Host-Specific  Bacteroides  Markers-Based Microbial Source Tracking in Aquaculture Areas

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Abstract

Various waterborne pathogens originate from human or animal feces and may cause severe gastroenteric outbreaks. Bacteroides spp. that exhibit strong host- or group-specificities are promising markers for identifying fecal sources and their origins. In the present study, 240 water samples were collected from two major aquaculture areas in Republic of Korea over a period of approximately 1 year, and the concentrations and occurrences of four host-specific Bacteroides markers (human, poultry, pig, and ruminant) were evaluated in the study areas. Host-specific Bacteroides markers were detected widely in the study areas, among which the poultry-specific Bacteroides marker was detected at the highest concentration (1.0–1.2 log ₁₀ copies L ⁻¹). During the sampling period, high concentrations of host-specific Bacteroides markers were detected between September and December 2015. The host-specific Bacteroides marker-combined geospatial map revealed the up-to-downstream gradient of fecal contamination, as well as the effects of land-use patterns on host-specific Bacteroides marker concentrations. In contrast to traditional bacterial indicators, the human-specific Bacteroides marker correlated with human specific pathogens, such as noroviruses ( r=0.337; P<0.001). The present results indicate that host-specific Bacteroides genetic markers with an advanced geospatial analysis are useful for tracking fecal sources and associated pathogens in aquaculture areas.

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Most cited references 43

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Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli.

Changsoo Lee, Jaai Kim, Seung Shin … (2006)

Real-time QPCR based methods for determination of plasmid copy number in recombinant Escherichia coli cultures are presented. Two compatible methods based on absolute and relative analyses were tested with recombinant E. coli DH5alpha harboring pBR322, which is a common bacterial cloning vector. The separate detection of the plasmid and the host chromosomal DNA was achieved using two separate primer sets, specific for the plasmid beta-lactamase gene (bla) and for the chromosomal d-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. Since both bla and dxs are single-copy genes of pBR322 and E. coli chromosomal DNA, respectively, the plasmid copy number can be determined as the copy ratio of bla to dxs. These methods were successfully applied to determine the plasmid copy number of pBR322 of E. coli host cells. The results of the absolute and relative analyses were identical and highly reproducible with coefficient of variation (CV) values of 2.8-3.9% and 4.7-5.4%, respectively. The results corresponded to the previously reported values of pBR322 copy number within E. coli host cells, 15-20. The methods introduced in this study are convenient to perform and cost-effective compared to the traditionally used Southern blot method. The primer sets designed in this study can be used to determine plasmid copy number of any recombinant E. coli with a plasmid vector having bla gene.

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Comparison of droplet digital PCR to real-time PCR for quantitative detection of cytomegalovirus.

R T Hayden, Z. Gu, J. Ingersoll … (2013)

Quantitative real-time PCR (QRT-PCR) has been widely implemented for clinical viral load testing, but a lack of standardization and relatively poor precision have hindered its usefulness. Digital PCR offers highly precise, direct quantification without requiring a calibration curve. Performance characteristics of real-time PCR were compared to those of droplet digital PCR (ddPCR) for cytomegalovirus (CMV) load testing. Tenfold serial dilutions of the World Health Organization (WHO) and the National Institute of Standards and Technology (NIST) CMV quantitative standards were tested, together with the AcroMetrix CMV tc panel (Life Technologies, Carlsbad, CA) and 50 human plasma specimens. Each method was evaluated using all three standards for quantitative linearity, lower limit of detection (LOD), and accuracy. Quantitative correlation, mean viral load, and variability were compared. Real-time PCR showed somewhat higher sensitivity than ddPCR (LODs, 3 log(10) versus 4 log(10) copies/ml and IU/ml for NIST and WHO standards, respectively). Both methods showed a high degree of linearity and quantitative correlation for standards (R(2) ≥ 0.98 in each of 6 regression models) and clinical samples (R(2) = 0.93) across their detectable ranges. For higher concentrations, ddPCR showed less variability than QRT-PCR for the WHO standards and AcroMetrix standards (P < 0.05). QRT-PCR showed less variability and greater sensitivity than did ddPCR in clinical samples. Both digital and real-time PCR provide accurate CMV load data over a wide linear dynamic range. Digital PCR may provide an opportunity to reduce the quantitative variability currently seen using real-time PCR, but methods need to be further optimized to match the sensitivity of real-time PCR.

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Development of Bacteroides 16S rRNA gene TaqMan-based real-time PCR assays for estimation of total, human, and bovine fecal pollution in water.

Alice C Layton, Larry McKay, Dan Williams … (2006)

Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r2= 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.

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Author and article information

Journal

Journal ID (nlm-ta): Microbes Environ

Journal ID (iso-abbrev): Microbes Environ

Title: Microbes and Environments

ISSN (Print): 1342-6311

ISSN (Electronic): 1347-4405

Publication date (Print): June 2018

Publication date (Electronic): 01 June 2018

Volume: 33

Issue: 2

Pages: 151-161

Affiliations

[1 ] Department of Environmental Health Sciences, Graduate School of Public Health, Seoul National University 1 Gwanak-ro, Gwanak-gu, Seoul Republic of Korea

[2 ] N-Bio, Seoul National University 1 Gwanak-ro, Gwanak-gu, Seoul Republic of Korea

[3 ] Department of Civil and Environmental Engineering, Dongguk University 1 Pildong-ro, Jung-gu, Seoul Republic of Korea

[4 ] Department of Biology, College of Sciences, Kyung Hee University 26 Kyungheedae-ro, Dongdaemun-gu, Seoul Republic of Korea

[5 ] Department of Seafood Science and Technology, Gyeongsang National University 38 Cheondaegukchi-gil, Tongyeong-si, Gyeongsangnam-do Republic of Korea

[6 ] Department of Environmental Engineering & Biotechnology, Mokpo National Maritime University 91, Haeyangdaehak-ro, Mokpo-si, Jeollanam-do Republic of Korea

[7 ] Institute of Health and Environment, Graduate School of Public Health, Seoul National University 1 Gwanak-ro, Gwanak-gu, Seoul Republic of Korea

[8 ] Center for Human and Environmental Microbiome, Seoul National University 1 Gwanak-ro, Gwanak-gu, Seoul Republic of Korea

Author notes

[* ]Corresponding author. E-mail: gko@ 123456snu.ac.kr ; Tel: +82–2–880–2731; Fax: +82–2–745–9104.

[†]

These authors contributed equally to this work.

Article

Publisher ID: 33_151

DOI: 10.1264/jsme2.ME17166

PMC ID: 6031393

PubMed ID: 29863059

SO-VID: 3fcb0fa4-161a-4896-89b5-74b2022c51fb

License:

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Host-Specific Bacteroides Markers-Based Microbial Source Tracking in Aquaculture Areas

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Most cited references 43

Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli.

Comparison of droplet digital PCR to real-time PCR for quantitative detection of cytomegalovirus.

Development of Bacteroides 16S rRNA gene TaqMan-based real-time PCR assays for estimation of total, human, and bovine fecal pollution in water.

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