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

International Journal of Hygiene and Environmental Health 226 (2020) 113482

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

T

Natcha Chyerochanaa,1, Akechai Kongprajuga,1, Pornjira Somnarkb, Pinida Leelapanang Kamphaengthongc, Skorn Mongkolsuka,d, Kwanrawee Sirikanchanaa,d,∗ a

Research Laboratory of Biotechnology, Chulabhorn Research Institute, Bangkok, 10210, Thailand Applied Biological Sciences, Chulabhorn Graduate Institute, Chulabhorn Royal Academy, Bangkok, 10210, Thailand c Water Quality Management Bureau, Pollution Control Department, Ministry of Natural Resources and Environment, Bangkok, 10400, Thailand d Center of Excellence on Environmental Health and Toxicology, CHE, Ministry of Education, Bangkok, 10400, Thailand b

A R T I C LE I N FO

A B S T R A C T

Keywords: Water quality monitoring Faecal indicators Microbial source tracking Bacteriophages Enterococci

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 nonhuman 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.

1. Introduction Increasing numbers of microbiologically impaired water bodies due to faecal pollution have been reported worldwide (Graciaa et al., 2018; Luby et al., 2008; Pollution Control Department, 2019). Faecal materials potentially carry pathogenic bacteria, viruses or protozoa, which can cause adverse health effects (Zhang et al., 2019). Various sources of faecal contamination into water resources include human, farm animals and wildlife sources; however, the higher risks to public health are related to water contaminated by human faecal pollution, which could carry human waterborne pathogens (Harwood et al., 2014; Soller et al., 2014). Microbial source tracking (MST) is a research field that utilises

host-specific gut microorganisms to differentiate faecal pollution from human or animal sources (Teaf et al., 2018; Zhang et al., 2019). Methods for analysing MST microorganisms are categorised into library-dependent and library-independent methods. The library-dependent methods, both genotypic (e.g. PFGE and ribotyping) and phynotypic (e.g. antibiotic resistance and carbon utilisation), require the availability of fingerprint databases for microorganisms of known faecal sources (US EPA, 2011). To circumvent time and cost consumption for developing databases, the library-independent methods detect the presence of source-specific microorganisms using either culture or genomic-based methods (US EPA, 2011). Bacteriophages infecting enterococci are groups of bacterial viruses that have been

∗

Corresponding author. Research Laboratory of Biotechnology, Chulabhorn Research Institute, Bangkok, 10210, Thailand. E-mail address: [email protected] (K. Sirikanchana). 1 Contributed equally to this manuscript. https://doi.org/10.1016/j.ijheh.2020.113482 Received 14 November 2019; Received in revised form 17 January 2020; Accepted 4 February 2020 1438-4639/ © 2020 Elsevier GmbH. All rights reserved.

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performance evaluations and monitoring studies using enterococcus qPCR assays have mainly been conducted in the United States due to the available policy and regulations (Dorevitch et al., 2017; Jennings et al., 2018; Shrestha and Dorevitch, 2019). However, information related to the abundance of enterococci as detected by the qPCR assay and its relationship with the enterococci standard culture method in tropical regions, e.g. Thailand, is currently limited. The availability of such information could provide insights into the potential adoption of molecular assays for microbial water quality monitoring in tropical countries in the future. Consequently, the objectives of this study were to 1) monitor the spatial and temporal distributions of AIM06 and SR14 phages as human-specific faecal indicators in a tropical river and to 2) determine the abundance and the relationship of the qPCR and culture detection methods with enterococci as a standard microbial water quality parameter in tropical freshwater.

isolated and have shown their ability to function as culture and libraryindependent MST markers in several geographical regions, including Puerto Rico (Bonilla et al., 2010; Santiago-Rodriguez et al., 2013; Santiago-Rodríguez et al., 2010), Portugal (Santiago-Rodríguez et al., 2010), the UK (Purnell et al., 2011), the United States (Vijayavel et al., 2014) and Thailand (Wangkahad et al., 2017). Particularly in Thailand, bacteriophages infecting E. faecalis strains AIM06 (DSM100702) and SR14 (DSM100701) have expressed their high performance as humanspecific MST markers with 100% specificity and 90% sensitivity to human sewage (Wangkahad et al., 2017). The morphologies of these bacteriophages were examined using transmission electron microscopy and were determined to be from the Siphoviridae, Podoviridae and Myoviridae families (Booncharoen et al., 2018). The decay rates of AIM06 and SR14 phages were investigated in different water matrices to study the effects of water constituents, including filtered and nonfiltered freshwater and seawater with low and high pollution levels (Booncharoen et al., 2018). Both human-specific bacteriophages were successfully evaluated for their performance and monitored for their abundance along the coastal seawater of Thailand (Kongprajug et al., 2019a). Therefore, to serve as effective MST markers in environmental waters, it is urgent to assess the presence and the abundance of humanspecific AIM06 and SR14 phages in freshwater environments. Although source-specific faecal identifiers have been proven useful to accurately identify public health risks and to facilitate water quality restoration and water resource management (Benham et al., 2011; Environment Agency, 2008), there are currently no regulatory requirements for water quality assessments. On the contrary, traditional faecal indicator bacteria (FIB) are standard water quality parameters for monitoring microbial water quality in many countries (Ashbolt et al., 2001; Fujioka et al., 2015; National Environment Board, 1994). As one of the FIB groups, enterococci are facultatively anaerobic, nonspore forming, Gram-positive bacteria that are normally found in human and animals’ intestinal tracts and faeces, although some species reside in soil, food, water and plants (Hardie and Whiley, 1997). In particular, E. faecalis and E. faecium are the predominant species in human gastrointestinal tracts and faeces (Devriese and Pot, 1995; Layton et al., 2010; Murray, 1990). The European Union Bathing Water Directive 2006/7/EC requires the use of intestinal enterococci as faecal indicators in both inland waters and coastal and transitional waters (European Union, 2006). Intestinal enterococci are measured using the most probable number method (ISO 7899–1) or the membrane filtration method (ISO 7899–2) as specified in the Bathing Water Directive (European Union, 2006). In 2012, the United States issued the Recreational Water Quality Criteria (RWQC) as federal guidelines for states to develop their water quality standards for primary contact recreational waters, including bathing, swimming, surfing, water skiing, tubing, water play and equivalent activities (US EPA, 2012a). The United States also included enterococci in the 2012 RWQC for both marine and freshwaters. The standard method for enumerating enterococci is a membrane filtration technique using the US EPA method 1600 utilising membrane-Enterococcus Indoxyl- Beta -D-Glucoside agar (mEI) (US EPA, 2002). Although culture methods for FIB have been stated in the regulatory protocol for most countries, there has been recent interest in molecular methods for FIB detection in place of the traditional culturing methods. In particular, a quantitative PCR technique has been of great interest due to its advantage of providing more rapid results, deeming it a significant factor in timely informing bathers to prevent public health risks from microbial contamination (Oliver et al., 2014). The United States 2012 RWQC includes the detection of enterococci DNA with the quantitative polymerase chain reaction (qPCR) assay Method 1611 as a voluntary option for adoption (US EPA, 2012a). This method detects part of the 23S DNA region of all species in the enterococci group. Although there have been discussions and evaluations of the readiness for adopting molecular detection methods in certain countries, e.g. Canada (Henrich et al., 2016) and the UK (Oliver et al., 2014), method

2. Materials and methods 2.1. Sample collection Freshwater samples (n = 48) were collected from 12 sampling stations (sites TC01 to TC28) along the 325-km Tha Chin River in Central Thailand, as previously described (Kongprajug et al., 2019b, 2019c; Somnark et al., 2018) (Supplemental Table S1). The Tha Chin River receives pollution due to multiple activities along the river, including activities from human communities, agricultural areas and animal husbandry facilities. Specifically, the middle and downstream sections of the river were reported as the third and fifth, respectively, for most deteriorated water quality in 2018 among all 65 freshwater sources in Thailand according to the water quality index calculated from dissolved oxygen (DO), biochemical oxygen demand (BOD), total coliform bacteria (TCB), faecal coliform bacteria (FCB) and ammonia (Pollution Control Department (PCD), 2019). Four sampling campaigns were launched from July 2017 to March 2018, two of which were in the wet season and two in the dry season. Quality control samples, including two field blanks and two field duplicates at site TC13, were also collected for analysis. Samples for microbiological analyses were collected at 30-cm below the surface according to Thailand's surface water collection protocol (National Environment Board, 1994). All samples were stored on ice and transported to the laboratory within 8 h. 2.2. Enumeration of bacteriophages of enterococci Magnesium chloride precipitation was used for bacteriophage concentration according to a published protocol with slight modifications (Contreras-Coll et al., 2002). Briefly, 10 mL of 1 M magnesium chloride was added to a 1-L water sample followed by an addition of 3.5 mL of 1 M dipotassium hydrogen phosphate dropwise while stirring and 2 M sodium hydroxide to adjust to pH 8.6. Next, the flocs were formed, while the solution was slowly stirred for 15 min. Then, the suspension was centrifuged at 5000×g for 30 min before the pellet was re-suspended in 25–35 mL of a 0.1% peptone salt solution. The concentrated water sample was enumerated for phage counts using a double layer agar assay. E. faecalis strains AIM06 (DSM100702) and SR14 (DSM100701) were grown at the starting concentration of 0.05 OD600 from an overnight culture. The culture reached a log phase within 2–3 h in a shaking incubator at 37 °C in tryptic soy broth (TSB; Becton Dickenson, Franklin Lakes, NJ, USA). A mixture of 1 mL concentrated water sample, 1.5 mL bacterial host, 4 mL semi-solid tryptic soy agar (TSA) (0.6% agar strength) and calcium chloride at a 2.6 mg/mL final concentration was overlaid onto a TSA plate (1.5% agar strength) and incubated invertedly at 37 °C for 4–5 h. Plaques from a total of 10 plates per one water sample were counted and calculated for bacteriophage concentrations. The sample's limit of detection (SLOD) in PFU per 100 mL original water sample was calculated for each water sample according to the limit of one plaque per 10 mL concentrated water 2

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the GenBac3 qPCR assay (Kongprajug et al., 2019b). Only two samples were determined as being inhibited, and then the 10 ng DNA template was used for the enterococcus qPCR run. In addition, a total of nine method blank samples were produced by processing sterile laboratory water through all laboratory steps as quality controls. The multi-target synthetic DNA standards of a size of 538 bp (Invitrogen, USA) were used to generate standard curves for the enterococci qPCR assay (Supplemental Fig. S1). The enterococcus qPCR target was designed according to the E. faecalis ATCC 29212 genome (GenBank accession number CP008816.1). DNA concentrations ranging from 5 × 101 to 5 × 106 gene copies per reaction, as measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), were prepared for a standard curve. Four individual instrumental runs were performed, during which each reaction was conducted in triplicate, and a mixed model was used to calculate the number of DNA copies (Sivaganesan et al., 2010). The analysis of covariance (ANCOVA) also revealed insignificant differences in slopes among these instrument runs (p > 0.05). The common slope from standard curves plotting Cq versus the log10 concentration of the DNA standard was determined. PCR amplification efficiency (Ep) was calculated as (10(−1/Slope) - 1) × 100. The limit of detection (LOD; copies per reaction), which was determined as the lowest concentration of the 10 standard replicates that showed a standard deviation of Cq of less than 0.5, was investigated. Lastly, the limit of quantification (LOQ; copies per reaction), which was determined as the lowest concentration of the standards in the standard curve, was characterised.

sample. 2.3. Method recovery of bacteriophages of enterococci To account for any possible losses during sample processing, representative bacteriophages were spiked into each water sample, and the sample's recovery percentages were calculated. Using E. faecalis strains AIM06 and SR14 as bacterial hosts, respectively, bacteriophages A1 and S1 were prepared as described elsewhere (Booncharoen et al., 2018; Wangkahad et al., 2015). Briefly, a single plaque on the surface of a TSA double-layer agar plate was cut carefully and transferred to a 1.5 ml microcentrifuge tube containing 1-mL TSB followed by pulsevortexing. The homogeneous phage solution was then further propagated by the double layer agar assay. Plaques of bacteriophages in soft agar were harvested by scraping off the surface of agar plates. The surface agar was vortexed and centrifuged at about 90×g for 25 min at 4 °C to settle the cellular debris and agar. The supernatant containing bacteriophages was filtered through a 0.22-μm pore size polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany). The filtrate, which contained a bacteriophage suspension, was aliquoted and stored at 4 °C until use. To proceed, two 1-L split samples were separately spiked with 6 × 104 PFU of E. faecalis bacteriophages A1 and S1. Then, ten mL of the spiked sample was withdrawn for phage enumeration with the double layer agar assay, while the remainder of the sample was precipitated as described prior to processing with the double layer agar assay. A phage recovery rate was calculated as (phage amount after precipitation/phage amount before precipitation) × 100%. Phages were also spiked in phosphate buffer saline (PBS), and the recovery rates were compared.

2.6. Method recovery of enterococci qPCR assay A 1-L split sample was spiked with one mL stock containing 107–108 CFU/mL of the E. faecalis host strain AIM06 prior to the water concentration and the DNA extraction steps as described. The enterococci qPCR assay recovery was calculated as [(gene copy amount after the process in a spiked sample – gene copy amount after the process in an unspiked sample)/gene copy amount of one mL stock] × 100%.

2.4. Water concentration and DNA extraction The acidification-filtration concentration method was conducted prior to the DNA extraction of filtered membranes as previously stated (Kongprajug et al., 2019b). In brief, 1 L of the water sample was preacidified to pH 3.5 ± 0.2 before the filtration of each of 250 mL sample through a 0.45-μm-pore- size HAWP membrane (Merck Millipore, Germany). Four filtered membranes per water sample were separately extracted using a Quick-DNA Faecal Soil Microbe Miniprep kit (Zymo Research, USA), and DNA eluent of each filtered membrane was combined to represent the extracted DNA of each water sample. The DNA extracts were stored at −80 °C until use.

2.7. Physicochemical and microbiological water quality parameters Other water quality measurements comprised onsite measurements of pH, temperature, salinity and conductivity and laboratory analyses of DO, BOD, total suspended solids (TSS), total dissolved solids (TDS), total phosphorus (TP), phosphate, TCB, FCB, E. coli, enterococcus and sewage-specific microbial source tracking markers HF183 (qPCR Bacteroidales DNA) and CPQ056 (qPCR crAssphage DNA) as previously described (Kongprajug et al., 2019b, 2019c). Enterococci was analysed using the US EPA Method 1600 (US EPA, 2002). Briefly, 10 mL of a 10fold serially diluted water sample was filtered with a 0.45-μm-pore-size cellulose nitrate filter (Sartorius Stedim, Goettingen, Germany), and the filtered membrane was plated onto mEI agar (Becton Dickinson, Franklin Lakes, NJ, USA). Next, the plates were incubated under aerobic conditions for 24 h at 41 °C ± 0.5 °C. The method detected colonies of E. faecalis, E. faecium and E. avium and their variants. These parameters were used for a correlation analysis with an abundance of AIM06 and SR14 phages and enterococcus DNA copies.

2.5. qPCR assay and standard curves for enterococci Primers and a hydrolysis probe for an enterococci qPCR assay were used according to the US EPA Method 1611 (US EPA, 2012b). The sequences of the forward primer, reverse primer and probe were 5′-GAGAAA-TTC-CAA-ACG-AAC-TTG-3′, 5′-CAG-TGC-TCT-ACC-TCC-ATCATT-3′ and [6-FAM]-5′-TGG-TTC-TCT-CCG-AAA-TAG-CTT-TAG-GGCTA-TAMRA, respectively. The qPCR protocol was conducted according to the MIQE guidelines (Bustin et al., 2009). Each 20-μL qPCR reaction mixture was composed of 0.8 μL of each 10 μM forward and reverse primer, 0.4 μL of 10 μM probe, 4 μL of DNA template (normalised to 40 ng of total DNA), 10 μL of the 2 × iTaq Universal Probes Supermix (Bio-Rad Laboratories, USA) and 4 μL of 1 μg/μL bovine serum albumin (BSA). All qPCR reactions were performed using an ABI StepOnePlus Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, USA). The qPCR cycling steps comprised an initial denaturation at 95 °C for 3 min followed by 40 cycles of denaturation at 95 °C for 20 s and a combined annealing and elongation step at 60 °C for 1 min. The qPCR run of each sample was performed in duplicate, and Cq values were averaged when both values had standard deviations of no more than 0.5; otherwise, additional runs were conducted. Positive and no-template controls (NTCs) were included for all instrumental runs. An inhibition effect was previously identified using the dilution method of

2.8. Statistical analysis All statistical analyses were performed with an R programme (R Core Team, 2017). For data sets containing all positive data, a normal distribution analysis was performed with the Shapiro-Wilk test. For non-normally distributed data, a comparison of two independent data sets was analysed using the Mann-Whitney test, and a paired difference test was conducted using the Wilcoxon singed-rank test. Significant difference testing for more than two non-normal data sets was performed using the Kruskal-Wallis test. 3

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For data sets containing data below the detection limits—so-called non-detects—a non-parametric survival analysis was performed (Helsel, 2012). Descriptive statistics were calculated using KaplanMeier estimates. Normality was tested using Shapiro-Francia Goodnessof-Fit test (Millard, 2013). The significance of the differences was determined using the paired Prentice–Wilcoxon test for paired comparisons, and the Generalised Wilcoxon test was used for group comparisons. Kendall's tau (rank correlation) using the U-Score with adjusted p-values using the Holm method was performed for the correlation analysis. The clustering model selection and the optimal number of clusters were analysed by implementing the Expectation-Maximisation algorithm to calculate the maximum likelihood estimator. 3. Results 3.1. Distributions of bacteriophages of enterococci A total of 48 samples were monitored for human-specific AIM06 and SR14 phages from 12 sampling stations during four sampling campaigns, i.e. Jul 2017, Aug 2017, Feb 2018 and Mar 2018. AIM06 phages were detected in 87.5% of all samples (n = 42 of 48), while SR14 phages were positively found in 81.3% of all samples (n = 39 of 48) with SLODs of 0.2–0.3 PFU/100 mL. Both phages were co-present in 92.9% of phage-positive samples, whereas only AIM phages were detectable at sampling stations TC07 during the third event (Feb 2018) and TC01 and TC09 during the final event (Mar 2018). Both phages showed positive detection in all four sampling events from stations TC04, TC22, TC23 and TC25, while AIM06 phages were also found in all events from stations TC07 and TC09 (Fig. 1). Seasonally, both AIM06 and SR14 phages were detected in 95.8% of samples in the wet season (Jul 2017 and Aug 2017; n = 24) but were found in 79.2% and 66.7% in the dry season (Feb 2018 and Mar 2018; n = 24), respectively. The absolute abundance of AIM06 and SR14 phages when incorporating the samples’ recovery efficiency for detectable samples in all four sampling events ranged from 0.25 to 221.94 and from 0.25 to 76.66 PFU/100 mL with median values of 1.47 and 1.93 PFU/100 mL, respectively (Fig. 2 and Supplemental Table S2). For co-presented samples, both phages were present in each sample with no significantly different concentration (paired Prentice-Wilcoxon test; p = 0.484). The maximum concentrations of AIM06 and SR14 phages in the wet seasons (24.34 and 37.73 PFU/100 mL, respectively) were lower than those in the dry seasons (221.94 and 76.66 PFU/100 mL, respectively). Significant differences were found between sampling events in Aug 2017 (wet season) and Feb 2018 (dry season) for both AIM06 and SR14 phages (paired-Prentice Wilcoxon test; p = 0.013 and 0.014, respectively), between sampling events in Jul 2017 and Aug 2017 (both wet seasons) for AIM06 phages (paired-Prentice Wilcoxon test; p = 0.036) and between sampling events Feb 2018 and Mar 2018 (both dry seasons) for SR14 phages (paired-Prentice Wilcoxon test; p = 0.042) (Figs. 2 and 3 and Supplemental Table S3). In addition, the precipitation records for one and seven days prior to sampling demonstrated no significant difference among the four sampling events as reported by Kongprajug et al. (2019b). Moreover, no significant difference among the 12 stations was observed for median values of the abundance of both AIM06 and SR14 phages for all four sampling events (Generalised Wilcoxon test; p = 0.1 and 0.5, respectively).

Fig. 1. Sampling map and positive detection rates from four sampling campaigns of AIM06 phage (in blue) and of SR14 phage (in red). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Table S4). The data sets were not normally distributed (Shapiro-Wilk test; p < 0.01), so a non-parametric analysis was conducted. The median values for the recovery percentages of bacteriophages A1 in PBS, S1 in PBS, A1 in river water and S1 in river water were 97.7, 87.9, 94.8 and 93.1, respectively. There was no significant difference between the recovery percentage medians in PBS and in environmental water for A1 or S1 (Mann-Whitney test; p = 0.31 and 0.17, respectively). In each environmental sample, the paired difference test of the recovery between both bacteriophages showed no significant difference (Wilcoxon signed-rank test; n = 48; p = 0.56). The recovery percentage medians among different events were significantly different for both A1 and S1 bacteriophages (Kruskal-Wallis test; p = 0.05 and 0.01, respectively), while those among varying sampling stations were not significantly different (Kruskal-Wallis test; p = 0.59 and 0.74, respectively). The recovery values of both phages in field duplicate samples (TC13-Rep1 and TC13-Rep2) also showed consistency with the coefficient of variation (CV), which ranged from 1.2-15.1% to 0.2–21.0% (n = 4) for bacteriophages A1 and S1, respectively (Supplemental Table S4). The bacteriophages’ spikes in this experiment were high enough that the natural bacteriophages showed a negligible contribution to the recovery calculation (median values of 0.02 and 0.03% for bacteriophages A1 and S1, respectively).

3.2. Recovery efficiencies of bacteriophages of enterococci in freshwater Bacteriophage recovery was characterised in all water samples to account for the actual concentration calculation in water. Initially, recovery efficiencies of bacteriophages A1 and S1, representing AIM06 and SR14 phages, respectively, were evaluated and ranged in PBS from 90.9 to 98.7% and 53.8–93.8% (n = 5), respectively. In river water, the recovery percentages of bacteriophages A1 and S1 were in a range from 57.9 to 99.6% and 49.6–99.9% (n = 48), respectively (Supplemental

3.3. Distribution and recovery efficiencies of enterococci DNA copies The standard curve characteristics of the enterococci qPCR assay comprised a common slope of −3.538, R2 of 0.997, qPCR efficiency of 91.71% and LOD and LOQ of 50 copies per reaction. Quality controls, 4

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Fig. 2. Temporal distribution of AIM06 phage (a), and SR14 phage (b) during four sampling events (i.e., Jul 2017, Aug 2017, Feb 2018, Mar 2018). Box plots represent the estimated 25th to 75th percentiles, with the median in between. Whiskers exhibit the maximum and minimum values. Horizontal lines indicate the maximum sample's limit of detection (SLOD). Asterisk signs indicate significant difference between two events (p < 0.05, Paired-Prentice Wilcoxon Test).

Fig. 4. Temporal distribution of enterococci DNA copies (a), and enterococci CFU (b) during four sampling events (i.e., Jul 2017, Aug 2017, Feb 2018, Mar 2018). Box plots represent the estimated 25th to 75th percentiles, with the median in between. Whiskers exhibit the maximum and minimum values. Asterisk signs indicate significant difference between two events (p < 0.05, Paired t-test).

Fig. 3. Spatial distribution of AIM06 phages (a) and SR14 phages (b) along twelve sampling stations (stations TC01 to TC28). Numbers in parentheses indicate the number of positive samples. Hollow data points indicate negative data, or data below the samples' detection limit (SLOD).

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et al., 2019). 3.4. Distributions of enterococci CFU An abundance of enterococci CFU in the Tha Chin River was previously reported (Kongprajug et al., 2019b), but for comparison purposes, it was then re-illustrated in detail (Figs. 4b and 5b and Supplemental Table S2). Enterococci CFU was detected in all river water samples at a range from 100.00 to 1593.00 CFU/100 mL with a median of 426.70 CFU/100 mL (n = 48). Ranges of enterococci CFU in the wet season were 186.70 to 1543.00 CFU/100 mL with a median of 418.40 CFU/100 mL (n = 24). In the dry season, enterococci CFU was detected at 100.00 to 1593.00 CFU/100 mL with a median of 461.70 CFU/100 mL (n = 24); however, no significant difference was observed between any sampling events for enterococci CFU (paired ttest and Wilcoxon signed rank test; p > 0.05) (Fig. 5b and Supplemental Table S3). Moreover, the median values of enterococci CFU were significantly different only between pairs of TC09 vs. TC23 and TC09 vs. TC28 (one-way ANOVA with Tukey's multiple comparison test; adjusted p < 0.05). 3.5. Correlation and multivariate analysis between microbial and physicochemical parameters A correlation analysis was performed among AIM06 phages, SR14 phages, enterococci DNA copies, enterococci CFU and other physicochemical and microbiological parameters from the same set of river water samples (Supplemental Fig. S2). AIM06 and SR14 phages did not show a correlation with the human-specific DNA markers CPQ056 and HF183 when analysed by incorporating non-detected data, which is likely due to high numbers of nondetects in CPQ056 and HF183 markers (Supplemental Figs. S3a–S3d); however, AIM06 and SR14 showed a strong correlation (Kendall's tau = 0.600) (Fig. 6a). Enterococci DNA copies showed a moderate correlation with enterococci CFU (Kendall's tau = 0.445) (Fig. 6b) and with certain physicochemical (i.e. TDS and DO) and microbiological (i.e. TCB MPN, TCB CFU, FCB MPN and E. coli CFU) parameters (Supplemental Fig. S2 and Fig. 6c and d). Enterococci CFU also demonstrated a moderate correlation with microbiological parameters (Supplemental Fig. S2 and Fig. 6e and f). Moreover, the recovery efficiencies of AIM06 and SR14 phages did not display a significant correlation with any of the water quality parameters (data not shown). In addition, the model-based clustering analysis, as indicated by the Bayesian information criterion (BIC), demonstrated that the EVE model (Scrucca et al., 2016) was the best fit model with two optimal clusters. By taking into account all 20 water quality parameters, the clustering analysis demonstrated that the levels of water quality from downstream stations were closely related regardless of sampling events, as shown in one cluster, while samples from upstream stations were placed in another cluster (Fig. 7).

Fig. 5. Spatial distribution of enterococci DNA copies (a), and enterococci CFU (modified from (Kongprajug et al., 2019b) (b) along twelve sampling stations (stations TC01 to TC28).

including nine method blanks, two field blanks and 27 NTCs from nine instrumental runs, demonstrated no contamination of enterococci DNA. Enterococci DNA copies were detected in all river water samples at a range from 3.24 to 6.32 log10 copies/100 mL with a mean concentration ± standard deviation of 4.65 ± 0.82 log10 copies/100 mL and a median of 4.58 log10 copies/100 mL (n = 48) (Fig. 4a and Supplemental Table S2). Ranges of enterococci DNA copies in the wet season were 3.24–6.21 log10 copies/100 mL with a mean concentration ± standard deviation of 4.45 ± 0.74 log10 copies/100 mL and a median of 4.21 log10 copies/100 mL (n = 24). In the dry season, enterococci DNA copies were detected at 3.30 to 6.32 log10 copies/ 100 mL with a mean concentration ± standard deviation of 4.86 ± 0.82 log10 copies/100 mL and a median of 4.73 log10 copies/ 100 mL (n = 24). No significant difference was observed except between Jul 2017 (wet season) and Mar 2018 (dry season) and between Aug 2017 (wet season) and Mar 2018 (dry season) (paired t-test; p = 0.013 and 0.041) (Figs. 4a and 5a and Supplemental Table S3). The mean values of enterococci DNA copies were significantly different between stations located in lower and upper sections of the Tha Chin River, i.e. pairs of TC01 vs. TC15, TC23 and TC28; pairs of TC04 vs. TC23 and TC28; pairs of TC07 vs. TC 15, TC22, TC23 and TC28; and pairs of TC09 vs. TC15, TC22, TC23 and TC28 (one-way ANOVA with Tukey's multiple comparison test; adjusted p < 0.05). Furthermore, nine representative water samples from sampling events in Feb 2018 and Mar 2018 were selected for assessing the method recovery efficiencies for enterococci DNA copies (Supplemental Table S5). The recovery percentages were in a range from 1282 to 2739% from the sampling event in Feb 2018 and in a range from 2274 to 3489% from the sampling event in Mar 2018. Despite the high percentages, these recovery efficiencies were in the ranges of the recovery acceptance criteria as stated in the US EPA Method 1611 (US EPA, 2012b). The high recovery percentages could have been due to the effects of suspended solids and organic matters that led to differences in DNA extraction and PCR inhibition removal efficiencies in spiked and unspiked samples as well as in spiking stocks (Claassen et al., 2013; Petcharat

4. Discussion This study is the first investigation of the abundance of humanspecific enterococci phages in freshwater in Thailand. The Tha Chin river appeared to be moderately polluted due to an abundance of up to two orders of magnitude (in PFU/100 mL), which is similar to those found in influents of wastewater treatment plants (WWTPinf) in the Bangkok area (Wangkahad et al., 2017). These phages were detected at higher levels of up to four orders of magnitude (in PFU/100 mL) in human sewage samples collected from untreated hospital wastewaters and septic tank samples (n = 10) and in polluted urban canals (n = 13) (Wangkahad et al., 2017). In coastal water along the upper part of the Gulf of Thailand (n = 31), these enterococci phages were detected up to three orders of magnitude (in PFU/100 mL) (Kongprajug et al., 2019a). All studies in Thailand demonstrated a percent co-presence of both phages ranging from 77.8% of phage-positive samples in coastal 6

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Fig. 6. Correlation plot, Kendall's tau correlation coefficients, and linear regression equations of parameters in river samples: AIM06 phages and SR14 phages (a), enterococci DNA copies and enterococci CFU (b), enterococci DNA copies and TCB MPN (c), enterococci DNA copies and FCB MPN (d) enterococci CFU and TCB MPN (e), and enterococci CFU and FCB MPN (f).

Fig. 7. Model-based clustering analysis of water samples from all twelve stations and four events (n = 48). 7

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no significant correlation between human-specific phages and FIOs, as shown in this study. The 2017 statistical records also demonstrated high swine and cattle populations in raising facilities along downstream locations (Information and Communication Technology Centre, 2017a, 2017b; 2017c). Moreover, the AIM06 and SR14 phages were present in all sampling stations along the river, similar to the human-specific crAssphage gene marker (Kongprajug et al., 2019c), which was shown to be present at a higher abundance than the human-specific HF183 gene marker (Kongprajug et al., 2019b).

water (Kongprajug et al., 2019a) to 92.9% in freshwater (this study), with no significant difference in abundance of both phages. Consequently, it is recommended that only one phage could be selected for monitoring as a human-specific faecal indicator. Furthermore, AIM06 and SR14 phages were present at similar levels as other human-specific bacteriophages of E. faecalis isolated and detected in other geographical regions in WWTPinf and in environmental water samples (Bonilla et al., 2010; Santiago-Rodríguez et al., 2010; Vijayavel et al., 2014), which supports their worldwide application as MST markers. Moreover, because different laboratory protocols and instruments could lead to discrepancies among studies and laboratories, it is crucial to characterise recovery efficiencies within laboratories for their own absolute quantification calculations (Mackowiak et al., 2018; Petcharat et al., 2019). Bacteriophages A1 in the Siphoviridae family and S1 in the Podoviridae family were selected as representatives for phages infecting E. faecalis host strains AIM06 and SR14, respectively, due to their highest persistence in environmental waters (Booncharoen et al., 2018). Identical spiking phage stocks and recovery protocols were performed for freshwater samples (n = 48), demonstrating a 57.9–99.6% AIM06 phage recovery and a 49.6–99% SR14 phage recovery (this study), and for seawater samples (n = 9), showing a 30.8–65.9% AIM06 phage recovery and a 29.5–92.9% SR14 phage recovery (unpublished data). This study also included molecular detection monitoring of enterococci in freshwater in Thailand. Moderate correlations were observed between enterococci DNA copies or enterococci CFU and other faecal indicator bacteria. According to Thailand's current Surface Water Quality Standards (National Environment Board, 1994), freshwater for recreational activities should have a TCB and an FCB of no more than 5000 and 1000 MPN/100 mL, respectively. This would correspond to 4.60 and 4.37 log10 copies/100 mL of enterococci DNA copies and 473 and 396 CFU/100 mL of enterococci CFU using the regression equations determined during this study. Consequently, 45.83% and 56.25% of samples exceeded both enterococci DNA copies and enterococci CFU values corresponding to regulatory TCB and FCB, respectively, which would require further corrective actions for water quality improvement. Furthermore, the correlation coefficient between enterococci DNA copies and enterococci CFU for river water samples in this study (Kendall's tau = 0.445; n = 48) was similar to that reported in seawater in the United States (maximum Kendall's tau = 0.46; n = 75), while that in more polluted combined sewer discharges demonstrated a higher correlation (Kendall's tau = 0.72; n = 28) (Jennings et al., 2018). The correlation between the two methods was also reported as a moderate correlation (Pearson correlation coefficient r = 0.60, n = 123) in coastal estuarine water in the United States as well (Gonzalez and Noble, 2014). Although the culture method for detecting enterococci in this study was the mEI agar and the abovementioned studies used the Enterolert® commercial kit in an MPN unit (IDEXX Laboratories, Inc.), the detected abundance of enterococci was generally similar or sometimes lower when using the mEI agar method, as reported for freshwater samples (n = 64) (Eckner, 1998; Kinzelman et al., 2003). The reason that the correlations between the qPCR and culture detection methods for enterococci were not stronger than reported might have been due to the intrinsic difference of the method as the qPCR assay detects genes possessed by all enterococci species, while the mEI agar assay generally recovers certain species, including E. faecalis, E. faecium and E. avium and their variants (US EPA, 2012b, 2002). The monitoring results of enterococci in the qPCR and culture methods and of AIM06 and SR14 phages in the Tha Chin River revealed continuing human faecal pollution along the river, and no significant difference in phage abundance was observed between upstream and downstream stations. Although enterococci detected by the qPCR and culture assays presented higher levels in downstream than upstream stations, their abundance may have been due to non-human faecal pollution, as supported by a moderate correlation between enterococci CFU and universal (non-host specific) and swine-specific MST markers indicated in the research by Kongprajug et al. (2019b), as well as due to

5. Conclusion This study underscores the importance of monitoring MST markers in addition to microbial water quality parameters, which could facilitate effective water quality management and public health risk reduction from exposure to faecally-contaminated water. Information from this study contributes to a comprehensive characterisation of AIM06 and SR14 phages as available, low-cost MST tools for pollution source identification and for freshwater and coastal water quality management. Moreover, the relationship between the enterococci qPCR detection method and the enterococci culture method has been characterised, which could promote its future use as a microbial water quality indicator in tropical regions. Declaration of competing interest The author(s) declare(s) that there is no conflict of interest regarding the publication of this article. Acknowledgements We would like to acknowledge the funding sources: the Thailand Research Fund (contract no. SRI5930305) and the Chulabhorn Research Institute. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ijheh.2020.113482. References Ashbolt, N.J.N., Grabow, W.W.O.K., Snozzi, M., 2001. Chapter 13 Indicators of microbial water quality. In: Fewtrell, L., Bartram, J. (Eds.), Water Quality: Guidelines, Standards and Health. IWA Publishing, London, UK, pp. 289–316. Benham, B., Krometis, L., Yagow, G., Kline, K., Dillaha, T., 2011. Chapter 14 applications of microbial source tracking in the TMDL process. In: Hagedorn, C., Blanch, A.R., Harwood, V.J. (Eds.), Microbial Source Tracking: Methods, Applications, and Case Studies 2. Springer, New York, NY. Bonilla, N., Santiago, T., Marcos, P., Urdaneta, M., Santo Domingo, J., Toranzos, G.A., 2010. Enterophages, a group of phages infecting Enterococcus faecalis, and their potential as alternate indicators of human faecal contamination. Water Sci. Technol. 61, 293–300. Booncharoen, N., Mongkolsuk, S., Sirikanchana, K., 2018. Comparative persistence of human sewage-specific enterococcal bacteriophages in freshwater and seawater. Appl. Microbiol. Biotechnol. 102, 6235–6246. Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J.F., Kubista, M., Mueller, R.D., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J., Wittwer, C.T., 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611–622. Claassen, S., du Toit, E., Kaba, M., Moodley, C., Zar, H.J., Nicol, M.P., 2013. A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples. J. Microbiol. Methods 94 (2), 103–110. Contreras-Coll, N., Lucena, F., Mooijman, K., Havelaar, A., Pierzo, V., Boque, M., Gawler, A., Holler, C., Lambiri, M., Mirolo, G., Moreno, B., Niemi, M., Sommer, R., Valentin, B., Wiedenmann, A., Young, V., Jofre, J., 2002. Occurrence and levels of indicator bacteriophages in bathing waters throughout Europe. Water Res. 36, 4963–4974. Devriese, L., Pot, B., 1995. Chapter 10. The genus Enterococcus. In: Wood, B.J.B., Holzapfel, W.H. (Eds.), The Genera of Lactic Acid Bacteria. Chapman & Hall, pp. 327–367. Dorevitch, S., Shrestha, A., DeFlorio-Barker, S., Breitenbach, C., Heimler, I., 2017. Monitoring urban beaches with qPCR vs. culture measures of fecal indicator bacteria: implications for public notification. Environ. Health 16, 45.

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