Development of polymerase chain reaction techniques for the detection of waterborne pathogens in environmental waters

Abstract

Currently water quality indicator bacteria (Escherichia coli, enterococci) are utilized to determine the sanitary quality of recreational waters. These indicators are normal inhabitants of the intestinal tract of warm-blooded animals and their presence in recreational water is considered indicative of fecal contamination. However, in tropical climates E. coli and enterococci appear to be naturally present in tropical fresh waters and soil. This has questioned the use of E. coli and enterococci as water quality indicators for tropical climates and demonstrates the need for alternative means to determine the sanitary quality of recreational waters. The development of the polymerase chain reaction (PCR) method has offered an attractive alternative to the problematic cultural isolation of indicator bacteria and pathogens. The study utilize PCR to detect waterborne pathogens and indicator bacteria in environmental waters. In addition survival experiments were conducted to determine the persistence of PCR-target gene sequences in ocean water. A pair of PCR primers were chosen targeting a Salmonella spp. gene sequence. These primers were specific for 25 Salmonella spp. and did not cross react with 27 different non Salmonella bacteria. In seeding experiments the sensitivity of these primers was 12 CFU (Salmonella typhimurium) in primary treated sewage and 1 CFU in ocean water. In addition, field studies were conducted using PCR and standard culture method. A total of six field samples were tested with these primers and 3 demonstrated the presence of Salmonella spp. Two of the PCR positive samples were positive for culturally viable Salmonella spp. A set of PCR primers were developed to detect Bacteroides spp., the most common inhabitant of the human intestinal tract. These primers were specific for 4 Bacteroides spp. and did not cross react with 27 non Bacteroides isolates. These primers were able to detect Bacteroides spp. in a variety of environmental waters. Survival experiments using E. coli and Salmonella typhimurium were conducted in ocean water and demonstrated a loss of E. coli and S. typhimurium gene sequences after exposure for 14 and 16 days, respectively. In summary this research supports the use of PCR as a water quality monitoring tool.