New Methods for Recovery and Detection of Emerging Pathogens in Recreational Water and Wastewater

A study was conducted in 2009 at Boquerón Beach, PR to examine the occurrence of pathogens and indicators in sources that potentially impact the recreational beach. To analyze large volume samples for pathogen and indicator occurrence, new methods were used incorporating ultrafiltration (UF) for sample concentration, with standard methods and quantitative polymerase chain reaction (qPCR) for pathogens and indicators. The first objective was to concentrate samples and recover pathogens for analysis; the second objective was to use these methods in a pathogen/indicator occurrence study. Four sample sites – a publicly-owned treatment works (POTW), a wastewater treatment package plant, a lagoon receiving urban and agricultural runoff, and the swimming beach - were monitored every Saturday and Sunday during the beach season. Dead-end ultrafiltration (DEUF) was used to concentrate samples in the field. To establish method performance, both reagent water and seawater matrices were used. 200L sample volumes were collected and concentrated to ∼1L. Indicators included E. coli (culture and qPCR), Enterococcus (culture and qPCR), male-specific coliphages (Method 1601), C perfringens (m-CP agar), and Bacteroidales (qPCR). Pathogens included total culturable viruses (TCV; BGM cell culture and qPCR), adenoviruses (A549 cell culture and qPCR), norovirus by qPCR, Salmonella (culture and qPCR), Campylobacter (culture), E. coli O157 and non-O157 Shiga toxin positive E. coli (culture and qPCR), Giardia/Cryptosporidium (Method 1623), and infectious Cryptosporidium (HCT8 cell culture).