Evaluation of the repeatability and reproducibility of a suite of PCR-based microbial source tracking methods

Many PCR-based methods for microbial source tracking (MST) have been developed and validated within research laboratories, but require inter-laboratory validation before implementation. As part of the State of California Source Identification Protocol Project (SIPP), a blinded set of challenge filters were analyzed by three to five laboratories with a suite of PCR-based methods utilizing standardized methods. Repeatability (within lab agreement) and reproducibility (between lab agreement) of results were assessed by multiple metrics and compared to previously observed values for other environmental methods. Repeatability and reproducibility were found to be generally comparable to previously observed values for other methods (Median CV .001-.033 and .020-.106, respectively). Variance component analysis showed contribution of laboratory to total variability to be larger but of similar magnitude to inherent intra-laboratory variability among laboratories using standardized methods. Results among laboratories using non-standardized protocols for the same methods were also observed to have >2 log differences at times. These findings verify the repeatability and reproducibility of these MST methods and highlight the need for standardization of protocols and consumables prior to implementation of larger scale microbial source tracking studies involving multiple laboratories

A multi-beach study of \u3ci\u3eStaphylococcus aureus\u3c/i\u3e, MRSA, and enterococci in seawater and beach sand

Incidences of Staphylococcus aureus and methicillin resistant S. aureus (MRSA) have risen worldwide prompting a need to better understand routes of human exposure and whether standard bacterial water quality monitoring practices adequately account for this potential threat. Beach water and sand samples were analyzed during summer months for S. aureus, enterococci, and MRSA at three southern California beaches (Avalon, Doheny, Malibu Surfrider). S. aureus frequently was detected in samples of seawater (59%, n = 328) and beach sand (53%, n = 358). MRSA sometimes was detected in seawater (1.6%, n = 366) and sand (2.7%, n = 366) at relatively low concentrations. Site specific differences were observed, with Avalon Beach presenting the highest concentrations of S. aureus and Malibu Surfrider the lowest in both seawater and sand. S. aureus concentrations in seawater and sand were correlated to each other and to a variety of other parameters. Multiple linear regression on the combined beach data indicated that significant explanatory variables for S. aureus in seawater were S. aureus in sand, water temperature, enterococci in seawater, and the number of swimmers. In sand, S. aureus concentrations were related to S. aureus in seawater, water temperature, enterococci in seawater, and inversely to surf height classification. Only the correlation to water temperature held for individually analyzed beaches and for S. aureus concentrations in both seawater and sand. To provide context for these results, the prevalence of S. aureus in sand was compared to published fomite studies, and results suggested that beach prevalence was similar to that in homes

A multi-beach study of Staphylococcus aureus, MRSA, and enterococci in seawater and beach

Available online xxx Surfrider). S. aureus frequently was detected in samples of seawater (59%, n ¼ 328) and beach sand (53%, n ¼ 358). MRSA sometimes was detected in seawater (1.6%, n ¼ 366) and sand (2.7%, n ¼ 366) at relatively low concentrations. Site specific differences were observed, with Avalon Beach presenting the highest concentrations of S. aureus and Malibu Surfrider the lowest in both seawater and sand. S. aureus concentrations in seawater and sand were correlated to each other and to a variety of other parameters. Multiple linear regression on the combined beach data indicated that significant explanatory variables for S. aureus in seawater were S. aureus in sand, water temperature, enterococci in seawater, and the number of swimmers. In sand, S. aureus concentrations were related to S. aureus in seawater, water temperature, enterococci in seawater, and inversely to surf height classification. Only the correlation to water temperature held for individually analyzed beaches and for S. aureus concentrations in both seawater and sand. To provide context for these results, the prevalence of S. aureus in sand was compared to published fomite studies, and results suggested that beach prevalence was similar to that in homes. Published by Elsevier Ltd

Comparison of PCR and quantitative real-time PCR methods for the characterization of ruminant and cattle fecal pollution sources

The State of California has mandated the preparation of a guidance document on the application of fecal source identification methods for recreational water quality management. California contains the fifth highest population of cattle in the United States, making the inclusion of cow-associated methods a logical choice. Because the performance of these methods has been shown to change based on geography and/or local animal feeding practices, laboratory comparisons are needed to determine which assays are best suited for implementation. We describe the performance characterization of two end-point PCR assays (CF128 and CF193) and five real-time quantitative PCR (qPCR) assays (Rum2Bac, BacR, BacCow, CowM2,andCowM3)reported to be associated with either ruminant or cattle feces. Each assay was tested against a blinded set of 38 reference challenge filters (19 duplicate samples) containing fecal pollution from 12 different sources suspected to impact water quality. The abundance of each host-associated genetic marker was measured for qPCR-based assays in both target and non-target animals and compared to quantities of total DNA mass, wet mass of fecal material, as well as Bacteroidales, and enterococci determined by 16S rRNA qPCR and culture-based approaches (enterococci only).Ruminant- and cow-associated genetic markers were detected in all filters containing a cattle fecal source. However, some assays cross reacted with non-target pollution sources. A large amount of variability was evident across laboratories when protocols were not fixed suggesting that protocol standardization will be necessary for widespread implementation. Finally, performance metrics indicate that the cattle-associatedCowM2qPCR method combined with either the BacR orRum2Bacruminantassociated methods are most suitable for implementation

Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study

A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman®, HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman® was found to be the most effective marker of human fecal contamination in this California-based study

Improved HF183 Quantitative Real-Time PCR Assay for Characterization of Human Fecal Pollution in Ambient Surface Water Samples

Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters