CrAssphage as a Potential Human Sewage Marker for Microbial

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CrAssphage as a Potential Human Sewage Marker for Microbial Source Tracking in Southeast Asia Akechai Kongprajug, Skorn Mongkolsuk, and Kwanrawee Sirikanchana Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.9b00041 • Publication Date (Web): 14 Feb 2019 Downloaded from http://pubs.acs.org on February 14, 2019

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Environmental Science & Technology Letters

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CrAssphage as a Potential Human Sewage Marker for Microbial Source

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Tracking in Southeast Asia

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Akechai Kongprajug †, Skorn Mongkolsuk †,§, and Kwanrawee Sirikanchana †,§*

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Research Laboratory of Biotechnology, Chulabhorn Research Institute, Bangkok, Thailand

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†

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10210

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§

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Education, Bangkok, Thailand 10400

Center of Excellence on Environmental Health and Toxicology, CHE, Ministry of

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*Corresponding author: Research Laboratory of Biotechnology, Chulabhorn Research Institute,

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54 Kampangpetch 6 Road, Laksi, Bangkok, Thailand 10210. Phone: +66 2553 8555 ext 8369.

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Fax: +66 2553 8572. Email: [email protected]. ORCID ID 0000-0001-7273-4060

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ABSTRACT:

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The human gut bacteriophage crAssphage has been proposed as a human-specific

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microbial source tracking (MST) marker for impacted water bodies. However, its global use as a

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human-specific MST marker requires validation in a tropical region. In this study, a crAssphage

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qPCR marker (CPQ_056) was detected in 21 sewage samples in Thailand with 100% sensitivity.

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The marker was detected in sewage from hospitals and residential buildings at 5.28–7.38 log10

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copies/100 mL and in four influent and four effluent samples of municipal wastewater treatment

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plants at 4.23–6.19 and 3.78–4.89 log10 copies/100 mL, respectively. Furthermore, a 99.2% 1 ACS Paragon Plus Environment


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specificity (n=127) was observed using feces from swine, cattle, chicken, duck, goat, sheep,

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buffalo, and fish, with cross-detection only occurring for one composite swine sample. The

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crAssphage marker was present in 56.25% (27 out of 48) of river samples at 3.20–7.29 log10

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copies/100 mL. The concentrations of the crAssphage marker and a prevalidated human-specific

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Bacteroidales marker (HF183/BFDrev) did not differ significantly in any of the sewage or

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wastewater samples, whereas the crAssphage marker abundance was higher in river samples.

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This initial validation of the crAssphage gene as a human-specific MST marker in Southeast

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Asia will promote its inclusion in an MST toolbox.

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INTRODUCTION

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Microbial source tracking (MST) is a form of microbial marker detection used in polluted

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water bodies to identify specific sources of fecal pollution, e.g., human sewage, animal manure,

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or bird droppings. The ability of MST to differentiate pollution sources has proved useful for the

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water resource management and water quality restoration of many impaired water bodies.1,2

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Human sewage contamination, either by direct or indirect disposal, poses a particularly high risk

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to public health.3–5 However, because discrepancies in the performance of human-specific

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markers have been observed in different geographical areas, regional validation of MST assays is

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required prior to their application.6,7

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CrAssphage is a bacteriophage that was first discovered in human fecal metagenomes8

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from multiple samples using the novel cross-assembly (crAss) approach.9 Because of the high

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abundance of crAssphage in the human gut, its potential as a human-specific MST marker was

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initially evaluated using metagenomic approaches10 and later with molecular methods.11–14 Two

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crAssphage markers, CPQ_056 and CPQ_064, were reported to be detected at equal abundance

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in sewage and environmental waters12 and to show strong correlation in environmental

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samples.14,15 Both markers have been monitored in limited geographical areas, including the

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USA and Australia.12–16 To encourage the use of these novel markers for the global application

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of MST, validation studies are needed, especially in regions with different climates. The

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objectives of this study were to 1) evaluate the performance of a crAssphage marker (i.e.,

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CPQ_056) compared with the validated HF183/BFDrev assay17 for sewage-specific markers in

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Thailand and 2) compare the abundances of both markers in sewage samples, municipal

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wastewater treatment influents and effluents, and polluted environmental samples.

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MATERIALS AND METHODS

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Sample Collection and DNA Extraction. Nonhuman fecal and human sewage samples were

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collected in the central region of Thailand in the Chao Phraya and Tha Chin river basins as

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previously described.6 One hundred twenty-seven composite nonhuman fecal samples were

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collected from freshly excreted feces (within 2 h after excretion) on the ground of agricultural

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farms for the following types (n; number of composite samples prepared by onsite manual

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agitation of approximately one gram of feces from at least 20 individuals): swine (n=39), cattle

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(n=35), chickens (n=21), ducks (n=5), goats (n=10), sheep (n=4), and buffaloes (n=6). Fish fecal

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samples (n=7) were collected from floating feces in river fish cages and experimental fish tanks

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from May to August 2018, while the other animal samples from agricultural farms were

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collected from January to September 2016. One hundred milliliters of raw sewage (n=21) was

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collected by grab sampling from August to October 2018 from influent sumps of residential

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buildings with at least 100 residents and from hospitals with at least 80 inpatient beds. Up to two 3 ACS Paragon Plus Environment


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liters of municipal wastewater treatment plants influents (WWTPinf; n=4) and effluents

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(WWTPeff; n=4) from the Chon Buri Province was collected by grab sampling during November

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2018. The Tha Chin river is among the five most deteriorated rivers in Thailand, with total

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coliforms (TC) and fecal coliforms (FC) ranging from 102.4 to 106 most probable numbers

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(MPN)/100 mL.18 Four sampling events from twelve sampling stations on the Tha Chin river

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(n=48) were performed at 30 cm below the water surface (Table S1) from July 2017 to February

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2018 as previously described.17 Field blanks, which were sterile demineralized water processed

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in the field, together with field duplicates, were also collected. The sewage, wastewater, and

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river samples were preacidified and filtered with 0.45-µm-pore-size HAWP membranes (Merck

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Millipore, Germany), and DNA extraction was performed using a Quick-DNA Fecal Soil

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Microbe Miniprep kit (Zymo Research, USA) as previously described.6,17 The DNA extracts

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were stored at -80 °C until use. Notably, the WWTPinf samples were centrifuged to separate the

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supernatant and sediment portions, prior to DNA extraction from sediment and supernatant-

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filtered membranes. Because DNA extracts from both the sediment and supernatant portions

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were derived from a similar volume of original wastewater, direct comparisons of markers in

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both portions were performed.

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Water Quality Parameters. The following physicochemical water quality parameters of river

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samples were measured as previously described

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suspended solids (TSS),20 total dissolved solids (TDS),21 dissolved oxygen (DO),22 phosphate

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phosphorus,23 and total phosphate.24 A membrane filtration method was used to detect fecal

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indicator bacteria (FIB), i.e., TC and E. coli,25 and enterococci.26 A multiple tube technique was

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also performed to evaluate TC

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biochemical oxygen demand (BOD),19 total

and FC.28 A double-layer agar assay was used to assess the


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presence of bacteriophages of enterococci AIM06 and SR14 as human sewage-specific MST

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markers.29,30

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qPCR Assays. Primers and probes for qPCR assays are shown in Table S2. The qPCR protocol

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was conducted according to the MIQE guidelines.31 For nonhuman feces and human sewage

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samples, a 20-µL reaction mixture comprised 0.8 µL of each 10 µM forward and reverse primer,

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0.4 µL of 10 µM probe, 5 µL of DNA template (normalized to 20 ng of total DNA), 10 µL of 2×

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iTaq Universal Probes Supermix (Bio-Rad, USA), and 3 µL of sterile distilled water. For river

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samples, each 20-µL reaction mixture was composed of 0.8 µL of each 10 µM forward and

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reverse primer, 0.4 µL of 10 µM probe, 4 µL of DNA template (normalized to 40 ng of total

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DNA), 10 µL of the 2× iTaq Universal Probes Supermix, and 4 µL of 1 µg/µL bovine serum

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albumin (BSA). All qPCR reactions were performed using an ABI StepOnePlus Real-Time PCR

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System (Applied Biosystems, Thermo Fisher Scientific, USA) with the following steps: initial

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denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 20 s and a

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combined annealing and elongation step at 60 °C for 1 min. All DNA reaction were performed in

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duplicate. Cq values were averaged for subsequent analysis when both values had standard

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deviations of no more than 0.5, otherwise additional runs were conducted. Positive and no-

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template controls (NTCs) were included in every instrumental run, as were extraction blanks, in

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which sterile RO water was processed through a Quick-DNA Fecal Soil Microbe Miniprep kit

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(Zymo Research). For samples showing inhibition as identified using the dilution method, 10-

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fold dilutions were used.6,17 Cloning and sequence analysis for positive qPCR results were

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performed as previously described.6

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qPCR Standard Curves. Synthetic DNA standards were used to generate standard curves as

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previously described for the GenBac3 and HF183 qPCR assay.17 For the crAssphage marker, 5 ACS Paragon Plus Environment


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standard plasmids were designed according to the crAssphage genomic region (GenBank

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accession number JQ995537) at position 14731−14856.12 The target sequence was inserted in the

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pMA-T plasmid by Invitrogen (USA). DNA concentrations ranging from 5 × 101 to 5 × 106 gene

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copies per reaction, as measured with a NanoDrop 2000 spectrophotometer (ThermoFisher

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Scientific), were prepared for a standard curve. Four individual instrumental runs, were

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performed in which each reaction was conducted in triplicate, and a mixed model was used to

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calculate the number of DNA copies.17,32 Analysis of covariance (ANCOVA) also revealed

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nonsignificant differences in slopes among these instrument runs (p>0.05). All statistical

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analyses were conducted in R33, and are described in Supporting Information File 1. Assay limits

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for qPCR, including limit of detection (LOD), and limit of quantification (LOQ), were also

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explained in Supporting Information File 1.

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RESULTS AND DISCUSSION

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qPCR Validation using Nonhuman Fecal Composites and Untreated Sewage Samples. The

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standard curve characteristics and assay limits for crAssphage and HF183 assays are shown in

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Table S3. CrAssphage showed higher performance criteria values (i.e., specificity, sensitivity,

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accuracy, and positive and negative predictive values) than HF183 for tracking pollution from

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human sources in Thailand (Table 1). Cross-detection of the crAssphage marker was observed

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for one swine fecal sample. Previous studies have reported false-positive results using feces from

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poultry, gulls, dogs, and cats and cattle wastewater.12–14 Although this study did not include dog,

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cat, and gull fecal samples in the specificity testing, gulls are not prevalent in the Tha Chin and

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Chao Phraya watersheds, with only migrating gulls present a during specific periods. The log10

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concentrations of both markers in sewage samples followed a normal distribution (Shapiro-Wilk 6 ACS Paragon Plus Environment

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test, p>0.05) and showed no significant differences (paired t test; p>0.05) (Figure 1 and Table

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S4).

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Although a lower abundance of crAssphage sequences has been reported in human

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sewage metagenomic databases in Asia than in the USA and Europe,10 a lower sensitivity of

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metagenomic database searches than of the molecular detection of crAssphage was previously

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documented.34 Therefore, this study is the first to determine the crAssphage marker distribution

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in human sewage in Southeast Asia using molecular laboratory techniques. Furthermore, to

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ensure that the comparison of the performance of the prior HF183 assay using freshly extracted

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DNA and that of the present crAssphage assay using similar sets of stored DNA samples from

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nonhuman samples was not biased, the quality of representative DNA samples was tested using

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the universal Bacteroidales (GenBac3) assay (Tables S2 and S3). No significant difference in the

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number of GenBac3 marker copies was observed in the recent and prior qPCR runs from the

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similar sets of DNA extracts up to a maximum storage time of 16 months (Table S5; Wilcoxon

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signed rank test; p>0.05). In addition, the quality controls using extraction blanks showed that no

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contamination occurred during sample processing.

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Abundance and Removal Efficiencies in Wastewater. The abundances of the crAssphage and

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HF183 markers was measured in WWTPinf, including supernatant and sediment samples (n=4

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each), and WWTPeff (n=4) (Table 1; Figure S1). No significant difference between the two

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marker concentrations was observed in each sample (paired Prentice-Wilcoxon test; p>0.05).

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The ratios of the concentrations of WWTPinf in the sediment to supernatant portions were 0.01–

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1.61 and 2.08–7.11 for the crAssphage and HF183 markers, respectively. The removal

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efficiencies of the wastewater treatment plants ranged from no removal (actually increased) to

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99.4% for crAssphage and from 76.54 to >99.9% for HF183. Because the HF183 concentration 7 ACS Paragon Plus Environment


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data set contained one negative sample (i.e., below LOQ), a nonparametric survival analysis

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incorporating nondetected data was performed.35–38 WWTPinf collected by combined sewer

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systems could receive runoff with fecal contamination from nonhuman sources, e.g., birds, stray

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dogs and cats. Therefore, WWTP samples were not considered ‘sole human sewage’ and not

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included in the performance evaluation (e.g., specificity and sensitivity) of the crAssphage and

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HF183 markers (Table 1).

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Abundance in Impacted River Water and Correlation among Parameters. The frequency of

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detection and the abundance of the crAssphage marker in 48 river samples were compared with

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the published data for the HF183 marker17 (Figure 2). Of ten co-occurring samples, only two had

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crAssphage abundances 0–1 log lower than those of HF183, while the remaining showed

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crAssphage marker abundances 1–3 orders of magnitude higher than those of HF183. Overall,

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the abundance of crAssphage was significantly higher than that of the HF183 marker in each

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assayed river sample (paired Prentice-Wilcoxon test; p Cq,LOD). For the HF183 marker, there were 10, 7, and 31 samples (i.e., 20.83, 14.58, and 64.58%, respectively) that were quantifiable, DNQ, and not detectable, respectively. Cq values of technical duplicates were always agreeable. 21 ACS Paragon Plus Environment


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Table 1. Performance Criteria Evaluation and Abundance of the crAssphage and HF183 Markers in this Study Compared with that in Other Studies

Specificitya

crAssphage HF183 crAssphage12 crAssphage15 crAssphage13 crAssphage14 (this study) (this study) 0.992f 0.850g 0.986h NA 0.927i 0.950j

Sensitivityb

1.000

1.000

1.000

NA

1.000

1.000

0.993

0.870

NAk

NA

NA

NA

Positive predictive valued Negative predictive valuee Abundance in sewage (log10 copies/100 mL)

0.955

0.530

NA

NA

NA

NA

1.000

1.000

NA

NA

NA

NA

5.28–7.38; n=21

NA

NA

NA

NA

Abundance in WWTPinf (log10 copies/100 mL)

4.23–6.19; n=4

4.77– 7.65; n=21 3.88– 5.66; n=4

2.49–4.37; n=9

NA

8.08–8.98; n=8

8.43 (average); n=12

Abundance in WWTPeff (log10 copies/100 mL) Abundance in river samples (log10 copies/100 mL)

3.78–4.89; n=4

3.25– 4.51l; n=3 3.13– 4.32m; n=10

NA

NA

NA

NA

1.82–2.20

4.02–6.04; n=30

2.60–3.91; n=24

2.40–5.04; n=19

Accuracyc

3.20–7.29; n=27

aSpecificity

is calculated from true negative/(true negative + false positive) is calculated from true positive/(true positive + false negative) cAccuracy is calculated from (true positive + true negative)/(true positive + false positive + true negative + false negative) dPositive predictive value is calculated from true positive/(true positive + false positive) eNegative predictive value is calculated from true negative/(true negative + false negative) fCross-detection with one swine composite sample at 7.38 log copies/g feces. The 126-bp 10 sequence showed 98% identity (3 mismatches) with the reference sequence JQ9955378 gCross-detection with swine (n=4; 5.5.75–6.18 log copies/g feces), cattle (n=6; 4.98–5.82 log 10 10 copies/g feces), chicken (n=3; 5.45–6.14 log10 copies/g feces), duck (n=3; 5.83–6.28 log10 copies/g feces), goat (n=2; 5.61–6.46 log10 copies/g feces) and sheep (n=1; 5.47 log10 copies/g feces) fecal samples hCross-detection with two gull samples and one dog sample from the USA iCross-detection with six poultry litter composite samples from Tampa, Florida, USA jCross-detection with six cat feces and 2 cattle wastewater samples from Australia kNot available lOne WWTP sample was negative for the HF183 marker (

CrAssphage as a Potential Human Sewage Marker for Microbial

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