate was observed for characteristically colored bacterial colonies associated with V. vulnificus or V. parahaemolyticus. As V. vulnificus and V. cholerae both appear as turquoise colonies on Chromagar Vibrio media, all turquoise colonies were replated onto cellobiose-collistin agar media to confirm V. vulnificus species. The CC agar cultures were incubated for 24 hours at 37uC and yellow-colored colonies were considered presumptive V. vulnificus. Tryptic soy broth, supplemented with 5% sodium chloride, was then 
inoculated with individual presumptive colonies of V. vulnificus or V. parahaemolyticus and incubated at 37uC for 24 hours and stored with 30% glycerol at 280uC. Materials and Methods Sampling sites Three sampling sites were selected based on their importance for human use in the Chesapeake Bay, Maryland Coastal Bays region. Two sites, Sandy Point State Park and St. Martin’s River, were characterized by frequent recreational use; and one site, the Pocomoke Sound, was characterized by heavy commercial fishing use. Sandy Point State Park includes an artificial beach on the western shore of the Chesapeake mid-Bay region, at the base of the Chesapeake Bay Bridge. It is open year round and frequented by approximately 768,000 visitors annually, many of whom visit the park’s beach during the summer. St. Martin’s River is a tributary of the Maryland Coastal Bays with approximately 10,000 residents. Land-use in the St. Martin’s River watershed is approximately 10% residential, 48% agricultural, and 34% forested. The Pocomoke Sound is a major embayment of the Chesapeake Bay’s Eastern Shore. It is influenced by agricultural practices, including high-density concentrated poultry feeding operations, and is a popular destination for commercial PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19630872 and recreational fishing. No specific permissions were required for each sampling location, as they are public access Vibrio species confirmation Vibrio DNA template was obtained by producing crude cell lysates by boiling 1 mL aliquots of TSB cultures in 2 mL microcentrifuge tubes at 100uC for 10 minutes. A Bio-rad CFX96 Touch Real-Time PCR Detection System was used to confirm the species of isolates with primers designed to detect Vibrio vulnificus or V. parahaemolyticus. Following initial confirmation,