Integrated analyses of fecal indicator bacteria, microbial source tracking markers, and pathogens for Southeast Asian beach water quality assessment.

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.

Distributions of enterococci and human-specific bacteriophages of enterococci in a tropical watershed.

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.

Human and animal microbial source tracking in a tropical river with multiple land use activities.

The enhancement and restoration of the water quality of deteriorating surface water resources can be challenging, particularly for rivers with multiple usages, such as agriculture, animal husbandry, human residence, and industries. Recently, the performance of DNA-based microbial source tracking (MST) indicators detected by end-point and quantitative PCR assays for identifying sources of fecal pollution from human sewage, swine, and cattle and non-host-specific (universal) fecal pollution in the Tha Chin River basin, Thailand, was evaluated. The present study monitored these validated MST markers and various physicochemical and microbial water quality parameters in samples collected from twelve stations along the Tha Chin River during four sampling events in the wet and dry seasons. No significant difference in precipitation was observed between the wet and dry samplings. Universal markers (both PCR and qPCR) were detected in all 48 samples, indicating persistent and continuing fecal contamination. The sewage- and swine-specific qPCR marker concentrations did not vary among the sampling events, whereas cattle-specific qPCR markers were detected only in the wet season. Animal-specific markers were detected in the lower Tha Chin River section, which is characterized by intensive animal farming. Sewage-specific markers were also found in the lower section and near an upstream residential area. The high agreement (87.5-100%) between the PCR and qPCR results suggested that PCR could serve as a lower-cost MST screening test that requires less technical expertise. A multivariate analysis conducted using the survival analysis procedure to include censored data also emphasized the high pollution in the lower section of the river at all sampling events. Universal and swine-specific markers showed moderate correlations with microbial indicators, including total coliforms, fecal coliforms, E. coli, and enterococci. None of the MST markers or microbial parameters were associated with the measured physicochemical parameters. This study provides the first evaluation of MST markers for monitoring surface freshwater in Thailand, and the findings might aid the pollution surveillance of impaired water bodies and the development of strategies for improving their water quality.

PCR data and comparative performance of Bacteroidales microbial source tracking genetic markers.

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.

Performance evaluation of Bacteroidales genetic markers for human and animal microbial source tracking in tropical agricultural watersheds.

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.