Bridging the Gap between Molecular and Traditional Assessments of Recreational Water Quality

Recreational water quality monitoring of fecal indicator bacteria (FIB), such as E. coli and Enterococcus sp., currently relies on culture methods that require at least 18 hours for results. Given the short duration of many contamination events, regulatory agencies have demanded rapid methods for more accurate protection of public health. Recently, much work has focused on developing QPCR assays for FIB. Work presented examines the relationships between QPCR- and culture-based assays for FIB in recreational water from several perspectives. Equivalency was compared between culture- and QPCR-measurements of Enterococcus sp. concentrations in beach water samples collected over two summers at two popular southern California beaches. QPCR- and culture-based concentrations were significantly correlated, and management decisions based on the two agreed up to 75% of the time. Persistence of DNA, the QPCR-measured endpoint, versus metabolically active cells, the endpoint of culture-based methods, was tested in seawater mesocosms inoculated with fecal material. QPCR and culture-based measurements conducted in replicate over time revealed that the DNA and cell signals were found to decay at similar rates with few and short-lived significant differences, helping to alleviate concerns that extended DNA persistence may lead to overestimations of FIB concentrations. Additional work was conducted to assess the use of a new QPCR assay to quantify the difficult to culture alternate marker of fecal contamination, fecal Bacteroides spp. The efficacy of the fecal Bacteroides spp. QPCR assay was compared to cultured FIB in lab-created samples and ambient (stormwater) samples. QPCR-measured fecal Bacteroides spp. concentrations were found to be as strongly correlated with contamination by sewage influent as current FIB. Findings also suggest that fecal Bacteroides spp. concentrations may be useful in distinguishing human from animal fecal contamination. Overall, results demonstrate the promise of QPCR-based methods as an improved tool for water quality monitoring. However, technical hurdles remain before QPCR-based monitoring can be implemented. The most important of these is sample processing to remove QPCR-inhibitory compounds. Across this set of studies, approximately 30% of samples experienced some level of inhibition. QPCR should prove a useful tool for water quality management with the advent of standardized approaches for addressing inhibition

Correlation between Quantitative PCR and Culture-Based Methods for Measuring Enterococcus spp. over Various Temporal Scales at Three California Marine Beaches

ABSTRACT Several studies have examined how fecal indicator bacteria (FIB) measurements compare between quantitative PCR (qPCR) and the culture methods it is intended to replace. Here, we extend those studies by examining the stability of that relationship within a beach, as affected by time of day and seasonal variations in source. Enterococcus spp. were quantified at three southern California beaches in the morning and afternoon using two qPCR assays, membrane filtration, and defined-substrate testing. While qPCR and culture-based measurements were consistently and significantly correlated, strength of the correlation varied both among and within beaches. Correlations were higher in the morning (0.45 < ρ < 0.74 [ P < 0.002]) than in the afternoon (0.18 < ρ < 0.45 [ P < 0.021]) and higher when the fecal contamination was concentrated (0.38 < ρ < 0.83 [ P < 0.001]) than when it was diffuse (0.19 < ρ < 0.34 [ P < 0.003]). The ratios of culture-based and qPCR results (CFU or most probable number [MPN] per calibrator cell equivalents [CCE]) also varied spatially and temporally. Ratios ranged between 0.04 and 0.85 CFU or MPN per CCE and were lowest at the beach affected by diffuse pollution. Patterns in the ratios over the course of the day were dissimilar across beaches, increasing with time at one beach and decreasing at another. The spatial and temporal variability we observed indicate that the empirical relationship between culture-based and qPCR results is not universal, even within a beach

Performance of viruses and bacteriophages for fecal source determination in a multi-laboratory, comparative study

An inter-laboratory study of the accuracy of microbial source tracking (MST) methods was conducted using challenge fecal and sewage samples that were spiked into artificial freshwater and provided as unknowns (blind test samples) to the laboratories. The results of the Source Identification Protocol Project (SIPP) are presented in a series of papers that cover 41 MST methods. This contribution details the results of the virus and bacteriophage methods targeting human fecal or sewage contamination. Human viruses used as source identifiers included adenoviruses (HAdV), enteroviruses (EV), norovirus Groups I and II (NoVI and NoVII), and polyomaviruses (HPyVs). Bacteriophages were also employed, including somatic coliphages and F-specific RNA bacteriophages (FRNAPH) as general indicators of fecal contamination. Bacteriophage methods targeting human fecal sources included genotyping of FRNAPH isolates and plaque formation on bacterial hosts Enterococcus faecium MB-55, Bacteroides HB-73 and Bacteroides GB-124. The use of small sample volumes (<= 50 ml) resulted in relatively insensitive theoretical limits of detection (10-50 gene copies or plaques x 50 ml(-1)) which, coupled with low virus concentrations in samples, resulted in high false-negative rates, low sensitivity, and low negative predictive values. On the other hand, the specificity of the human virus methods was generally close to 100% and positive predictive values were similar to 40-70% with the exception of NoVs, which were not detected. The bacteriophage methods were generally much less specific toward human sewage than virus methods, although FRNAPH II genotyping was relatively successful, with 18% sensitivity and 85% specificity. While the specificity of the human virus methods engenders great confidence in a positive result, better concentration methods and larger sample volumes must be utilized for greater accuracy of negative results, i.e. the prediction that a human contamination source is absent

Dramatic Improvements in Beach Water Quality Following Gull Removal

Gulls are often cited as important contributors of fecal contamination to surface waters, and some recreational beaches have used gull control measures to improve microbial water quality. In this study, gulls were chased from a Lake Michigan beach using specially trained dogs, and water quality improvements were quantified. Fecal indicator bacteria and potentially pathogenic bacteria were measured before and during gull control using culture methods and quantitative polymerase chain reaction (qPCR). Harassment by dogs was an effective method of gull control: average daily gull populations fell from 665 before to 17 during intervention; and a significant reduction in the density of a gull-associated marker was observed (<i>p</i> < 0.001). <i>Enterococcus</i> spp. and <i>Escherichia coli</i> densities were also significantly reduced during gull control (<i>p</i> < 0.001 and <i>p</i> = 0.012, respectively for culture methods; <i>p</i> = 0.012 and <i>p</i> = 0.034, respectively for qPCR). Linear regression results indicate that a 50% reduction in gulls was associated with a 38% and 29% decrease in <i>Enterococcus</i> spp. and <i>E. coli</i> densities, respectively. Potentially human pathogenic bacteria were detected on 64% of days prior to gull control and absent during gull intervention, a significant reduction (<i>p</i> = 0.005). This study demonstrates that gull removal can be a highly successful beach remedial action to improve microbial water quality