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Pinpointing water pollution sources using host-specific gastrointestinal microbes, known as microbial source tracking (MST), have significant benefits for countries with water quality management issues related to pollution. A recently discovered bacteriophage, crAssphage, shows promise as a human-specific MST marker. However, loss of genetic materials during the recovery and the detection processes could alter the ability to measure virus quantities in a water sample. This study determined the crAssphage recovery efficiencies in water sources, including seawater, freshwater, and influent and effluent from a wastewater treatment plant, by spiking natural crAssphage concentrates prior to DNA extraction and quantitative PCR analysis. The results showed that river and seawater with no or low crAssphage background experienced no recovery loss. Evaluating recovery efficiencies in samples with high crAssphage backgrounds posed a challenge due to the inability to prepare high crAssphage titers. This study highlights the importance of intra-laboratory assessment of recovery efficiency in environmental samples for retrieving absolute crAssphage quantification with correction of bias among water samples and increase in data accuracy. PRACTITIONER POINTS: In laboratory assessment of recovery efficiency is crucial for bias correction and data accuracy for absolute crAssphage quantification in water samples. No loss in crAssphage recovery was observed in river and seawater that contained no or low crAssphage backgrounds. Inability to prepare high crAssphage spike concentrations remains the major limitation for evaluating recovery in samples with high crAssphage backgrounds. The results underline the importance of evaluating method recovery in real environmental samples that reflect actual matrix effect. Absolute crAssphage quantification, as human-specific pollution marker, could be used for prioritizing water quality restoration and area-based management plan.

The degradation of coastal water quality from fecal pollution poses a health risk to visitors at recreational beaches. Fecal indicator bacteria (FIB) are a proxy for fecal pollution; however the accuracy of their representation of fecal pollution health risks at recreational beaches impacted by non-point sources is disputed due to non-human derivation. This study aimed to investigate the relationship between FIB and a range of culturable and molecular-based microbial source tracking (MST) markers and pathogenic bacteria, and physicochemical parameters and rainfall. Forty-two marine water samples were collected from seven sampling stations during six events at two tourist beaches in Thailand. Both beaches were contaminated with fecal pollution as evident from the GenBac3 marker at 88%-100% detection and up to 8.71 log10 copies/100 mL. The human-specific MST marker human polyomaviruses JC and BK (HPyVs) at up to 4.33 log10 copies/100 mL with 92%-94% positive detection indicated that human sewage was likely the main contamination source. CrAssphage showed lower frequencies and concentrations; its correlations with the FIB group (i.e., total coliforms, fecal coliforms, and enterococci) and GenBac3 diminished its use as a human-specific MST marker for coastal water. Human-specific culturable AIM06 and SR14 bacteriophages and general fecal indicator coliphages also showed less sensitivity than the human-specific molecular assays. The applicability of the GenBac3 endpoint PCR assay as a lower-cost prescreening step prior to the GenBac3 qPCR assay was supported by its 100% positive predictive value, but its limited negative predictive values required subsequent qPCR confirmation. Human enteric adenovirus and Vibrio cholerae were not found in any of the samples. The HPyVs related to Vibrio parahaemolyticus, Vibrio vulnificus, and 5-d rainfall records, all of which were more prevalent and concentrated during the wet season. More monitoring is therefore recommended during wet periods. Temporal differences but no spatial differences were observed, suggesting the need for a sentinel site at each beach for routine monitoring. The exceedance of FIB water quality standards did not indicate increased prevalence or concentrations of the HPyVs or Vibrio spp. pathogen group, so the utility of FIB as an indicator of health risks at tropical beaches maybe challenged. Accurate assessment of fecal pollution by incorporating MST markers could lead to developing a more effective water quality monitoring plan to better protect human health risks in tropical recreational beaches.

Identifying human sewage contamination via microbial source tracking (MST) marker genes has proven useful for effective water quality management worldwide; however, performance evaluations for these genes in tropical areas are limited. Therefore, this research assessed four human-associated MST marker genes in aquatic environments of Central Thailand: human polyomaviruses (JC and BK viruses [HPyVs]), bacteriophage crAssphage (CPQ_056),LachnospiraceaeLachno3, andBacteroidesBacV6-21. HPyV and crAssphage assays were highly sensitive and specific to sewage (n= 19), with no cross-detection in 120 swine, cattle, chicken, duck, goat, sheep, and buffalo composite fecal samples. Lachno3 and BacV6-21 demonstrated high sensitivity but moderate specificity; however, using both markers could improve specificity to >0.80 (max value of 1.00). The most abundant markers in sewage were Lachno3 and BacV6-21 (5.42-8.02 and non-detected-8.05 log10copies/100 mL), crAssphage (5.28-7.38 log10copies/100 mL), and HPyVs (3.66-6.53 log10copies/100 mL), respectively. HPyVs showed higher levels (up to 4.33 log10copies/100 mL) and higher detection rates (92.7%) in two coastal beaches (n= 41) than crAssphage (up to 3.51 log10copies/100 mL and 56.1%). HPyVs were also found at slightly lower levels (up to 5.10 log10copies/100 mL), but at higher detection rates (92.6%), in a freshwater canal (n= 27) than crAssphage (up to 5.21 log10copies/100 mL and 88.9%). Overall, both HPyVs and crAssphage are suggested as human sewage-associated MST markers in aquatic environments of Central Thailand. This study underlines the importance of characterizing and validating MST markers in host groups and environmental waters before including them in a water quality management toolbox.

The widely used microbial source tracking (MST) technique, quantitative polymerase chain reaction (qPCR), quantifies host-specific gene abundance in polluted water to identify and prioritize contamination sources. This study characterized the effects of a qPCR data analysis using the sample PCR efficiencies (the LinRegPCR model) on gene abundance and compared them with the standard curve-based method (the mixed model). Five qPCR assays were evaluated: the universal GenBac3, human-specific HF183/BFDrev and CPQ_056, swine-specific Pig-2-Bac, and cattle-specific Bac3qPCR assays. The LinRegPCR model increased the low-copy amplification, especially in the HF183/BFDrev assay, thus lowering the specificity to 0.34. Up to 1.41 log

We reported modified endpoint PCR results analyzed by universal and human-, swine-, and cattle-specific Bacteroidales gene markers with human sewage and animal fecal samples (i.e., swine, cattle, chicken, goat, sheep, buffalo, and duck) from Tha Chin and Chao Phraya watersheds. Annealing locations of PCR primers were illustrated by maps of 16s rRNA Bacteroidales genes. We also summarized previously published work on the performance of the PCR assays. For further discussion of the data presented here, please refer to Somnark et al., Performance evaluation of Bacteroidales genetic markers for human and animal microbial source tracking in tropical agricultural watersheds, Environ. Pollut. 236 (2018) 100–110. Keywords: Endpoint PCR, Fecal pollution, Microbial source tracking, Bacteroidales, Sensitivity, Specificity, Water quality

Coastal water quality is deteriorating worldwide. Water quality monitoring is therefore essential for public health risk evaluation and the management of water bodies. This study investigated the feasibility of using bacteriophages of Enterococcus faecalis as sewage-specific faecal indicators, together with physicochemical (dissolved oxygen, pH, temperature and total suspended solids) and biological parameters, to assess coastal water quality using multivariate analysis incorporating non-detects. The principal component and cluster analyses demonstrated that coastal water quality was mostly influenced by biological parameters, including Escherichia coli and total coliforms, which were found in all 31 sampling sites, and enterococci, which was found in all but two sampling sites. The enterococcal bacteriophages AIM06 and SR14 were detected in 17 and 18 samples at concentrations up to 1,815 and 2,790 PFU/100 mL, respectively. Both bacteriophages co-presented in approximately 80% of phage-positive samples, and the concentrations at each site were not significantly different. Overall, either bacteriophage could be used to differentiate high- and low-level coastal water pollution, as grouped by cluster analysis. This study is the first to investigate the suitability of sewage-specific bacteriophages of E. faecalis for monitoring coastal water quality and emphasises the importance of a multivariate analysis with non-detects to facilitate coastal water quality monitoring and management.

The bacteriophages of E. faecalis strains AIM06 (DSM100702) and SR14 (DSM100701) have previously been validated as human-specific microbial source tracking (MST) markers in Thailand. In this study, their spatial and temporal distribution in a freshwater river was investigated for the first time (n = 48). The abundance of enterococci as a standard microbial water quality parameter was evaluated by both the qPCR detection assay with primers and a hydrolysis probe according to the US EPA Method 1611 and the US EPA Method 1600 membrane filtration culture method. AIM06 and SR14 phages were detected by a double layer agar assay and were present in 87.5% and 81.3% of all samples with a co-presence of 92.9% of phage-positive samples. After spiking the representative phages, the ranges of recovery efficiencies were 57.9–99.6% and 49.6–99.9% (n = 48) for AIM06 and SR14 phages, respectively. The absolute abundance of AIM06 and SR14 phages ranged from 0.25 to 221.94 and from 0.25 to 76.66 PFU/100 mL, respectively. Enterococci DNA copies and CFU were detected in all samples ranging from 3.24 to 6.32 log10 copies/100 mL and 100.00 to 1593 CFU/100 mL, respectively. Enterococci in the qPCR assay also showed a moderate correlation with the culture method. The AIM06 and SR14 phage results indicated continuing human faecal pollution along the river with no significant different levels among stations. Interestingly, the higher levels of enterococci in downstream stations for both the qPCR and culture methods along with the significant correlation with other faecal indicator organisms and non-human MST markers implied non-human faecal pollution. In conclusion, this study provides insightful information that could lead to effective water quality management and public health risk reduction from exposure to faecally-contaminated water.

Microbial source tracking (MST) DNA-based assays have been used to successfully solve fecal pollution problems in many countries, particularly in developed nations. However, their application in developing countries has been limited but continues to increase. In this study, sixteen endpoint and quantitative PCR (qPCR) assays targeting universal and human-, swine-, and cattle-specific Bacteroidales gene markers were modified for endpoint PCR, evaluated for their performance with sewage and fecal samples from the Tha Chin watershed and subsequently validated with samples from the Chao Phraya watershed, Thailand. Sample sizes of 81 composite samples (from over 1620 individual samples) of farm animals of each type as well as 19 human sewage samples from the Tha Chin watershed were calculated using a stratified random sampling design to achieve a 90% confidence interval and an expected prevalence (i.e., desired assay's sensitivity) of 0.80. The best universal and human-, swine-, and cattle-specific fecal markers were BacUni EP, HF183/BFDrev EP, Pig-2-Bac EP, and Bac3 assays, respectively. The detection limits for these assays ranged from 30 to 3000 plasmid copies per PCR. The positive predictive values were high in universal and swine- and cattle-specific markers (85–100%), while the positive predictive value of the human-specific assay was 52.2%. The negative predictive values in all assays were relatively high (90.8–100%). A suite of PCR assays in Thailand was established for potential MST use in environmental waters, which supports the worldwide applicability of Bacteroidales gene markers. This study also emphasizes the importance of using a proper sample size in assessing the performance of MST markers in a new geographic region.