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Microbial community analysis and identification of alternative host-specific fecal indicators in fecal and river water samples using pyrosequencing

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Abstract

It is important to know the comprehensive microbial communities of fecal pollution sources and receiving water bodies for microbial source tracking. Pyrosequencing targeting the V1–V3 hypervariable regions of the 16S rRNA gene was used to investigate the characteristics of bacterial and Bacteroidales communities in major fecal sources and river waters. Diversity analysis indicated that cow feces had the highest diversities in the bacterial and Bacteroidales group followed by the pig sample, with human feces having the lowest value. The Bacteroidales, one of the potential fecal indicators, totally dominated in the fecal samples accounting for 31%–52% of bacterial sequences, but much less (0.6%) in the river water. Clustering and Venn diagram analyses showed that the human sample had a greater similarity to the pig sample in the bacterial and Bacteroidales communities than to samples from other hosts. Traditional fecal indicators, i.e., Escherichia coli, were detected in the human and river water samples at very low rates and Clostridium perfringens and enterococci were not detected in any samples. Besides the Bacteroidales group, some microorganisms detected in the specific hosts, i.e., Parasutterella excrementihominis, Veillonella sp., Dialister invisus, Megamonas funiformis, and Ruminococcus lactaris for the human and Lactobacillus amylovorus and Atopostipes sp. for the pig, could be used as potential host-specific fecal indicators. These microorganisms could be used as multiple fecal indicators that are not dependent on the absence or presence of a single indicator. Monitoring for multiple indicators that are highly abundant and host-specific would greatly enhance the effectiveness of fecal pollution source tracking.

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References

  • Ahmed, W., A. Goonetilleke, D. Powell, and T. Gardner. 2009. Evaluation of multiple sewage-associated bacteroides PCR markers for sewage pollution tracking. Water Res. 43, 4872–4877.

    Article  PubMed  CAS  Google Scholar 

  • Andersson, A.F., M. Lindberg, H. Jakobsson, F. Backhed, P. Nyren, and L. Engstrand. 2008. Comparative analysis of human gut microbiota by barcoded pyrosequencing. PLoS One 3, e2836.

    Article  PubMed  Google Scholar 

  • Ballard, S.A., E.A. Grabsch, P.D. Johnson, and M.L. Grayson. 2005. Comparison of three PCR primer sets for identification of vanB gene carriage in feces and correlation with carriage of vancomycinresistant Enterococci: Interference by vanB-containing anaerobic Bacilli. Antimicrob. Agents Chemother. 49, 77–81.

    Article  PubMed  CAS  Google Scholar 

  • Bernhard, A.E. and K.G. Field. 2000a. Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes. Appl. Environ. Microbiol. 66, 1587–1594.

    Article  PubMed  CAS  Google Scholar 

  • Bernhard, A.E. and K.G. Field. 2000b. A PCR assay to discriminate human and ruminant feces on the basis of host differences in bacteroides-prevotella genes encoding 16S rRNA. Appl. Environ. Microbiol. 66, 4571–4574.

    Article  PubMed  CAS  Google Scholar 

  • Carson, C.A., J.M. Christiansen, H. Yampara-Iquise, V.W. Benson, C. Baffaut, J.V. Davis, R.R. Broz, W.B. Kurtz, W.M. Rogers, and W.H. Fales. 2005. Specificity of a bacteroides thetaiotaomicron marker for human feces. Appl. Environ. Microbiol. 71, 4945–4949.

    Article  PubMed  CAS  Google Scholar 

  • Chun, J., J.H. Lee, Y. Jung, M. Kim, S. Kim, B.K. Kim, and Y.W. Lim. 2007. Eztaxon: A web-based tool for the identification of prokaryotes based on 16S ribosomal rRNA gene sequences. Int. J. Syst. Evol. Microbiol. 57, 2259–2261.

    Article  PubMed  CAS  Google Scholar 

  • Claesson, M.J., O. O’sullivan, Q. Wang, J. Nikkila, J.R. Marchesi, H. Smidt, W.M. de Vos, R.P. Ross, and P.W. O’Toole. 2009. Comparative analysis of pyrosequencing and a phylogenetic microarray for exploring microbial community structures in the human distal intestine. PLoS One 4, e6669.

    Article  PubMed  Google Scholar 

  • Cotta, M.A., T.R. Whitehead, M.D. Collins, and P.A. Lawson. 2004. Atopostipes suicloacale gen. nov., sp. nov., isolated from an underground swine manure storage pit. Anaerobe 10, 191–195.

    Article  PubMed  CAS  Google Scholar 

  • Dethlefsen, L., S. Huse, M.L. Sogin, and D.A. Relman. 2008. The pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep 16S rRNA sequencing. PLoS Biol. 6, e280.

    Article  PubMed  Google Scholar 

  • Dick, L.K., A.E. Bernhard, T.J. Brodeur, J.W. Santo Domingo, J.M. Simpson, S.P. Walters, and K.G. Field. 2005. Host distributions of uncultivated fecal bacteroidales bacteria reveal genetic markers for fecal source identification. Appl. Environ. Microbiol. 71, 3184–3191.

    Article  PubMed  CAS  Google Scholar 

  • Dowd, S.E., T.R. Callaway, R.D. Wolcott, Y. Sun, T. McKeehan, R.G. Hagevoort, and T.S. Edrington. 2008a. Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded flx amplicon pyrosequencing (btefap). BMC Microbiol. 8, 125.

    Article  PubMed  Google Scholar 

  • Dowd, S.E., Y. Sun, R.D. Wolcott, A. Domingo, and J.A. Carroll. 2008b. Bacterial tag-encoded FLX amplicon pyrosequencing (btefap) for microbiome studies: Bacterial diversity in the ileum of newly weaned salmonella-infected pigs. Foodborne Pathog. Dis. 5, 459–472.

    Article  PubMed  CAS  Google Scholar 

  • Drasar, B.S. and P.A. Barrow. 1985. Intestinal microbiology wokingham, Van Nostrand Reinhold, UK.

    Google Scholar 

  • Eckburg, P.B., E.M. Bik, C.N. Bernstein, E. Purdom, L. Dethlefsen, M. Sargent, S.R. Gill, K.E. Nelson, and D.A. Relman. 2005. Diversity of the human intestinal microbial flora. Science 308, 1635–1638.

    Article  PubMed  Google Scholar 

  • Finegold, S.M., S.E. Dowd, V. Gontcharova, C. Liu, K.E. Henley, R.D. Wolcott, E. Youn, and et al. 2010. Pyrosequencing study of fecal microflora of autistic and control children. Anaerobe 16, 444–453.

    Article  PubMed  CAS  Google Scholar 

  • Hill, J.E., W.M.U. Fernando, G.A. Zello, R.T. Tyler, W.J. Dahl, and A.G.V. Kessel. 2010. Improvement of the representation of bifidobacteria in fecal microbiota metagenomic libraries by application of the cpn60 universal primer cocktail. Appl. Environ. Microbiol. 76, 4550–4552.

    Article  PubMed  CAS  Google Scholar 

  • Jeong, J.Y., K.I. Gil, K.H. Lee, and J.O. Ka. 2008. Molecular identification of fecal pollution sources in water supplies by host-specific fecal DNA markers and terminal restriction fragment length polymorphism profiles of 16S rRNA gene. J. Microbiol. 46, 599–607.

    Article  PubMed  CAS  Google Scholar 

  • Jeong, J.Y., H.D. Park, K.H. Lee, J.H. Hwang, and J.O. Ka. 2010. Quantitative analysis of human and cow-specific 16S rRNA gene markers for assessment of fecal pollution in river waters by real-time PCR. J. Microbiol. Biotechnol. 20, 245–253.

    PubMed  CAS  Google Scholar 

  • Khoruts, A., J. Dicksved, J.K. Jansson, and M.J. Sadowsky. 2010. Changes in the composition of the human fecal microbiome after bacteriotherapy for recurrent clostridium difficile-associated diarrhea. J. Clin. Gastroenterol. 44, 354–360.

    PubMed  Google Scholar 

  • Kildare, B.J., C.M. Leutenegger, B.S. McSwain, D.G. Bambic, V.B. Rajal, and S. Wuertz. 2007. 16S rRNA-based assays for quantitative detection of universal, human-, cow-, and dog-specific fecal bacteroidales: A bayesian approach. Water Res. 41, 3701–3715.

    Article  PubMed  CAS  Google Scholar 

  • Konstantinov, S.R., H. Smidt, and W.M. de Vos. 2005. Representational difference analysis and real-time PCR for strain-specific quantification of Lactobacillus sobrius sp. Nov. Appl. Environ. Microbiol. 71, 7578–7581.

    Article  PubMed  CAS  Google Scholar 

  • Kreader, C.A. 1995. Design and evaluation of bacteroides DNA probes for the specific detection of human fecal pollution. Appl. Environ. Microbiol. 61, 1171–1179.

    PubMed  CAS  Google Scholar 

  • Layton, A., L. McKay, D. Williams, V. Garrett, R. Gentry, and G. Sayler. 2006. Development of bacteroides 16S rRNA gene taqman-based real-time PCR assays for estimation of total, human, and bovine fecal pollution in water. Appl. Environ. Microbiol. 72, 4214–4224.

    Article  PubMed  CAS  Google Scholar 

  • Ley, R.E., M. Hamady, C. Lozupone, P.J. Turnbaugh, R.R. Ramey, J.S. Bircher, M.L. Schlegel, T.A. Tucker, M.D. Schrenzel, R. Knight, and J.I. Gordon. 2008. Evolution of mammals and their gut microbes. Science 320, 1647–1651.

    Article  PubMed  CAS  Google Scholar 

  • Marti, R., P. Dabert, C. Ziebal, and A.M. Pourcher. 2010. Evaluation of Lactobacillus sobrius/L. amylovorus as a new microbial marker of pig manure. Appl. Environ. Microbiol. 76, 1456–1461.

    Article  PubMed  CAS  Google Scholar 

  • McLain, J.E., H. Ryu, L. Kabiri-Badr, C.M. Rock, and M. Abbaszadegan. 2009. Lack of specificity for PCR assays targeting human bacteroides 16S rRNA gene: Cross-amplification with fish feces. FEMS Microbiol. Lett. 299, 38–43.

    Article  PubMed  CAS  Google Scholar 

  • McLellan, S.L., S.M. Huse, S.R. Mueller-Spitz, E.N. Andreishcheva, and M.L. Sogin. 2009. Diversity and population structure of sewage-derived microorganisms in wastewater treatment plant influent. Environ. Microbiol. 12, 378–392.

    Article  PubMed  Google Scholar 

  • Mieszkin, S., J.P. Furet, G. Corthier, and M. Gourmelon. 2009. Estimation of pig fecal contamination in a river catchment by real-time PCR using two pig-specific bacteroidales 16S rRNA genetic markers. Appl. Environ. Microbiol. 75, 3045–3054.

    Article  PubMed  CAS  Google Scholar 

  • Miller, S.R., A.L. Strong, K.L. Jones, and M.C. Ungerer. 2009. Barcoded pyrosequencing reveals shared bacterial community properties along the temperature gradients of two alkaline hot springs in yellowstone national park. Appl. Environ. Microbiol. 75, 4565–4572.

    Article  PubMed  CAS  Google Scholar 

  • Nagai, F., M. Morotomi, H. Sakon, and R. Tanaka. 2009. Parasutterella excrementihominis gen. Nov., sp. Nov., a member of the family alcaligenaceae isolated from human faeces. Int. J. Syst. Evol. Microbiol. 59, 1793–1797.

    Article  PubMed  CAS  Google Scholar 

  • Okabe, S., N. Okayama, O. Savichtcheva, and T. Ito. 2007. Quantification of host-specific bacteroides-prevotella 16S rRNA genetic markers for assessment of fecal pollution in freshwater. Appl. Microbiol. Biotechnol. 74, 890–901.

    Article  PubMed  CAS  Google Scholar 

  • Quince, C., A. Lanzen, T.P. Curtis, R.J. Davenport, N. Hall, J.M. Head, L.F. Read, and W.T. Sloan. 2009. Accurate determination of microbial diversity from 454 pyrosequencing data. Nat. Methods 6, 639–641.

    Article  PubMed  CAS  Google Scholar 

  • Reischer, G.H., D.C. Kasper, R. Steinborn, A.H. Farnleitner, and R.L. Mach. 2007. A quantitative real-time PCR assay for the highly sensitive and specific detection of human faecal influence in spring water from a large alpine catchment area. Lett. Appl. Microbiol. 44, 351–356.

    Article  PubMed  CAS  Google Scholar 

  • Reischer, G.H., D.C. Kasper, R. Steinborn, R.L. Mach, and A.H. Farnleitner. 2006. Quantitative PCR method for sensitive detection of ruminant fecal pollution in freshwater and evaluation of this method in alpine karstic regions. Appl. Environ. Microbiol. 72, 5610–5614.

    Article  PubMed  CAS  Google Scholar 

  • Resnick, I.G. and M.A. Levin. 1981. Assessment of bifidobacteria as indicators of human fecal pollution. Appl. Environ. Microbiol. 42, 433–438.

    PubMed  CAS  Google Scholar 

  • Sakon, H., F. Nagai, M. Morotomi, and R. Tanaka. 2008. Sutterella parvirubra sp. nov. and Megamonas funiformis sp. nov., isolated from human faeces. Int. J. Syst. Evol. Microbiol. 58, 970–975.

    Article  PubMed  Google Scholar 

  • Sanapareddy, N., T.J. Hamp, L.C. Gonzalez, H.A. Hilger, A.A. Fodor, and S.M. Clinton. 2009. Molecular diversity of a north carolina wastewater treatment plant as revealed by pyrosequencing. Appl. Environ. Microbiol. 75, 1688–1696.

    Article  PubMed  CAS  Google Scholar 

  • Schloss, P.D., S.L. Westcott, T. Ryabin, J.R. Hall, M. Hartmann, E.B. Hollister, R.A. Lesniewski, and et al. 2009. Introducing mothur: Open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 75, 7537–7541.

    Article  PubMed  CAS  Google Scholar 

  • Tillett, D., D.L. Parker, and B.A. Neilan. 2001. Detection of toxigenicity by a probe for the microcystin synthetase a gene (mcyA) of the cyanobacterial genus microcystis: Comparison of toxicities with 16S rRNA and phycocyanin operon (phycocyanin intergenic spacer) phylogenies. Appl. Environ. Microbiol. 67, 2810–2818.

    Article  PubMed  CAS  Google Scholar 

  • Walk, S.T., E.W. Alm, L.M. Calhoun, J.M. Mladonicky, and T.S. Whittam. 2007. Genetic diversity and population structure of Escherichia coli isolated from freshwater beaches. Environ. Microbiol. 9, 2274–2288.

    Article  PubMed  Google Scholar 

  • Wang, Q., G.M. Garrity, J.M. Tiedje, and J.R. Cole. 2007. Naive bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73, 5261–5267.

    Article  PubMed  CAS  Google Scholar 

  • Yue, J.C. and M.K. Clayton. 2005. A similarity measure based on species proportions. Comm. Statist. Theory Methods 34, 2123–2131.

    Article  Google Scholar 

  • Zheng, G., H. Yampara-Iquise, J.E. Jones, and C. Andrew Carson. 2009. Development of Faecalibacterium 16S rRNA gene marker for identification of human faeces. J. Appl. Microbiol. 106, 634–641.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Water Quality Research, Gyeonggi-do Institute of Health and Environment, Suwon, 440-290, Republic of Korea

    Ju-Yong Jeong & Kyong-Hee Lee

  2. School of Civil, Environmental and Architectural Engineering, Korea University, Seoul, 136-713, Republic of Korea

    Hee-Deung Park

  3. Rural Development Administration (RDA), Korean Agricultural Culture Collection (KACC), National Academy of Agricultural Science (NAAS), Suwon, 441-853, Republic of Korea

    Hang-Yeon Weon

  4. Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-742, Republic of Korea

    Ju-Yong Jeong & Jong-Ok Ka

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  1. Ju-Yong Jeong
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  2. Hee-Deung Park
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Correspondence to Jong-Ok Ka.

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Jeong, JY., Park, HD., Lee, KH. et al. Microbial community analysis and identification of alternative host-specific fecal indicators in fecal and river water samples using pyrosequencing. J Microbiol. 49, 585–594 (2011). https://doi.org/10.1007/s12275-011-0530-6

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  • DOI: https://doi.org/10.1007/s12275-011-0530-6

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