Improved simultaneous quantification of multiple waterborne pathogens and fecal indicator bacteria with the use of a sample process control

Zhang, Qian; Ishii, Satoshi

doi:10.1016/j.watres.2018.03.023

Cited by 28 publications

(19 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Nonquantifiable samples or samples with low levels (Ͻ10 3 to 10 4 GC/g) of sewageassociated marker genes found in nonhuman fecal samples should not be seen as a pitfall. This is because in the event of contamination by a confounding source in urban areas, such low levels will be difficult to detect due to loss of marker genes through sample concentration and DNA extraction processes and also dilution factor, which will likely mask their detection (34). Further, individual animals were also sporadically positive, further reducing the likelihood that a detectable signal would interfere with interpretation of results.…”

Section: Discussionmentioning

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

View full text Add to dashboard Cite

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...

show abstract

Section: Discussionmentioning

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

View full text Add to dashboard Cite

show abstract

“…Substantial effort has been devoted in recent years to the development and implementation of standardized protocols and quality control metrics for fecal indicator assessment by qPCR [ 113 , 167 , 168 ]. A notable feature shared by these protocols and other recent recommendations for improved reliability of molecular detection methods is a reliance on numerous controls throughout the procedure [ 128 , 137 ]. While the use of positive and negative controls is standard for most analytical techniques, requirements outlined in standardized qPCR protocols generally include multiple serial dilutions of standard reference material to construct calibration curves, sample processing controls (SPC), method extraction blanks (MEB), internal amplification controls (IAC), and no template controls (NTC), with two or three replicates of each sample, control, and standard dilution series concentration analyzed on each instrument run [ 113 ].…”

Section: Molecular Methods: Challenges and Advancesmentioning

confidence: 99%

Microbial Indicators of Fecal Pollution: Recent Progress and Challenges in Assessing Water Quality

Holcomb

Stewart

2020

Curr Envir Health Rpt

104

View full text Add to dashboard Cite

Purpose of Review Fecal contamination of water is a major public health concern. This review summarizes recent developments and advancements in water quality indicators of fecal contamination. Recent Findings This review highlights a number of trends. First, fecal indicators continue to be a valuable tool to assess water quality and have expanded to include indicators able to detect sources of fecal contamination in water. Second, molecular methods, particularly PCR-based methods, have advanced considerably in their selected targets and rigor, but have added complexity that may prohibit adoption for routine monitoring activities at this time. Third, risk modeling is beginning to better connect indicators and human health risks, with the accuracy of assessments currently tied to the timing and conditions where risk is measured. Summary Research has advanced although challenges remain for the effective use of both traditional and alternative fecal indicators for risk characterization, source attribution and apportionment, and impact evaluation.

show abstract

“…The MF‐qPCR was performed on two technical replicates of DNA from each sample after pre‐amplifying the target genes with a specific target amplification. A more detailed explanation of this method can be found elsewhere (Ishii et al, 2013; Sandberg et al, 2017; Ahmed et al, 2018; Zhang and Ishii, 2018). …”

Section: Methodsmentioning

confidence: 99%

Antibiotic Resistance Genes in Freshwater Trout Farms in a Watershed in Chile

et al. 2019

Self Cite

View full text Add to dashboard Cite

Point sources such as wastewater treatment plants, terrestrial agriculture, and aquaculture may release antibiotic residues, antibiotic resistant bacteria, and antibiotic resistance genes (ARGs) into aquatic ecosystems. However, there is a lack of quantitative studies attributing environmental ARG abundance to specific sources. The goal of this study was to evaluate the role of freshwater trout farms in the release and dissemination of ARGs into the environment. Sediment samples upstream and downstream from five rainbow trout farms were collected over time in southern Chile. A microfluidic quantitative polymerase chain reaction approach was used to quantify an ARG array covering different mechanisms of resistance, and data were analyzed using principal component analysis (PCA) and linear mixed regression models. Surveys were also conducted to obtain information about management practices, including antibiotic use, at the farms. Florfenicol and oxytetracycline were used at these farms, although at different rates. A total of 93 samples were analyzed. In the PCA, strB, sul1, sul2, qacG, tet(A), tet(B), tet(C), tet(W), and floR grouped together. A statistically significant increase in abundance of qacG, strB, sul1, and several tet genes was found downstream from the farms compared with upstream sites, and retention ponds had the highest ARG abundance at each site. Antibiotic resistance gene levels returned to baseline at an average distance of 132.7 m downstream from the farms. Although results from this study indicate an influence of trout farms on the presence of ARGs in the immediate environment, the extent of their contribution to ARG dissemination is unknown and deserves further investigation. Core Ideas Point sources contribute to antimicrobial resistance (AMR) in the environment. Antibiotic resistance genes (ARG) were quantified in river sediment samples. Freshwater trout farms in southern Chile increased ARG abundance in the watershed. The extent of the contribution of the trout farms on ARG dissemination is uncertain. Epidemiological methods are needed to attribute environmental AMR to specific point sources.

show abstract

Improved simultaneous quantification of multiple waterborne pathogens and fecal indicator bacteria with the use of a sample process control

Cited by 28 publications

References 36 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

Microbial Indicators of Fecal Pollution: Recent Progress and Challenges in Assessing Water Quality

Antibiotic Resistance Genes in Freshwater Trout Farms in a Watershed in Chile

Contact Info

Product

Resources

About