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Application of high-throughput 16S rRNA sequencing to identify fecal contamination sources and to complement the detection of fecal indicator bacteria in rural groundwater

Application of high-throughput 16S rRNA sequencing to identify fecal contamination sources and to... | | 393 © 2019 The Authors Journal of Water and Health 17.3 2019 Application of high-throughput 16S rRNA sequencing to identify fecal contamination sources and to complement the detection of fecal indicator bacteria in rural groundwater Paul Naphtali, Mahi M. Mohiuddin, Athanasios Paschos and Herb E. Schellhorn ABSTRACT Paul Naphtali Residents in rural communities across Canada collect potable water from aquifers. Fecal contaminants Mahi M. Mohiuddin from sewage and agricultural runoffs can penetrate aquifers, posing a public health risk. Standard Athanasios Paschos Herb E. Schellhorn (corresponding author) methods for detecting fecal contamination test for fecal indicator bacteria (FIB), but the presence of Department of Biology, McMaster University, these do not identify sources of contamination. In contrast, DNA-based diagnostic tools can achieve this Hamilton, ON, Canada important objective. We employed quantitative polymerase chain reaction (qPCR) and high-throughput E-mail: schell@mcmaster.ca DNA sequencing to trace fecal contamination sources in Wainfleet, a rural Ontario township that has This article has been made Open Access thanks to been under the longest active boil water advisory in Canada due to FIB contamination in groundwater the generous support of a global network of libraries as part of the Knowledge Unlatched Select wells. Using traditional methods, we identified FIBs indicating persistent fecal pollution in well initiative. waters. We used 16S rRNA sequencing to profile groundwater microbial communities and identified Campylobacteraceae as a fecal contamination DNA marker in septic tank effluents (STEs). We also identified Turicibacter and Gallicola as a potential cow and chicken fecal contamination marker, respectively. Using human specific Bacteroidales markers, we identified leaking septic tanks as the likely primary fecal contamination source in some of Wainfleet’s groundwater. Overall, the results support the use of sequencing-based methods to augment traditional water quality testing methods and help end-users assess fecal contamination levels and identify point and non-point pollution sources. Key words amplicon sequencing, fecal marker, fecal pollution, groundwater, qPCR INTRODUCTION Fecal pollutants from sewage and agricultural runoff can because of the diversity and low abundance of pathogens penetrate decaying groundwater wells and render the well in water. Escherichia coli and Enterococcus, the standard water unsafe to drink (USEPA ). Fecal contaminants fecal indicator bacteria (FIB), are present in high densities may contain waterborne pathogens that transfer into aquatic within the intestine of warm-blooded animals. FIB detection environments and cause infectious disease (Harwood et al. act as proxies for high fecal contamination levels (Field & ). Waterborne pathogen detection remains a challenge Samadpour ). Public officials in rural communities enact boil water advisories upon FIB detection to lower This is an Open Access article distributed under the terms of the Creative the risk of waterborne disease outbreaks. Commons Attribution Licence (CC BY 4.0), which permits copying, While FIBs indicate fecal contamination, the source adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/). of contamination and the true abundance of pathogens doi: 10.2166/wh.2019.295 Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021 | | | 394 P. Naphtali et al. Tracing groundwater fecal contamination through high-throughput sequencing Journal of Water and Health 17.3 2019 cannot be identified using FIBs alone. Current practices for In this proof-of-principle study, we tested whether a monitoring water quality include the use of microbial source next-generation DNA sequencing approach can be used to tracking (MST) methods that target genetic markers such as trace fecal contamination sources in Wainfleet’s private the 16S rRNA gene to quantify and source fecal contamination well waters. Using both traditional methods of FIB detection (Field & Samadpour ). The HF183 16S rRNA sequence, and 16S rRNA amplicon sequencing, we quantified fecal belonging to a human Bacteroidales 16S rRNA gene fragment, contamination levels and identified likely fecal pollution was the first genetic DNA marker used to detect human fecal sources. We also measured the concentration of human contamination in drinking water (Bernhard & Field ). Bacteroidales gene markers as a proxy of sewage-based con- Quantitative polymerase chain reaction (qPCR) assays are tamination. Information obtained from our analyses can now commonly used to quantify the HF183 marker as an augment traditional methods of water quality monitoring indicator of human fecal pollution (Seurinck et al. ). by providing additional information on fecal pollution Human and animal-associated DNA markers can also be markers in potable waters. used to measure point and non-point source contamination inputs in freshwater environments (Staley et al. ). METHODS In addition to MST-based approaches, next-generation DNA sequencing is now being used to identify fecal con- Study site description tamination sources and to examine the co-occurrence of fecal and source water bacteria in respective environments Wainfleet is situated in the southwest portion of the Niagara (Unno et al. ). Next-generation sequencing based Region (42.92 N, 79.38 W). Residents obtain potable water methods are also used to identify FIBs and pathogens in using on-site groundwater wells. Many residences are built freshwater reservoirs (Mohiuddin et al. , ). Aquatic in low-lying areas close to Lake Erie. Of the 107 residences microbiota containing taxa belonging to fecal bacteria are surveyed in March 2005 that use on-site groundwater wells, more likely to be contaminated through fecal sources (Cao 44 had septic tanks that are 20 years old, and 49% of the et al. ). A 16S rRNA gene-based sequencing approach residences do not comply with current provincial building can also identify additional human fecal markers, such as codes (Niagara Region Report ). Most homeowners Lachnospiraceae, for further characterization with other install septic tanks to discharge septic tank effluents genetic methods such as oligotyping (McLellan et al. ). (STEs) into the underlying soils through tile beds. Many of Despite the decreasing cost and increasing usefulness of the plots have an area too small to install functioning next-generation sequencing methods to identify fecal con- septic tanks to current building standards. Besides the tamination sources, MST protocols have not yet, however, potential for leaking, concentrated raw sewage seeps integrated next-generation DNA sequencing as a standard through the underlying bed and into aquifers that supply for monitoring Canadian drinking water sources. wells (Niagara Region Report ). Wainfleet, a rural Ontario Township by Lake Erie, is under the longest active boil water advisory in Canada. A previous monitoring study on fecal contamination in Groundwater tap sample collection 280 private residential groundwater wells determined that 50% contained detectable FIBs (Niagara Region Report We identified nine test sampling sites (Sites A–K, Figure 1) ). The town’s boil water advisory provides an opportu- and 21 wells based on FIB detection in the previous inde- nity to test next-generation sequencing DNA methods in pendent study (Niagara Region Report ). We received MST monitoring. Combining next-generation DNA sequen- written consent from the township and identified volunteers cing approaches with traditional MST-based methods can based on the sampling site selection process. We collected augment current water quality monitoring approaches by tap water samples every month from April to November incorporating new fecal-specific DNA markers to quantify 2015 and grouped the samples based on season (spring, host-specific contamination. summer, and fall). A total of 48 samples were collected Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021 | | | 395 P. Naphtali et al. Tracing groundwater fecal contamination through high-throughput sequencing Journal of Water and Health 17.3 2019 Figure 1 | Location of sampling sites. from nine test sites. For each sampling event, town technical of well water into 100 mL of 1× PBS solution (pH 7.0), to staff collected groundwater tap samples from homeowners a10 -fold dilution. One hundred mL of the well water by filling 500 mL autoclaved plastic bottles (Nalgene). The sample and PBS diluted samples were passed through water samples were then kept on ice, transported to the 0.45 μm pore-size 47-mm-diameter sterile mixed cellulose laboratory, and processed within 6 h of collection. ester membrane filters (Thermo Fisher Scientific, Burling- ton, ON, Canada). Each membrane filter was placed on differential coliform (DC, Oxoid) and mEI agar (BD Septic tank effluent and manure sample collection Difco) plate and incubated at 42 C for 24 h. After incubation, E. coli and Enterococcus spp. colony forming STEs were collected from two septic tanks owned by two units (CFUs) for filters containing stock and diluted well homeowners that participated in this study (fall 2015). water samples were enumerated. To determine the concen- Three biological replicates for each sewage sample were tration of FIBs (CFU/mL) in water samples, we multiplied collected on three consecutive days. Manure samples were the CFU count by its associated dilution factor. Detection collected from manure mounds stored by a concentrated of one (or more) CFU per 100 mL of drinking water samples animal feeding operation for chickens, a cow farm, a horse were considered positive for FIBs (deemed unsafe for hobby farm, and a pig farm. Similar to septic samples, drinking (Health-Canada )). three biological replicates for each manure type were collected on three consecutive days. All samples were transferred into 500 mL autoclaved bottles, kept in ice DNA extraction and library generation for sequencing and transported to the laboratory. The samples were then stored at –80 C until further analysis. DNA was extracted from water samples as described earlier (Mohiuddin et al. ). Briefly, 500 mL of each ground- FIB detection assay water sample was passed through 0.45-m pore-size 47-mm- diameter sterile mixed cellulose ester membrane filters. FIBs in water samples were detected using standard methods The filters were then cut into fragments (1 cm size) with (APHA ). To enumerate E. coli and Enterococcus spp., sterile scissors and the cut fragments were aseptically trans- 100-fold serial dilutions were prepared by transferring 1 mL ferred with sterile forceps into 1.7 mL microfuge tubes for Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021 | | | 396 P. Naphtali et al. Tracing groundwater fecal contamination through high-throughput sequencing Journal of Water and Health 17.3 2019 DNA extraction. DNA was extracted from the filters using a library were checked using BioAnalyzer and High Soil DNA Isolation Kit (Norgen Biotek, Thorold, ON, Sensitivity DNA Kit (Agilent, Mississauga, ON, Canada). Canada) according to manufacturers’ instruction and iso- Each library was then quantified by qPCR and sequencing lated DNA was stored at –20 C for further analysis. DNA was performed on the Illumina MiSeq platform which quantity and purity were measured using the Nanodrop generated 2 × 300 bp paired-end sequences. 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Burlington, ON, Canada). Manure samples stored at –80 C Processing and analysis of 16S rRNA gene sequences were taken out overnight to thaw the samples and 200 mg of thawed samples were used for DNA extraction using a Stool Paired-end sequences generated by Illumina MiSeq were de- Nucleic Acid Isolation Kit (Norgen Biotek, Thorold, ON, multiplexed and quality-filtered using methods described Canada). Similar to manure samples, septic tank samples earlier (Bokulich et al. ; Mohiuddin et al. ). Only were taken out overnight to thaw the samples and 10 mL full amplicons with 464 bp sequences were considered for of the thawed samples were centrifuged at 10,000× g for further analysis. Before downstream analysis, all sequences 10 min at 4 C. After centrifugation, the pellet was resus- were trimmed using a quality score threshold (Q score) of pended with resuspension buffer provided with Stool 25 over at least 75% of the sequence read. Sequences that Nucleic Acid Isolation Kit (Norgen Biotek, Thorold, contained ambiguous bases, errors in barcode sequences, ON, Canada) and DNA was extracted according to >10 consecutive low-quality base pairs and >2 nt mis- manufacturers’ instructions. matches from the primer sequences were also removed After DNA extraction, PCR assays were conducted to (Bokulich et al. ). Processed sequence reads were then amplify the V3–V4 region of the 16S rRNA gene using analyzed using software package QIIME v.1.9.0 (Caporaso an optimized primer pair (S-D-Bact-0341-b-102 S-17/S-D- et al. ). Sequences were clustered into Operational Bact-0785-a-A-21) evaluated elsewhere (Klindworth et al. Taxonomic Units (OTUs) sharing 97% nucleotide sequence ). Reaction mixes of 25.0 μL were prepared as follows: identity and a minimum query alignment length of 50% to 2þ 2.5 μL10× PCR buffer minus Mg (Invitrogen, Burlington, the Greengenes 2013 reference database (McDonald et al. ON, Canada), 0.5 μL of 100 mM dNTP solution, 1.0 μL ) with a sequence similarity threshold of 97% and a each of the forward and reverse primer (1.0 μM each) with minimum query alignment length of 50% using UCLUST unique Illumina adapter sequences attached to them, (Edgar ). The resulting OTU table was then rarified 1.0 μL 10 mg/mL UV-treated BSA solution, 0.75 μL25 mM to 5,000 OTU counts/sample before statistical analyses MgCl solution (Invitrogen, Burlington, ON, Canada), were performed. A potential DNA marker of a human or 0.25 μL Taq DNA Polymerase (Invitrogen), 2.0 μL of DNA animal-specific fecal or manure contaminant was identified template, and 16.0 μL ddH O. The target DNA was ampli- if it was detected in 10% abundance in a fecal sample but fied using the T100 Thermal Cycler (Bio-Rad, Mississauga, 1% abundance in all other fecal samples. The relative ON, Canada) and conducted as follows: Initial denaturation abundance of the potential markers was determined in the at 95 C for 3 min, 40 cycles of denaturation at 94 C for groundwater samples across the sampling months. 30 s, annealing at 50 C for 30 s, and elongation at 72 C for 1 min, and a final extension step at 72 C for 10 min. Preparation of DNA standards for quantification of All PCR products were examined in 0.9% agarose gels, human fecal pollution the desired band was cut from the gel and DNA was extracted from the cut band with the Nucleospin PCR and The HF183 primer pair (HF183F and Bac708R) (Bernhard Gel Cleanup Kit (Macherey-Nagel, Bethelhem, PA, USA). & Field ) that flanks the fragments in the 16S rRNA All amplicon extracts were pooled in equal mass amounts gene of human Bacteroidales populations was used to and sent to the Farncombe Metagenomics Facility at prepare standards for quantification of human-based fecal McMaster University for further processing and sequencing. pollution. First, a conventional PCR assay was performed Product size and the presence of primer dimers in each to generate a 500 bp product. The amplified product was Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021 | | | 397 P. Naphtali et al. Tracing groundwater fecal contamination through high-throughput sequencing Journal of Water and Health 17.3 2019 then cloned into a vector which served as standard in qPCR (Bio-Rad), and 0.5 μL each of the forward and reverse assay. For conventional PCR, 50.0 μL reaction volumes were primer (10 μM). Thermal cycling conditions included the fol- prepared. The reaction mix contained 5.0 μL of Thermopol lowing steps: Initial denaturation at 95 C for 30 s, 40 cycles Buffer (10 × , NEB), 1.0 μL (10.0 μM each) each of the for- of denaturation at 95 C for 30 s and 60 C for 10 s, and then 0 0 ward (HF183F: 5 -ATCATGAGTTCACATGTCCG-3 ) and a melt curve analysis from 65 to 95 C at increments of 0 0 reverse primer (Bac708R: 5 -CAATCGGAGTTCTTCGTG-3 ), 0.5 C for 5 s per increment. The CFX Analyzer (Bio-Rad, 1.0 μL of dNTP mix (NEB, 10 mM), 1.0 μLof5 U Taq Poly- Mississauga, ON, Canada) was used to generate a standard merase (NEB), and 2.0 μL of DNA template. All PCR assays curve and a melt curve and quantify qPCR copy numbers. were run with the conditions as follows: initial denaturation for 5 min at 95 C, 35 cycles of denaturation at 95 C for 30 s, annealing at 52 C, and extension at 72 C for 1 min, RESULTS and a final extension step at 72 C for 6 min. PCR products were loaded onto 1% agarose gels and amplicons were Detection of FIB in groundwater wells extracted from the gel using the Nucleospin PCR and Gel Cleanup Kit (Macherey-Nagel, Bethelhem, PA, USA). As a preliminary assessment of fecal contamination levels To generate plasmid DNA standards, the TOPO TA Cloning in Wainfleet’s well waters, we examined FIB detection Reaction (Invitrogen, Burlington, ON, Canada) was used. frequency in tap water collected from private wells using Briefly, 2.0 μL of the amplicons were ligated into the Health Canada recommended guidelines (see above under TOPO vector, according to the manufacturer’s instructions. ‘Methods’). Sixty-one per cent of the groundwater samples Transformants were isolated using blue-white screening collected from 21 wells within the boil water advisory in technique and subsequently purified. Purified colonies 2015 contained either E. coli or Enterococcus (Figure 2). were then inoculated into 3.0 mL of LB broth supplemented with 50.0 μg/mL kanamycin for overnight incubation at 37 C with shaking at 220 rpm. DNA was extracted from 1.5 mL aliquots of the cultures with a Plasmid DNA Miniprep Kit (Norgen Biotek, Thorold, ON, USA). DNA concentrations were quantified using the Qubit Fluorometer 2.0 (Invitrogen, Burlington, ON, USA). DNA extracts were 1 7 diluted such that a range of 10 –10 plasmid copies of the marker was present in the standards. Quantification of human Bacteroidales marker in sewage and groundwater samples Quantification of human specific Bacteroidales marker was performed using the same forward primer used for the conventional PCR (HF183F) and a different reverse primer 0 0 (5 -TACCCCGCCTACTATCTAATG-3 )(Seurinck et al. ). The newly amplified product had a length of 82 bp. qPCR assays were conducted using 2.0 μL of groundwater DNA extracts and plasmid standard in 10.0 μL reaction mix volumes. All DNA templates were diluted 10-fold to mini- mize qPCR inhibition. Each reaction mix contained 1.0 μL Figure 2 | Detection of E. coli and Enterococcus in well water samples (n ¼ 48) at of DNA template, 5.0 μL of the SsoFast EvaGreen SuperMix locations in the boil water advisory zone in 2015. Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021 | | | 398 P. Naphtali et al. Tracing groundwater fecal contamination through high-throughput sequencing Journal of Water and Health 17.3 2019 Positive E. coli detection ranged from 60.0% to 70.0%. Identification of potential STE and animal manure Enterococcus detection rates had a large seasonal range contamination markers from 10.0% to 81.0%, with the detection rate lowest in the spring and highest in the summer (Figure 2). Groundwater To identify potential fecal contamination markers in ground- samples collected from two residential wells, B and K, also water, we parsed for sewage and manure-associated bacteria contained much higher mean E. coli and Enterococcus in the 16S rRNA sequencing data. We defined a host-specific than the other groundwater wells (Figure 3). fecal marker as a microbial group detected at 10.0% rela- tive abundance in one type of fecal matter but detected at 1.0% abundance in the other fecal sources. In the two Basic sequencing data STE samples, the mean abundance of Campylobacteraceae was 32.5% (Figure 4). At the genus level, sequences anno- As a first step towards identifying potential fecal contami- tated to Sulphospirillum and Arcobacter were the most nation sources in the FIB-positive well waters, we profiled abundant members of Campylobacteraceae. In contrast, the microbial communities from 21 groundwater samples, average abundance of Campylobacteraceae was 1% for all six samples from two STEs, and 12 manure samples from four animal manure samples. Gallicola and Turicibacter four different animal farms using 16S rRNA sequencing. were the most abundant genera in chicken and cow Sequence reads obtained from septic tank samples were manure, comprising 42.2% and 9.4% of 16S rRNA sequences classified into 972 (±89) OTUs. The number of reads respectively. In contrast, these markers were detected at obtained from animal manure and groundwater samples 1.0% abundance among the pig and horse manure and were higher than septic tank samples and reads from STE samples (Figure 4). No genetic marker was identified animal manure and groundwater wells were classified into in pig and horse manure based on the classification criterion. 2,987 (±315) and 3,101 (±287) OTUs respectively. Abundance of STE and manure-based OTUs in groundwater samples To identify possible fecal contaminants in well water, we determined the relative abundance of the human and Figure 4 | Relative abundance of potential manure and sewage markers in waste samples collected in Wainfleet. Error bars represent standard deviation of Figure 3 | E. coli and Enterococcus levels in wells waters B and K compared with the the mean. Three biological replicates for each of the samples was used to other well water sites. Error bars represent standard deviation of the mean. calculate mean and standard deviation. Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021 | | | 399 P. Naphtali et al. Tracing groundwater fecal contamination through high-throughput sequencing Journal of Water and Health 17.3 2019 Figure 5 | Relative abundance of potential STE and animal-specific contamination Figure 6 | Presence of human Bacteroidales marker in selected septic tank and markers in well water sites within the active boil water advisory zone. groundwater wells within the boil water advisory zone in Wainfleet. Error bars Error bars represent standard deviation of the mean. represent standard deviation of the mean. animal-associated markers we identified in the reference wells without E. coli detection also tested positive for the fecal samples. Groundwater wells sampled in July, HF183 marker. Groundwater well A, treated with UV light September, and November had the highest relative abundance before sampling, contained more HF183 copies than other of STE-based Campylobacteraceae sequences (Figure 5). None groundwater wells (Figure 6). Groundwater well I had a of the chicken and cow markers had a relative abundance slightly lower HF183 marker level than B, F, and K. above2.0%, with the relativeabundance below1.0%in wells collected in July, August, and November (Figure 5). DISCUSSION Identification of human fecal contamination The presence of FIBs in drinking waters is a major health concern. Using national guidelines for drinking water and To determine whether the human-specific fecal contami- the results of a previous study as a reference, we sought to nation was present in the groundwater samples, we determine whether high levels of FIBs are still present in conducted qPCR assays of the HF183 human Bacteroidales the Wainfleet well waters. Our analyses suggest that FIBs marker in selected wells with or without FIB detection. We are still present in over half of the tested well water sites prepared a qPCR standard curve that quantifies the human and, therefore, the quality of drinking water may still be a Bacteroidales marker in each run (Figure S1A). Each standard concern in the majority of the households within the town- curve was robust for HF183 quantification (Figure S1, avail- ship. Many of the sampled groundwater wells are located able with the online version of this paper). Melt curve near the shores of Lake Erie, a low-lying region where analysis of the standard curve and all groundwater samples most of the E. coli and total coliform exceedances were pre- that were examined after the qPCR assay yielded a single viously observed (Niagara Region Report ). Some well peak at 84 C (Figure S1B). water samples also contained far higher FIB counts than The HF183 marker genome was more abundant in others. Mean E. coli and Enterococcus levels in some STE samples than in groundwater wells (Figure 6). qPCR groundwater wells were three and two orders of magnitude of the human Bacteroidales marker in groundwater wells higher than the average FIB counts for the groundwater B, F and K resulted in positive detection, containing 30–50 wells collected across all the other locations. These wells genome copies/100 mL (Figure 6). A subset of groundwater with higher than average FIB counts are located near Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021 | | | 400 P. Naphtali et al. Tracing groundwater fecal contamination through high-throughput sequencing Journal of Water and Health 17.3 2019 septic tanks that may leak raw sewage into the aquifer (Nia- were also present in other manure types. However, analyses gara Region Report ). Poor well maintenance also of additional samples that include both biological and facilitates sewage leaching into the groundwater (Howard technical replicates are required to confirm the findings of et al. ). Altogether, chronic FIB contamination in this proof-of-principle study. A higher sequencing depth, in individual groundwater wells like B and K require further conjunction with shotgun metagenomic sequencing, may investigation to confirm potential fecal contamination sources. facilitate the identification of novel or rare (but nonetheless important) taxa that can be used to identify contamination Characterization of fecal microbiota and identification at the species level. DNA markers unique to horse or pig of potential fecal markers manure are yet to be established. The robust resolution of horse and pig-associated markers with next-generation The presence of host-associated fecal contamination sequencing represents opportunities for future investigation. may help to trace fecal contamination sources in complex environmental samples. As a proof-of-principle experiment, Fecal source identification in residential groundwater we examined whether we could complement FIB detection sites using 16S rRNA sequencing with 16S rRNA sequencing methods by identifying sewage and manure-associated markers. We selected a maximum The co-detection of animal and/or human associated mar- detection rate of 1.0% limit to ensure the specificity of the kers in drinking water provides evidence of possible fecal marker to its host. In cow manure, we identified Turicibacter contamination by that source. After identifying human and spp., a member of the Firmicutes phylum present in animal-associated markers in our reference fecal samples, high abundance at the genus level, agreeing with previous we screened for possible fecal contamination in private cow microbiome profiles (Kim et al. ). In chicken well waters by profiling the well water microbial commu- manure, Gallicola – also a member of the Firmicutes nities. The mean Campylobacteraceae relative abundance phylum – was the most abundant genus. Both markers was highest in November and September, but lowest in were also detected at 0.1% abundance in other manure August. The large standard deviations in Campylobactera- types and sewage samples. These genera may act as DNA ceae abundances within the sampling months suggest that markers of host-associated fecal contamination in the some groundwater sites contain more fecal contamination town’s groundwater wells. than others. The relative abundance of Campylobacteraceae In the two STE sites, we identified the Campylobactera- sequences still far exceeded the relative abundance of ceae family (member of the Proteobacteria phylum) as fecal chicken and cow fecal markers, Gallicola and Turicibacter. pollution marker, comprising 32.5% of 16S rRNA gene While we cannot exclude the possibility of cow or chicken sequences at the family level. The predominance of farm contamination in September and October, the higher Campylobacteraceae 16S rRNA gene sequences in STEs is mean relative abundance of Campylobacteraceae across identified in other septic tank systems (Tomaras et al. the groundwater wells in every month except August raises ). Although we only collected STEs from two septic the likelihood of STE contamination in the residential tanks, both septic tank microbiomes contained a similar well water. abundance of Campylobacteraceae sequences. Campylobac- Interestingly, well waters collected in August contained teraceae was also absent in the animal manure samples, the lowest abundance of the three host-associated markers. suggesting that the Campylobacteraceae microbes are likely Except for well water site K, E. coli counts did not exceed exclusive to STEs in the township. 100 CFUs/100 mL (raw data), suggesting diffuse fecal While we identified host-associated markers for chicken contamination among the well water samples collected manure, cow manure, and STEs, we could not establish the in August. In other sampling months, there was a far presence of host-associated markers for horse and pig higher abundance of Campylobacteraceae sequences in manure. Although Clostridium and Turicibacter were more the autumn. The detection of E. coli counts could be due abundant in horse and pig manure samples, these sequences to leaking septic tanks from individual sites. Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021 | | | 401 P. Naphtali et al. Tracing groundwater fecal contamination through high-throughput sequencing Journal of Water and Health 17.3 2019 Quantification of human-based fecal contamination presence of DNA from dead (or inactivated) Bacteroidales (inactivated through UV treatment) which was amplified To confirm the presence of STE contamination in selected during the qPCR assay. Other groundwater wells, like G well sites, we used the HF183 marker to quantify human- and J, did not contain a detectable HF183 marker, removing based contamination in DNA extracted from STE and the possibility of STE contamination in those wells. Cultur- groundwater samples. We first validated the use of the able FIBs were also absent in G and J, corroborating the HF183 marker by preparing standard and melt curves of absence of STE-based contamination. the HF183 qPCR assay. Although qPCR assays were con- ducted with 100 bp fragments to minimize spurious fluorescence, we observed high R value and robust CONCLUSIONS E-value, suggesting the quality of the amplification reactions (Figure S1A). Furthermore, the melt curve analysis shows A prior study found extensive FIB contamination through- that a single product was detected by the qPCR assay out a rural community (Niagara Region Report ). In (Figure S1B). Furthermore, we detected the HF183 marker this study, we complemented cultured-based methods with in the two STE sites at 10-fold higher concentration than 16S sequencing and qPCR to re-assess fecal contamination groundwater samples. These results validate quantifying levels and, further, to identify potential contamination the entire HF183 amplicon in DNA extracted from the sources in Wainfleet. We found FIB contamination in the town’s well water as a proxy of STE contamination. well waters we tested, with E. coli counts as high as We also detected the HF183 DNA marker in B and K, 10 CFU/mL. In addition, the identification of additional groundwater wells containing the highest E. coli CFUs. STE DNA markers to Campylobacteraceae and the human Curiously, groundwater wells I and F, which did not contain Bacteroidales is consistent with the idea that human waste E. coli, were also positive for the HF183 marker. A may impact residential groundwater wells. The low abun- weak correlation between E. coli counts and HF183 dance (and absence) of animal manure-associated DNA marker concentrations was established in residential areas markers – Turicibacter in cow manure and Gallicola in (Nshimyimana et al. ) and the Great Lakes beach chicken manure – reduces the possibility that observed sands (Staley et al. ). The weak correlation (Pearson cor- contamination is due to animal sources. These results relation; r ¼ 0.33) between E. coli counts and HF183 marker indicate that profiling microbial communities using 16S concentrations indicates that E. coli levels are not a reliable rRNA sequencing can augment culture-based methods in indicator of human fecal contamination in well waters. This contamination analysis studies. The use of next-generation is primarily due to the factors that affect viability of E. coli sequencing methods can specifically facilitate the assess- in well water and contamination of well waters through ment of groundwater quality by detecting host-associated sources other than humans. Within Wainfleet, groundwater markers and quantifying relative contributions of likely wells B and K may receive E. coli from Lake Erie where fecal sources to groundwater contamination. surface water flows into the aquifer. However, these ground- To trace fecal contamination sources in water sources water wells also receive human contamination loads that more robustly, amplicon sequencing at higher depths may come from leaking septic tanks, evidenced by the (more reads per sample) may be useful in identification of positive HF183 detection in septic tanks and the wells. novel or rare taxa. Shotgun metagenomic sequencing Interestingly, the groundwater well sample collected from may also be used which, at a higher depth, may facilitate well A contained almost five times the HF183 marker concen- the identification of FIB at the species level. While deeper tration as groundwater wells B and K. Well A’s homeowner sequencing depth may facilitate species identification, such installed a UV treatment system in residence to inactivate as the Campylobacter spp. that comprise STEs and possible fecal microbes. While E. coli and Enterococcus were absent pig and horse-associated markers within Clostridium and in the UV treated well water, the HF183 marker was still pre- other genera, the cost of such in-depth analyses are fairly sent in UV treated well water. This could be due to the high. Highly focused DNA sampling programs that target Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021 | | | 402 P. Naphtali et al. Tracing groundwater fecal contamination through high-throughput sequencing Journal of Water and Health 17.3 2019 Harwood, V. J., Staley, C., Badgley, B. D., Borges, K. & Korajkic, well water sites having high FIB levels may allow detailed A.  Microbial source tracking markers for detection of identification contamination inputs that include potential fecal contamination in environmental waters: relationships pathogens that, unlike E. coli, are difficult to culture. between pathogens and human health outcomes. 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First received 3 December 2018; accepted in revised form 14 January 2019. Available online 1 March 2019 Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Water and Health Unpaywall

Application of high-throughput 16S rRNA sequencing to identify fecal contamination sources and to complement the detection of fecal indicator bacteria in rural groundwater

Naphtali, Paul; Mohiuddin, Mahi M.; Paschos, Athanasios; Schellhorn, Herb E.
Journal of Water and Health – Mar 1, 2019
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10.2166/wh.2019.295
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| | 393 © 2019 The Authors Journal of Water and Health 17.3 2019 Application of high-throughput 16S rRNA sequencing to identify fecal contamination sources and to complement the detection of fecal indicator bacteria in rural groundwater Paul Naphtali, Mahi M. Mohiuddin, Athanasios Paschos and Herb E. Schellhorn ABSTRACT Paul Naphtali Residents in rural communities across Canada collect potable water from aquifers. Fecal contaminants Mahi M. Mohiuddin from sewage and agricultural runoffs can penetrate aquifers, posing a public health risk. Standard Athanasios Paschos Herb E. Schellhorn (corresponding author) methods for detecting fecal contamination test for fecal indicator bacteria (FIB), but the presence of Department of Biology, McMaster University, these do not identify sources of contamination. In contrast, DNA-based diagnostic tools can achieve this Hamilton, ON, Canada important objective. We employed quantitative polymerase chain reaction (qPCR) and high-throughput E-mail: schell@mcmaster.ca DNA sequencing to trace fecal contamination sources in Wainfleet, a rural Ontario township that has This article has been made Open Access thanks to been under the longest active boil water advisory in Canada due to FIB contamination in groundwater the generous support of a global network of libraries as part of the Knowledge Unlatched Select wells. Using traditional methods, we identified FIBs indicating persistent fecal pollution in well initiative. waters. We used 16S rRNA sequencing to profile groundwater microbial communities and identified Campylobacteraceae as a fecal contamination DNA marker in septic tank effluents (STEs). We also identified Turicibacter and Gallicola as a potential cow and chicken fecal contamination marker, respectively. Using human specific Bacteroidales markers, we identified leaking septic tanks as the likely primary fecal contamination source in some of Wainfleet’s groundwater. Overall, the results support the use of sequencing-based methods to augment traditional water quality testing methods and help end-users assess fecal contamination levels and identify point and non-point pollution sources. Key words amplicon sequencing, fecal marker, fecal pollution, groundwater, qPCR INTRODUCTION Fecal pollutants from sewage and agricultural runoff can because of the diversity and low abundance of pathogens penetrate decaying groundwater wells and render the well in water. Escherichia coli and Enterococcus, the standard water unsafe to drink (USEPA ). Fecal contaminants fecal indicator bacteria (FIB), are present in high densities may contain waterborne pathogens that transfer into aquatic within the intestine of warm-blooded animals. FIB detection environments and cause infectious disease (Harwood et al. act as proxies for high fecal contamination levels (Field & ). Waterborne pathogen detection remains a challenge Samadpour ). Public officials in rural communities enact boil water advisories upon FIB detection to lower This is an Open Access article distributed under the terms of the Creative the risk of waterborne disease outbreaks. Commons Attribution Licence (CC BY 4.0), which permits copying, While FIBs indicate fecal contamination, the source adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/). of contamination and the true abundance of pathogens doi: 10.2166/wh.2019.295 Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021 | | | 394 P. Naphtali et al. Tracing groundwater fecal contamination through high-throughput sequencing Journal of Water and Health 17.3 2019 cannot be identified using FIBs alone. Current practices for In this proof-of-principle study, we tested whether a monitoring water quality include the use of microbial source next-generation DNA sequencing approach can be used to tracking (MST) methods that target genetic markers such as trace fecal contamination sources in Wainfleet’s private the 16S rRNA gene to quantify and source fecal contamination well waters. Using both traditional methods of FIB detection (Field & Samadpour ). The HF183 16S rRNA sequence, and 16S rRNA amplicon sequencing, we quantified fecal belonging to a human Bacteroidales 16S rRNA gene fragment, contamination levels and identified likely fecal pollution was the first genetic DNA marker used to detect human fecal sources. We also measured the concentration of human contamination in drinking water (Bernhard & Field ). Bacteroidales gene markers as a proxy of sewage-based con- Quantitative polymerase chain reaction (qPCR) assays are tamination. Information obtained from our analyses can now commonly used to quantify the HF183 marker as an augment traditional methods of water quality monitoring indicator of human fecal pollution (Seurinck et al. ). by providing additional information on fecal pollution Human and animal-associated DNA markers can also be markers in potable waters. used to measure point and non-point source contamination inputs in freshwater environments (Staley et al. ). METHODS In addition to MST-based approaches, next-generation DNA sequencing is now being used to identify fecal con- Study site description tamination sources and to examine the co-occurrence of fecal and source water bacteria in respective environments Wainfleet is situated in the southwest portion of the Niagara (Unno et al. ). Next-generation sequencing based Region (42.92 N, 79.38 W). Residents obtain potable water methods are also used to identify FIBs and pathogens in using on-site groundwater wells. Many residences are built freshwater reservoirs (Mohiuddin et al. , ). Aquatic in low-lying areas close to Lake Erie. Of the 107 residences microbiota containing taxa belonging to fecal bacteria are surveyed in March 2005 that use on-site groundwater wells, more likely to be contaminated through fecal sources (Cao 44 had septic tanks that are 20 years old, and 49% of the et al. ). A 16S rRNA gene-based sequencing approach residences do not comply with current provincial building can also identify additional human fecal markers, such as codes (Niagara Region Report ). Most homeowners Lachnospiraceae, for further characterization with other install septic tanks to discharge septic tank effluents genetic methods such as oligotyping (McLellan et al. ). (STEs) into the underlying soils through tile beds. Many of Despite the decreasing cost and increasing usefulness of the plots have an area too small to install functioning next-generation sequencing methods to identify fecal con- septic tanks to current building standards. Besides the tamination sources, MST protocols have not yet, however, potential for leaking, concentrated raw sewage seeps integrated next-generation DNA sequencing as a standard through the underlying bed and into aquifers that supply for monitoring Canadian drinking water sources. wells (Niagara Region Report ). Wainfleet, a rural Ontario Township by Lake Erie, is under the longest active boil water advisory in Canada. A previous monitoring study on fecal contamination in Groundwater tap sample collection 280 private residential groundwater wells determined that 50% contained detectable FIBs (Niagara Region Report We identified nine test sampling sites (Sites A–K, Figure 1) ). The town’s boil water advisory provides an opportu- and 21 wells based on FIB detection in the previous inde- nity to test next-generation sequencing DNA methods in pendent study (Niagara Region Report ). We received MST monitoring. Combining next-generation DNA sequen- written consent from the township and identified volunteers cing approaches with traditional MST-based methods can based on the sampling site selection process. We collected augment current water quality monitoring approaches by tap water samples every month from April to November incorporating new fecal-specific DNA markers to quantify 2015 and grouped the samples based on season (spring, host-specific contamination. summer, and fall). A total of 48 samples were collected Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021 | | | 395 P. Naphtali et al. Tracing groundwater fecal contamination through high-throughput sequencing Journal of Water and Health 17.3 2019 Figure 1 | Location of sampling sites. from nine test sites. For each sampling event, town technical of well water into 100 mL of 1× PBS solution (pH 7.0), to staff collected groundwater tap samples from homeowners a10 -fold dilution. One hundred mL of the well water by filling 500 mL autoclaved plastic bottles (Nalgene). The sample and PBS diluted samples were passed through water samples were then kept on ice, transported to the 0.45 μm pore-size 47-mm-diameter sterile mixed cellulose laboratory, and processed within 6 h of collection. ester membrane filters (Thermo Fisher Scientific, Burling- ton, ON, Canada). Each membrane filter was placed on differential coliform (DC, Oxoid) and mEI agar (BD Septic tank effluent and manure sample collection Difco) plate and incubated at 42 C for 24 h. After incubation, E. coli and Enterococcus spp. colony forming STEs were collected from two septic tanks owned by two units (CFUs) for filters containing stock and diluted well homeowners that participated in this study (fall 2015). water samples were enumerated. To determine the concen- Three biological replicates for each sewage sample were tration of FIBs (CFU/mL) in water samples, we multiplied collected on three consecutive days. Manure samples were the CFU count by its associated dilution factor. Detection collected from manure mounds stored by a concentrated of one (or more) CFU per 100 mL of drinking water samples animal feeding operation for chickens, a cow farm, a horse were considered positive for FIBs (deemed unsafe for hobby farm, and a pig farm. Similar to septic samples, drinking (Health-Canada )). three biological replicates for each manure type were collected on three consecutive days. All samples were transferred into 500 mL autoclaved bottles, kept in ice DNA extraction and library generation for sequencing and transported to the laboratory. The samples were then stored at –80 C until further analysis. DNA was extracted from water samples as described earlier (Mohiuddin et al. ). Briefly, 500 mL of each ground- FIB detection assay water sample was passed through 0.45-m pore-size 47-mm- diameter sterile mixed cellulose ester membrane filters. FIBs in water samples were detected using standard methods The filters were then cut into fragments (1 cm size) with (APHA ). To enumerate E. coli and Enterococcus spp., sterile scissors and the cut fragments were aseptically trans- 100-fold serial dilutions were prepared by transferring 1 mL ferred with sterile forceps into 1.7 mL microfuge tubes for Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021 | | | 396 P. Naphtali et al. Tracing groundwater fecal contamination through high-throughput sequencing Journal of Water and Health 17.3 2019 DNA extraction. DNA was extracted from the filters using a library were checked using BioAnalyzer and High Soil DNA Isolation Kit (Norgen Biotek, Thorold, ON, Sensitivity DNA Kit (Agilent, Mississauga, ON, Canada). Canada) according to manufacturers’ instruction and iso- Each library was then quantified by qPCR and sequencing lated DNA was stored at –20 C for further analysis. DNA was performed on the Illumina MiSeq platform which quantity and purity were measured using the Nanodrop generated 2 × 300 bp paired-end sequences. 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Burlington, ON, Canada). Manure samples stored at –80 C Processing and analysis of 16S rRNA gene sequences were taken out overnight to thaw the samples and 200 mg of thawed samples were used for DNA extraction using a Stool Paired-end sequences generated by Illumina MiSeq were de- Nucleic Acid Isolation Kit (Norgen Biotek, Thorold, ON, multiplexed and quality-filtered using methods described Canada). Similar to manure samples, septic tank samples earlier (Bokulich et al. ; Mohiuddin et al. ). Only were taken out overnight to thaw the samples and 10 mL full amplicons with 464 bp sequences were considered for of the thawed samples were centrifuged at 10,000× g for further analysis. Before downstream analysis, all sequences 10 min at 4 C. After centrifugation, the pellet was resus- were trimmed using a quality score threshold (Q score) of pended with resuspension buffer provided with Stool 25 over at least 75% of the sequence read. Sequences that Nucleic Acid Isolation Kit (Norgen Biotek, Thorold, contained ambiguous bases, errors in barcode sequences, ON, Canada) and DNA was extracted according to >10 consecutive low-quality base pairs and >2 nt mis- manufacturers’ instructions. matches from the primer sequences were also removed After DNA extraction, PCR assays were conducted to (Bokulich et al. ). Processed sequence reads were then amplify the V3–V4 region of the 16S rRNA gene using analyzed using software package QIIME v.1.9.0 (Caporaso an optimized primer pair (S-D-Bact-0341-b-102 S-17/S-D- et al. ). Sequences were clustered into Operational Bact-0785-a-A-21) evaluated elsewhere (Klindworth et al. Taxonomic Units (OTUs) sharing 97% nucleotide sequence ). Reaction mixes of 25.0 μL were prepared as follows: identity and a minimum query alignment length of 50% to 2þ 2.5 μL10× PCR buffer minus Mg (Invitrogen, Burlington, the Greengenes 2013 reference database (McDonald et al. ON, Canada), 0.5 μL of 100 mM dNTP solution, 1.0 μL ) with a sequence similarity threshold of 97% and a each of the forward and reverse primer (1.0 μM each) with minimum query alignment length of 50% using UCLUST unique Illumina adapter sequences attached to them, (Edgar ). The resulting OTU table was then rarified 1.0 μL 10 mg/mL UV-treated BSA solution, 0.75 μL25 mM to 5,000 OTU counts/sample before statistical analyses MgCl solution (Invitrogen, Burlington, ON, Canada), were performed. A potential DNA marker of a human or 0.25 μL Taq DNA Polymerase (Invitrogen), 2.0 μL of DNA animal-specific fecal or manure contaminant was identified template, and 16.0 μL ddH O. The target DNA was ampli- if it was detected in 10% abundance in a fecal sample but fied using the T100 Thermal Cycler (Bio-Rad, Mississauga, 1% abundance in all other fecal samples. The relative ON, Canada) and conducted as follows: Initial denaturation abundance of the potential markers was determined in the at 95 C for 3 min, 40 cycles of denaturation at 94 C for groundwater samples across the sampling months. 30 s, annealing at 50 C for 30 s, and elongation at 72 C for 1 min, and a final extension step at 72 C for 10 min. Preparation of DNA standards for quantification of All PCR products were examined in 0.9% agarose gels, human fecal pollution the desired band was cut from the gel and DNA was extracted from the cut band with the Nucleospin PCR and The HF183 primer pair (HF183F and Bac708R) (Bernhard Gel Cleanup Kit (Macherey-Nagel, Bethelhem, PA, USA). & Field ) that flanks the fragments in the 16S rRNA All amplicon extracts were pooled in equal mass amounts gene of human Bacteroidales populations was used to and sent to the Farncombe Metagenomics Facility at prepare standards for quantification of human-based fecal McMaster University for further processing and sequencing. pollution. First, a conventional PCR assay was performed Product size and the presence of primer dimers in each to generate a 500 bp product. The amplified product was Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021 | | | 397 P. Naphtali et al. Tracing groundwater fecal contamination through high-throughput sequencing Journal of Water and Health 17.3 2019 then cloned into a vector which served as standard in qPCR (Bio-Rad), and 0.5 μL each of the forward and reverse assay. For conventional PCR, 50.0 μL reaction volumes were primer (10 μM). Thermal cycling conditions included the fol- prepared. The reaction mix contained 5.0 μL of Thermopol lowing steps: Initial denaturation at 95 C for 30 s, 40 cycles Buffer (10 × , NEB), 1.0 μL (10.0 μM each) each of the for- of denaturation at 95 C for 30 s and 60 C for 10 s, and then 0 0 ward (HF183F: 5 -ATCATGAGTTCACATGTCCG-3 ) and a melt curve analysis from 65 to 95 C at increments of 0 0 reverse primer (Bac708R: 5 -CAATCGGAGTTCTTCGTG-3 ), 0.5 C for 5 s per increment. The CFX Analyzer (Bio-Rad, 1.0 μL of dNTP mix (NEB, 10 mM), 1.0 μLof5 U Taq Poly- Mississauga, ON, Canada) was used to generate a standard merase (NEB), and 2.0 μL of DNA template. All PCR assays curve and a melt curve and quantify qPCR copy numbers. were run with the conditions as follows: initial denaturation for 5 min at 95 C, 35 cycles of denaturation at 95 C for 30 s, annealing at 52 C, and extension at 72 C for 1 min, RESULTS and a final extension step at 72 C for 6 min. PCR products were loaded onto 1% agarose gels and amplicons were Detection of FIB in groundwater wells extracted from the gel using the Nucleospin PCR and Gel Cleanup Kit (Macherey-Nagel, Bethelhem, PA, USA). As a preliminary assessment of fecal contamination levels To generate plasmid DNA standards, the TOPO TA Cloning in Wainfleet’s well waters, we examined FIB detection Reaction (Invitrogen, Burlington, ON, Canada) was used. frequency in tap water collected from private wells using Briefly, 2.0 μL of the amplicons were ligated into the Health Canada recommended guidelines (see above under TOPO vector, according to the manufacturer’s instructions. ‘Methods’). Sixty-one per cent of the groundwater samples Transformants were isolated using blue-white screening collected from 21 wells within the boil water advisory in technique and subsequently purified. Purified colonies 2015 contained either E. coli or Enterococcus (Figure 2). were then inoculated into 3.0 mL of LB broth supplemented with 50.0 μg/mL kanamycin for overnight incubation at 37 C with shaking at 220 rpm. DNA was extracted from 1.5 mL aliquots of the cultures with a Plasmid DNA Miniprep Kit (Norgen Biotek, Thorold, ON, USA). DNA concentrations were quantified using the Qubit Fluorometer 2.0 (Invitrogen, Burlington, ON, USA). DNA extracts were 1 7 diluted such that a range of 10 –10 plasmid copies of the marker was present in the standards. Quantification of human Bacteroidales marker in sewage and groundwater samples Quantification of human specific Bacteroidales marker was performed using the same forward primer used for the conventional PCR (HF183F) and a different reverse primer 0 0 (5 -TACCCCGCCTACTATCTAATG-3 )(Seurinck et al. ). The newly amplified product had a length of 82 bp. qPCR assays were conducted using 2.0 μL of groundwater DNA extracts and plasmid standard in 10.0 μL reaction mix volumes. All DNA templates were diluted 10-fold to mini- mize qPCR inhibition. Each reaction mix contained 1.0 μL Figure 2 | Detection of E. coli and Enterococcus in well water samples (n ¼ 48) at of DNA template, 5.0 μL of the SsoFast EvaGreen SuperMix locations in the boil water advisory zone in 2015. Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021 | | | 398 P. Naphtali et al. Tracing groundwater fecal contamination through high-throughput sequencing Journal of Water and Health 17.3 2019 Positive E. coli detection ranged from 60.0% to 70.0%. Identification of potential STE and animal manure Enterococcus detection rates had a large seasonal range contamination markers from 10.0% to 81.0%, with the detection rate lowest in the spring and highest in the summer (Figure 2). Groundwater To identify potential fecal contamination markers in ground- samples collected from two residential wells, B and K, also water, we parsed for sewage and manure-associated bacteria contained much higher mean E. coli and Enterococcus in the 16S rRNA sequencing data. We defined a host-specific than the other groundwater wells (Figure 3). fecal marker as a microbial group detected at 10.0% rela- tive abundance in one type of fecal matter but detected at 1.0% abundance in the other fecal sources. In the two Basic sequencing data STE samples, the mean abundance of Campylobacteraceae was 32.5% (Figure 4). At the genus level, sequences anno- As a first step towards identifying potential fecal contami- tated to Sulphospirillum and Arcobacter were the most nation sources in the FIB-positive well waters, we profiled abundant members of Campylobacteraceae. In contrast, the microbial communities from 21 groundwater samples, average abundance of Campylobacteraceae was 1% for all six samples from two STEs, and 12 manure samples from four animal manure samples. Gallicola and Turicibacter four different animal farms using 16S rRNA sequencing. were the most abundant genera in chicken and cow Sequence reads obtained from septic tank samples were manure, comprising 42.2% and 9.4% of 16S rRNA sequences classified into 972 (±89) OTUs. The number of reads respectively. In contrast, these markers were detected at obtained from animal manure and groundwater samples 1.0% abundance among the pig and horse manure and were higher than septic tank samples and reads from STE samples (Figure 4). No genetic marker was identified animal manure and groundwater wells were classified into in pig and horse manure based on the classification criterion. 2,987 (±315) and 3,101 (±287) OTUs respectively. Abundance of STE and manure-based OTUs in groundwater samples To identify possible fecal contaminants in well water, we determined the relative abundance of the human and Figure 4 | Relative abundance of potential manure and sewage markers in waste samples collected in Wainfleet. Error bars represent standard deviation of Figure 3 | E. coli and Enterococcus levels in wells waters B and K compared with the the mean. Three biological replicates for each of the samples was used to other well water sites. Error bars represent standard deviation of the mean. calculate mean and standard deviation. Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021 | | | 399 P. Naphtali et al. Tracing groundwater fecal contamination through high-throughput sequencing Journal of Water and Health 17.3 2019 Figure 5 | Relative abundance of potential STE and animal-specific contamination Figure 6 | Presence of human Bacteroidales marker in selected septic tank and markers in well water sites within the active boil water advisory zone. groundwater wells within the boil water advisory zone in Wainfleet. Error bars Error bars represent standard deviation of the mean. represent standard deviation of the mean. animal-associated markers we identified in the reference wells without E. coli detection also tested positive for the fecal samples. Groundwater wells sampled in July, HF183 marker. Groundwater well A, treated with UV light September, and November had the highest relative abundance before sampling, contained more HF183 copies than other of STE-based Campylobacteraceae sequences (Figure 5). None groundwater wells (Figure 6). Groundwater well I had a of the chicken and cow markers had a relative abundance slightly lower HF183 marker level than B, F, and K. above2.0%, with the relativeabundance below1.0%in wells collected in July, August, and November (Figure 5). DISCUSSION Identification of human fecal contamination The presence of FIBs in drinking waters is a major health concern. Using national guidelines for drinking water and To determine whether the human-specific fecal contami- the results of a previous study as a reference, we sought to nation was present in the groundwater samples, we determine whether high levels of FIBs are still present in conducted qPCR assays of the HF183 human Bacteroidales the Wainfleet well waters. Our analyses suggest that FIBs marker in selected wells with or without FIB detection. We are still present in over half of the tested well water sites prepared a qPCR standard curve that quantifies the human and, therefore, the quality of drinking water may still be a Bacteroidales marker in each run (Figure S1A). Each standard concern in the majority of the households within the town- curve was robust for HF183 quantification (Figure S1, avail- ship. Many of the sampled groundwater wells are located able with the online version of this paper). Melt curve near the shores of Lake Erie, a low-lying region where analysis of the standard curve and all groundwater samples most of the E. coli and total coliform exceedances were pre- that were examined after the qPCR assay yielded a single viously observed (Niagara Region Report ). Some well peak at 84 C (Figure S1B). water samples also contained far higher FIB counts than The HF183 marker genome was more abundant in others. Mean E. coli and Enterococcus levels in some STE samples than in groundwater wells (Figure 6). qPCR groundwater wells were three and two orders of magnitude of the human Bacteroidales marker in groundwater wells higher than the average FIB counts for the groundwater B, F and K resulted in positive detection, containing 30–50 wells collected across all the other locations. These wells genome copies/100 mL (Figure 6). A subset of groundwater with higher than average FIB counts are located near Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021 | | | 400 P. Naphtali et al. Tracing groundwater fecal contamination through high-throughput sequencing Journal of Water and Health 17.3 2019 septic tanks that may leak raw sewage into the aquifer (Nia- were also present in other manure types. However, analyses gara Region Report ). Poor well maintenance also of additional samples that include both biological and facilitates sewage leaching into the groundwater (Howard technical replicates are required to confirm the findings of et al. ). Altogether, chronic FIB contamination in this proof-of-principle study. A higher sequencing depth, in individual groundwater wells like B and K require further conjunction with shotgun metagenomic sequencing, may investigation to confirm potential fecal contamination sources. facilitate the identification of novel or rare (but nonetheless important) taxa that can be used to identify contamination Characterization of fecal microbiota and identification at the species level. DNA markers unique to horse or pig of potential fecal markers manure are yet to be established. The robust resolution of horse and pig-associated markers with next-generation The presence of host-associated fecal contamination sequencing represents opportunities for future investigation. may help to trace fecal contamination sources in complex environmental samples. As a proof-of-principle experiment, Fecal source identification in residential groundwater we examined whether we could complement FIB detection sites using 16S rRNA sequencing with 16S rRNA sequencing methods by identifying sewage and manure-associated markers. We selected a maximum The co-detection of animal and/or human associated mar- detection rate of 1.0% limit to ensure the specificity of the kers in drinking water provides evidence of possible fecal marker to its host. In cow manure, we identified Turicibacter contamination by that source. After identifying human and spp., a member of the Firmicutes phylum present in animal-associated markers in our reference fecal samples, high abundance at the genus level, agreeing with previous we screened for possible fecal contamination in private cow microbiome profiles (Kim et al. ). In chicken well waters by profiling the well water microbial commu- manure, Gallicola – also a member of the Firmicutes nities. The mean Campylobacteraceae relative abundance phylum – was the most abundant genus. Both markers was highest in November and September, but lowest in were also detected at 0.1% abundance in other manure August. The large standard deviations in Campylobactera- types and sewage samples. These genera may act as DNA ceae abundances within the sampling months suggest that markers of host-associated fecal contamination in the some groundwater sites contain more fecal contamination town’s groundwater wells. than others. The relative abundance of Campylobacteraceae In the two STE sites, we identified the Campylobactera- sequences still far exceeded the relative abundance of ceae family (member of the Proteobacteria phylum) as fecal chicken and cow fecal markers, Gallicola and Turicibacter. pollution marker, comprising 32.5% of 16S rRNA gene While we cannot exclude the possibility of cow or chicken sequences at the family level. The predominance of farm contamination in September and October, the higher Campylobacteraceae 16S rRNA gene sequences in STEs is mean relative abundance of Campylobacteraceae across identified in other septic tank systems (Tomaras et al. the groundwater wells in every month except August raises ). Although we only collected STEs from two septic the likelihood of STE contamination in the residential tanks, both septic tank microbiomes contained a similar well water. abundance of Campylobacteraceae sequences. Campylobac- Interestingly, well waters collected in August contained teraceae was also absent in the animal manure samples, the lowest abundance of the three host-associated markers. suggesting that the Campylobacteraceae microbes are likely Except for well water site K, E. coli counts did not exceed exclusive to STEs in the township. 100 CFUs/100 mL (raw data), suggesting diffuse fecal While we identified host-associated markers for chicken contamination among the well water samples collected manure, cow manure, and STEs, we could not establish the in August. In other sampling months, there was a far presence of host-associated markers for horse and pig higher abundance of Campylobacteraceae sequences in manure. Although Clostridium and Turicibacter were more the autumn. The detection of E. coli counts could be due abundant in horse and pig manure samples, these sequences to leaking septic tanks from individual sites. Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021 | | | 401 P. Naphtali et al. Tracing groundwater fecal contamination through high-throughput sequencing Journal of Water and Health 17.3 2019 Quantification of human-based fecal contamination presence of DNA from dead (or inactivated) Bacteroidales (inactivated through UV treatment) which was amplified To confirm the presence of STE contamination in selected during the qPCR assay. Other groundwater wells, like G well sites, we used the HF183 marker to quantify human- and J, did not contain a detectable HF183 marker, removing based contamination in DNA extracted from STE and the possibility of STE contamination in those wells. Cultur- groundwater samples. We first validated the use of the able FIBs were also absent in G and J, corroborating the HF183 marker by preparing standard and melt curves of absence of STE-based contamination. the HF183 qPCR assay. Although qPCR assays were con- ducted with 100 bp fragments to minimize spurious fluorescence, we observed high R value and robust CONCLUSIONS E-value, suggesting the quality of the amplification reactions (Figure S1A). Furthermore, the melt curve analysis shows A prior study found extensive FIB contamination through- that a single product was detected by the qPCR assay out a rural community (Niagara Region Report ). In (Figure S1B). Furthermore, we detected the HF183 marker this study, we complemented cultured-based methods with in the two STE sites at 10-fold higher concentration than 16S sequencing and qPCR to re-assess fecal contamination groundwater samples. These results validate quantifying levels and, further, to identify potential contamination the entire HF183 amplicon in DNA extracted from the sources in Wainfleet. We found FIB contamination in the town’s well water as a proxy of STE contamination. well waters we tested, with E. coli counts as high as We also detected the HF183 DNA marker in B and K, 10 CFU/mL. In addition, the identification of additional groundwater wells containing the highest E. coli CFUs. STE DNA markers to Campylobacteraceae and the human Curiously, groundwater wells I and F, which did not contain Bacteroidales is consistent with the idea that human waste E. coli, were also positive for the HF183 marker. A may impact residential groundwater wells. The low abun- weak correlation between E. coli counts and HF183 dance (and absence) of animal manure-associated DNA marker concentrations was established in residential areas markers – Turicibacter in cow manure and Gallicola in (Nshimyimana et al. ) and the Great Lakes beach chicken manure – reduces the possibility that observed sands (Staley et al. ). The weak correlation (Pearson cor- contamination is due to animal sources. These results relation; r ¼ 0.33) between E. coli counts and HF183 marker indicate that profiling microbial communities using 16S concentrations indicates that E. coli levels are not a reliable rRNA sequencing can augment culture-based methods in indicator of human fecal contamination in well waters. This contamination analysis studies. The use of next-generation is primarily due to the factors that affect viability of E. coli sequencing methods can specifically facilitate the assess- in well water and contamination of well waters through ment of groundwater quality by detecting host-associated sources other than humans. Within Wainfleet, groundwater markers and quantifying relative contributions of likely wells B and K may receive E. coli from Lake Erie where fecal sources to groundwater contamination. surface water flows into the aquifer. However, these ground- To trace fecal contamination sources in water sources water wells also receive human contamination loads that more robustly, amplicon sequencing at higher depths may come from leaking septic tanks, evidenced by the (more reads per sample) may be useful in identification of positive HF183 detection in septic tanks and the wells. novel or rare taxa. Shotgun metagenomic sequencing Interestingly, the groundwater well sample collected from may also be used which, at a higher depth, may facilitate well A contained almost five times the HF183 marker concen- the identification of FIB at the species level. While deeper tration as groundwater wells B and K. Well A’s homeowner sequencing depth may facilitate species identification, such installed a UV treatment system in residence to inactivate as the Campylobacter spp. that comprise STEs and possible fecal microbes. While E. coli and Enterococcus were absent pig and horse-associated markers within Clostridium and in the UV treated well water, the HF183 marker was still pre- other genera, the cost of such in-depth analyses are fairly sent in UV treated well water. This could be due to the high. Highly focused DNA sampling programs that target Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021 | | | 402 P. Naphtali et al. Tracing groundwater fecal contamination through high-throughput sequencing Journal of Water and Health 17.3 2019 Harwood, V. J., Staley, C., Badgley, B. D., Borges, K. & Korajkic, well water sites having high FIB levels may allow detailed A.  Microbial source tracking markers for detection of identification contamination inputs that include potential fecal contamination in environmental waters: relationships pathogens that, unlike E. coli, are difficult to culture. between pathogens and human health outcomes. FEMS Microbiol. Rev. 38 (1), 1–40. Health-Canada  Guidance on the Use of the Microbiological Drinking Water Quality Guidelines. Water and Air Quality ACKNOWLEDGEMENTS Bureau, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario, Canada. We thank members of the Schellhorn Lab for their help Howard, G., Pedley, S., Barrett, M., Nalubega, M. & Johal, K.  Risk factors contributing to microbiological contamination of and comments on the manuscript. We thank Trevor shallow groundwater in Kampala, Uganda. Water Res. 37 Imhoff (Wainfleet Township) for advice and support of (14), 3421–3429. this project. We gratefully acknowledge the financial Kim, M., Morrison, M. & Yu, Z.  Status of the phylogenetic support through the Niagara Region WaterSmart Program, diversity census of ruminal microbiomes. FEMS Microbiol. 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First received 3 December 2018; accepted in revised form 14 January 2019. Available online 1 March 2019 Downloaded from http://iwaponline.com/jwh/article-pdf/17/3/393/639178/jwh0170393.pdf by guest on 21 September 2021

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