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Improved detection of Rhodococcus coprophilus with a new quantitative PCR assay

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Abstract

Agricultural practices, such as spreading liquid manure or the utilisation of land as animal pastures, can result in faecal contamination of water resources. Rhodococcus coprophilus is used in microbial source tracking to indicate animal faecal contamination in water. Methods previously described for detecting of R. coprophilus in water were neither sensitive nor specific. Therefore, the aim of this study was to design and validate a new quantitative polymerase chain reaction (qPCR) to improve the detection of R. coprophilus in water. The new PCR assay was based on the R. coprophilus 16S rRNA gene. The validation showed that the new approach was specific and sensitive for deoxyribunucleic acid from target host species. Compared with other PCR assays tested in this study, the detection limit of the new qPCR was between 1 and 3 log lower. The method, including a filtration step, was further validated and successfully used in a field investigation in Switzerland. Our work demonstrated that the new detection method is sensitive and robust to detect R. coprophilus in surface and spring water. Compared with PCR assays that are available in the literature or to the culture-dependent method, the new molecular approach improves the detection of R. coprophilus.

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References

  • Anon (2006) Swiss Accreditation Service: Leitfaden zur Validierung mikrobiologischer Prüfverfahren und zur Abschätzung der Messunsicherheit im Bereich Lebensmittel- und Umweltmikrobiologie. Mitt Lebensm Hyg 97:73–106

    Google Scholar 

  • Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622

    Article  CAS  Google Scholar 

  • Gavini F, Pourcher AM, Neut C, Monget D, Romond C, Oger C, Izard D (1991) Phenotypic differentiation of bifidobacteria of human and animal origins. Int J Syst Bacteriol 41:548–557

    Article  CAS  Google Scholar 

  • Havelaar AH, Pot-Hogeboom WM, Furuse K, Pot R, Hormann MP (1990) F-specific RNA bacteriophages and sensitive host strains in faeces and wastewater of human and animal origin. J Appl Bacteriol 69:30–37

    Article  CAS  Google Scholar 

  • Jagals P, Grabow WOK, de Villiers JC (1995) Evaluation of indicators for assessment of human and animal faecal pollution of surface run-off. Water Sci Technol 31:235–241

    Google Scholar 

  • Layton A, McKay L, Williams D, Garrett V, Gentry R, Sayler G (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  CAS  Google Scholar 

  • Long SC, Shafer E, Arango C, Siraco D (2003) Evaluation of three source tracking indicator organisms for watershed management. J Water SRT Aqua 52:565–575

    Google Scholar 

  • Mara DD, Oragui JI (1981) Occurrence of Rhodococcus coprophilus and associated actinomycetes in feces, sewage, and freshwater. Appl Environ Microbiol 42:1037–1042

    CAS  Google Scholar 

  • Mara DD, Oragui JI (1983) Sorbitol-fermenting bifidobacteria as specific indicators of human faecal pollution. J Appl Bacteriol 55:349–357

    Article  CAS  Google Scholar 

  • Meays CL, Broersma K, Nordin R, Mazumder A (2004) Source tracking fecal bacteria in water: a critical review of current methods. J Environ Manage 73:71–79

    Article  Google Scholar 

  • Mendez J, Audicana A, Isern A, Llaneza J, Moreno B, Tarancon ML, Jofre J, Lucena F (2004) Standardised evaluation of the performance of a simple membrane filtration-elution method to concentrate bacteriophages from drinking water. J Virol Methods 117:19–25

    Article  CAS  Google Scholar 

  • Oragui JI, Mara DD (1983) Investigation of the survival characteristics of Rhodococcus coprophilus and certain fecal indicator bacteria. Appl Environ Microbiol 46:356–360

    CAS  Google Scholar 

  • Reischer GH, Kasper DC, Steinborn R, Mach RL, Farnleitner AH (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  CAS  Google Scholar 

  • Rowbotham TJ, Cross T (1977a) Ecology of Rhodococcus coprophilus and associated actionomycetes in fresh water and agricultural habitats. J Gen Microbiol 100:231–240

    Google Scholar 

  • Rowbotham TJ, Cross T (1977b) Rhodococcus coprophilus sp. nov.:an aerobic nocardioform actinomycete belonging to the ‘rhodochrous’ complex. J Gen Microbiol 100:123–138

    Google Scholar 

  • Savichtcheva O, Okabe S (2006) Alternative indicators of fecal pollution: relations with pathogens and conventional indicators, current methodologies for direct pathogen monitoring and future application perspectives. Water Res 40:2463–2476

    Article  CAS  Google Scholar 

  • Savill MG, Murray SR, Scholes P, Maas EW, McCormick RE, Moore EB, Gilpin BJ (2001) Application of polymerase chain reaction (PCR) and TaqMan PCR techniques to the detection and identification of Rhodococcus coprophilus in faecal samples. J Microbiol Methods 47:355–368

    Article  CAS  Google Scholar 

  • Scott TM, Rose JB, Jenkins TM, Farrah SR, Lukasik J (2002) Microbial source tracking: current methodology and future directions. Appl Environ Microbiol 68:5796–5803

    Article  CAS  Google Scholar 

  • Shanks OC, Santo Domingo JW, Lamendella R, Kelty CA, Graham JE (2006) Competitive metagenomic DNA hybridization identifies host-specific microbial genetic markers in cow fecal samples. Appl Environ Microbiol 72:4054–4060

    Article  CAS  Google Scholar 

  • Shanks OC, Atikovic E, Blackwood AD, Lu J, Noble RT, Domingo JS, Seifring S, Sivaganesan M, Haugland RA (2008) Quantitative PCR for detection and enumeration of genetic markers of bovine fecal pollution. Appl Environ Microbiol 74:745–752

    Article  CAS  Google Scholar 

  • Shanks OC, White K, Kelty CA, Hayes S, Sivaganesan M, Jenkins M, Varma M, Haugland RA (2010) Performance assessment PCR-based assays targeting bacteroidales genetic markers of bovine fecal pollution. Appl Environ Microbiol 76:1359–1366

    Article  CAS  Google Scholar 

  • Sinton LW, Finlay RK, Hannah DJ (1998) Distinguishing human from animal faecal contamination in water: a review. N Z J Mar Freshwater Res 32:323–348

    Article  Google Scholar 

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Acknowledgements

The present study was conducted in the laboratories of the Consumer Protection Directorate of the Federal Office of Public Health (FOPH; 3003 Bern, Switzerland) and was financed by research grant FOPH/07.006623. The authors would like to thank Dr. Dominik Moor (FOPH) for proofreading the article.

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Authors and Affiliations

  1. Federal Office of Public Health, 3003, Bern, Switzerland

    Melanie Wicki, Richard Felleisen, Marianne Liniger, Isabel Niederhauser & Andreas Baumgartner

  2. Swiss Tropical and Public Health Institute, 4002, Basel, Switzerland

    Melanie Wicki & Marcel Tanner

  3. University of Basel, 4000, Basel, Switzerland

    Melanie Wicki & Marcel Tanner

  4. Office of Environmental Protection and Energy, Basel-Landschaft, 4410, Liestal, Switzerland

    Adrian Auckenthaler

  5. University of Lausanne, 1015, Lausanne, Switzerland

    Caroline Loutre

Authors
  1. Melanie Wicki
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  2. Adrian Auckenthaler
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  3. Richard Felleisen
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  4. Marianne Liniger
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  5. Caroline Loutre
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  6. Isabel Niederhauser
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  7. Marcel Tanner
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  8. Andreas Baumgartner
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Correspondence to Melanie Wicki.

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Wicki, M., Auckenthaler, A., Felleisen, R. et al. Improved detection of Rhodococcus coprophilus with a new quantitative PCR assay. Appl Microbiol Biotechnol 93, 2161–2169 (2012). https://doi.org/10.1007/s00253-012-3888-4

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  • DOI: https://doi.org/10.1007/s00253-012-3888-4

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