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

Kongprajug, Akechai; Chyerochana, Natcha; Somnark, Pornjira; Kampaengthong, Pinida Leelapanang; Mongkolsuk, Skorn; Sirikanchana, Kwanrawee

doi:10.1016/j.ijheh.2019.01.005

Cited by 26 publications

(45 citation statements)

References 33 publications

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“…Thus, their presence in water bodies indicates fecal contamination has occurred from specific host groups. Most commonly used MST tools identify or quantify a DNA sequence(s) or marker gene(s) from the host-associated microorganisms by PCR or quantitative PCR (qPCR) assays (3)(4)(5)(6).…”

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confidence: 99%

Host Specificity and Sensitivity of Established and Novel Sewage-Associated Marker Genes in Human and Nonhuman Fecal Samples

Ahmed

Gyawali

Feng

et al. 2019

Appl Environ Microbiol

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Microbial source tracking (MST) methods measure fecal contamination levels and identify possible sources using quantitative PCR (qPCR) that targets host-associated fecal microorganisms. To date, most established MST assays for human sources, especially bacterial markers, have shown some nonhuman host cross-reactions. Recently developed assays, such as the crAssphage CPQ_056, Lachnospiraceae Lachno3, and Bacteroides BacV6-21, have more limited information on host sensitivity and host specificity for human or sewage sources, particularly in countries other than the United States. In this study, we rigorously evaluated six sewage-associated MST assays (i.e., Bacteroides HF183, human adenovirus [HAdV], human polyomavirus [HPyV], crAssphage CPQ_056, Lachno3, and BacV6-21) to show advantages and disadvantages of their applications for MST. A total of 29 human and 3 sewage samples and 360 nonhuman fecal samples across 14 hosts collected from a subtropical region of Australia were tested for marker host specificity, host sensitivity, and concentrations. All sewage samples were positive for all six marker genes tested in this study. Bacterial markers were more prevalent than viral markers in human feces. Testing against animal hosts showed human feces (or sewage)associated marker gene specificity was HAdV (1.00) Ͼ HPyV (0.99) Ͼ crAssphage CPQ_056 (0.98) Ͼ HF183 (0.96) Ͼ Lachno3 (0.95) Ͼ BacV6-21 (0.90), with marker concentrations in some animal fecal samples being 3 to 5 orders of magnitude lower than those in sewage. When considering host specificity, sensitivity, and concentrations in source samples, the HF183, Lachno3, and crAssphage CPQ_056 tests were the most suitable assays in this study for sewage contamination tracking in subtropical waters of Australia. IMPORTANCE Large financial investments are required to remediate fecal contamination sources in waterways, and accurate results from field studies are crucial to build confidence in MST approaches. Host specificity and sensitivity are two main performance characteristics for consideration when choosing MST assays. Ongoing efforts for marker assay validation will improve interpretation of results and could shed light on patterns of occurrence in nontarget hosts that might explain the underlying drivers of cross-reaction of certain markers. For field applications, caution should be taken to choose appropriate MST marker genes and assays based on available host specificity and sensitivity data and background knowledge of the contaminating sources in the study area. Since many waterborne pathogens are viruses, employing both viral and bacterial markers in investigations could provide insight into contamination dynamics and ecological behavior in the environment. Therefore, combined usage of marker assays is recommended for more accurate and informative sewage contamination detection and fecal source resolution. KEYWORDS microbial source trackingCitation Ahmed W, Gyawali P, Feng S, McLellan SL. 2019. Host specificity and sensitivity of established and novel sewageassoc...

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confidence: 99%

Host Specificity and Sensitivity of Established and Novel Sewage-Associated Marker Genes in Human and Nonhuman Fecal Samples

Ahmed

Gyawali

Feng

et al. 2019

Appl Environ Microbiol

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“…The volumes of canal water, WWTP inf , and WWTP eff in each reactor were 500 ml, whereas those of river water and seawater were 1,000 and 1,500 ml, respectively. The discrepancies in volumes among different water types were due to previously setup protocols for monitoring fecal indicators and microbial source trackers, including crAssphage, in our various studies (Kongprajug, Booncharoen, et al, ; Kongprajug, Chyerochana, et al, ; Kongprajug, Mongkolsuk, et al, ). Phosphate buffer solution (PBS; US EPA, ), as a representative of a water matrix containing no interfering substances, was also used to prepare three 500‐ml spiked reactors and one 500‐ml unspiked reactor.…”

Section: Methodsologymentioning

confidence: 84%

“…The spiked reactors were continuously stirred for 10 min. Next, spiked and unspiked reactors were pH adjusted to 3.50 prior to vacuum filtration with a 0.45‐μm‐pore‐size HAWP membrane (Merck Millipore; Kongprajug, Chyerochana, et al, ). Filtered membranes were cut, and DNA extracted using a Quick‐DNA Fecal Soil Microbe Miniprep kit (Zymo Research, Irvine, CA, USA; Somnark, Chyerochana, Kongprajug, Mongkolsuk, & Sirikanchana, ; Somnark, Chyerochana, Mongkolsuk, & Sirikanchana, ).…”

Section: Methodsologymentioning

confidence: 99%

“…Sample processing controls (SPC) for checking matrix interference were performed in all samples with salmon testes DNA (Shanks et al, ). PCR inhibition assessment was carried out with a dilution method using three dilutions of samples at 10, 20, and 40 ng of total DNA, as previously described (Kongprajug, Chyerochana, et al, ). When inhibition was identified, 20 ng of total DNA, instead of 40 ng, was used for qPCR analysis.…”

Section: Methodsologymentioning

confidence: 99%

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Assessing human‐specific CrAssphage recovery after acidification‐filtration concentrating method in environmental water

Kongprajug

Chyerochana

Sangkaew

et al. 2019

Water Environment Research

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

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“…Human-derived pollution poses higher microbial risks than animal wastewaters due to human pathogenic waterborne microorganisms (Soller et al, 2015, 2014). Microbial source tracking (MST) is a research area studying the microorganisms excreted in feces and urine, identifying their association with their host sources, e.g., humans and animals (Ahmed et al, 2020; Kongprajug et al, 2019a; Sirikanchana et al, 2014). Pinpointing pollution sources could lead to effective pollution mitigation and improved risk assessment (Ahmed et al, 2019b; Ballesté et al, 2020; Zhang et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Superior performance of human wastewater-associated viral markers compared to bacterial markers in tropical environments

Sangkaew

Kongprajug

Chyerochana

et al. 2020

Preprint

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Identifying human sewage contamination via microbial source tracking (MST) marker genes has proven useful for effective water quality management worldwide; however, performance evaluations for these genes in tropical areas are limited. Therefore, this research assessed four human-associated MST marker genes in aquatic environments of Central Thailand: human polyomaviruses (JC and BK viruses [HPyVs]), bacteriophage crAssphage (CPQ_056), Lachnospiraceae Lachno3, and Bacteroides BacV6-21. HPyV and crAssphage assays were highly sensitive and specific to sewage (n = 19), with no cross-detection in 120 swine, cattle, chicken, duck, goat, sheep, and buffalo composite fecal samples. Lachno3 and BacV6-21 demonstrated high sensitivity but moderate specificity; however, using both markers could improve specificity to >0.80 (max value of 1.00). The most abundant markers in sewage were Lachno3 and BacV6-21 (5.42-8.02 and non-detected-8.05 log10 copies/100 mL), crAssphage (5.28-7.38 log10 copies/100 mL), and HPyVs (3.66-6.53 log10 copies/100 mL), respectively. HPyVs showed higher levels (up to 4.33 log10 copies/100 mL) and higher detection rates (92.7%) in two coastal beaches (n = 41) than crAssphage (up to 3.51 log10 copies/100 mL and 56.1%). HPyVs were also found at slightly lower levels (up to 5.10 log10 copies/100 mL), but at higher detection rates (92.6%), in a freshwater canal (n = 27) than crAssphage (up to 5.21 log10 copies/100 mL and 88.9%). Overall, both HPyVs and crAssphage are suggested as human sewage-associated MST markers in aquatic environments of Central Thailand. This study underlines the importance of characterizing and validating MST markers in host groups and environmental waters before including them in a water quality management toolbox.

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

Cited by 26 publications

References 33 publications

Host Specificity and Sensitivity of Established and Novel Sewage-Associated Marker Genes in Human and Nonhuman Fecal Samples

Host Specificity and Sensitivity of Established and Novel Sewage-Associated Marker Genes in Human and Nonhuman Fecal Samples

Assessing human‐specific CrAssphage recovery after acidification‐filtration concentrating method in environmental water

Superior performance of human wastewater-associated viral markers compared to bacterial markers in tropical environments

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