Multitiered Approach Using Quantitative PCR To Track Sources of Fecal Pollution Affecting Santa Monica Bay, California

Abstract

The ubiquity of fecal indicator bacteria such as Escherichia coli and Enterococcus spp. in urban environments makes tracking of fecal contamination extremely challenging. A multitiered approach was used to assess sources of fecal pollution in Ballona Creek, an urban watershed that drains to the Santa Monica Bay (SMB) near Los Angeles, Calif. A mass-based design at six main-stem sites and four major tributaries over a 6-h period was used (i) to assess the flux of Enterococcus spp. and E. coli by using culture-based methods (tier 1); (ii) to assess levels of Enterococcus spp. by using quantitative PCR and to detect and/or quantify additional markers of human fecal contamination, including a human-specific Bacteroides sp. marker and enterovirus, using quantitative reverse transcriptase PCR (tier 2); and (iii) to assess the specific types of enterovirus genomes found via sequence analysis (tier 3). Sources of fecal indicator bacteria were ubiquitous, and concentrations were high, throughout Ballona Creek, with no single tributary dominating fecal inputs. The flux of Enterococcus spp. and E. coli averaged 10 9 to 10 10 cells h ؊1 and was as high at the head of the watershed as at the mouth prior to discharge into the SMB. In addition, a signal for the human-specific Bacteroides marker was consistently detected: 86% of the samples taken over the extent during the study period tested positive. Enteroviruses were quantifiable in 14 of 36 samples (39%), with the highest concentrations at the site furthest upstream (Cochran). These results indicated the power of using multiple approaches to assess and quantify fecal contamination in freshwater conduits to high-use, high-priority recreational swimming areas.The Santa Monica Bay (SMB), California, is home to some of the most popular beaches in the world. It is located adjacent to metropolitan Los Angeles, and more than 50 million beachgoers visit SMB shorelines every year-more than those visiting all other beaches in California combined (38). However, there are serious concerns about beach water quality because of continued exceedances of water quality thresholds based on fecal indicator bacteria such as total coliforms, fecal coliforms, or Escherichia coli and Enterococcus spp., particularly in areas impacted by urban runoff. Thirteen percent of the shoreline mile-days in the SMB exceeded water quality thresholds between 1995 and 2000, with over 50% of these exceedances located near storm drains (37). The public health risk associated with urban runoff has been directly demonstrated through epidemiology studies. Haile et al. (19) demonstrated that swimmers near storm drain discharges in the SMB had a higher likelihood of respiratory and/or gastrointestinal symptoms than swimmers more than 400 m from a storm drain.Despite the impairment of water quality and risks to human health, identification and elimination of the sources of bacteria responsible for the beach warnings remain elusive. The difficulty in identifying and eliminating the sources of bacteria results from thre...

Luminex detection of fecal indicators in river samples, marine recreational water, and beach sand

“…1,Fig. 2) to assess whether this system might be useful to multitiered analyses of coastal water quality (Noble et al, 2006).…”

“…4). DNA from damaged, viable-but-nonculturable (VBNC), and dead cells can be detected with molecular methods (Lipp et al, 2003;Lleo et al, 2003;Noble et al, 2006); therefore, Luminex can overcome the obstacle of oxygen sensitivity because live cells are not necessary for analysis. Overall, these results suggest that molecular analysis offers a convenient way to detect gut anaerobes such as Bacteroides spp.…”

“…Bacteroides spp. have been suggested as promising alternatives to E. coli and enterococci analysis (Allsop and Stickler, 1985;Bernhard and Field, 2000a;Bernhard and Field, 2000b;Fiksdal et al, 1985;Kreader, 1995), and these anaerobic gut microbes may provide a useful source tracking tool (Noble et al, 2006). Molecular methods primarily have been used for evaluation of this indicator because culturing requires maintenance of anaerobic conditions.…”

“…Rapid detection of multiple species could yield a "fingerprint" of water quality that would be a useful addition to fecal indicator enumeration. Such a multitiered approach (Boehm et al, 2003;Noble et al, 2006) could yield more information about the source of contamination, the potential health risk, and the best strategy for remediation. This approach could benefit environmental research, epidemiological studies, and routine water quality analysis.…”

Using microbiological tracers to assess the impact of winter land use restrictions on the quality of stream headwaters in a small catchment

“…In Ireland a library-independent method, developed by Kildare et al (2007) , has permitted its application to typical environmental settings and has designed host-specific qPCR assays based on the order Bacteroidales. Since this bacterial group are more prevalent in the gastrointestinal tract than the FIOs, and make up approximately one-third of the human faecal microflora (Noble et al, 2006), qPCR assays targeting variable regions of the Bacteroidales 16S rRNA gene allow for the discrimination between human and ruminant sources of faecal pollution.…”