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Identification of coagulase-negative Staphylococcus species by gas chromatography

  • M-È ParadisEmail author
  • D. Haine
  • S. Messier
  • J. Middleton
  • J. Perry
  • A. I. Ramirez
  • D. T. Scholl
Chapter

Abstract

Researching the impact and epidemiology of coagulase-negative Staphylococcus species (CNS) causing intramammary infections (IMI) require their identification at the species level. Gene sequencing is the gold standard but faster and less expensive methods could be useful. The Sherlock Microbial Identification System is an automated gas chromatographic (MIS-GC) system able to speciate CNS isolates in human clinical medicine by identifying the unique cellular fatty acid patterns in bacteria cell walls. Our objective was to validate the MIS-GC method for speciating CNS responsible for IMI in dairy cows. The CNS isolates examined include 429 isolates of Staphylococcus chromogenes, 195 Staphylococcus simulans, 108 Stapylococcus xylosus, 81 Staphylococcus haemolyticus and 42 Staphylococcus epidermididis obtained from the Canadian Bovine Mastitis Research Network culture collection and speciated using rpoB gene sequencing. Isolates were harvested from apparently normal mammary quarters before and after the dry period or during lactation. CNS isolates were divided in 2 groups within species. Speciation by MIS-GC of the first group was performed with a human-source CNS fatty-acid profile library and was used to construct a bovine-source library. MIS-GC speciation of the second group was performed with the new library. Repeatability of the technique was evaluated by re-culturing and re-testing a minimum of 50 isolates of each species. Using rpoB sequencing as gold standard, sensitivities for S. chromogenes, S. simulans, S. xylosus and S. epidermidis with the human library were 63% (n=215), 58% (n=89), 40% (n=55) and 52% (n=21) respectively, and 91%, 79%, 71% and 100% with the bovine library. Repeatability was 80% (n=100), 81% (n=97), 72% (n=53). Sensitivity for S. haemolyticus was 8% with the human library and could not be included in the bovine library. Its repeatability was 90% (n=40). The final bovine library tested on a random sample of S. chromogenes gave a sensitivity of 89%.

Keywords

intramammary infection fatty acid sensitivity repeatability 

References

  1. Birnbaum, D., Herwaldt, L., Low, D.E., Noble, M., Pfaller, M., Sherertz, R. and Chow, A.W., 1994. Efficacy of microbial identification system for epidemiologic typing of coagulase-negative staphylococci. Journal of Clinical Microbiology 32:2113-2119.PubMedGoogle Scholar
  2. Bradley, A.J., Leach, K.A., Breen, J.E., Green, L.E. and Green, M.J., 2007. Survey of the incidence and aetiology of mastitis on dairy farms in England and Wales. The Veterinary Record 160: 253-258.PubMedCrossRefGoogle Scholar
  3. Capurro, A., Artursson, K., Persson Waller, K., Bengtsson, B., Ericsson-Unnerstad, H. and Aspan, A., 2009. Comparison of a commercialized phenotyping system, antimicrobial susceptibility testing, and tuf gene sequence-based genotyping for species-level identification of coagulase-negative staphylococci isolated from cases of bovine mastitis. Veterinary Microbiology 134: 327-333.PubMedCrossRefGoogle Scholar
  4. Drancourt, M. and Raoult, D., 2002. rpoB gene-sequence-based identification of Staphylococcus species. Journal of Clinical Microbiology 40: 1333-1338.PubMedCrossRefGoogle Scholar
  5. Park, J.Y., Fox, L.K., Seo, K.S., McGuire, M.A., Park, Y.H., Rurangirwa, F.R., Sischo, W.M. and Bohach, G.A., 2011. Comparison of phenotypic and genotypic methods for the species identification of coagulase-negative staphylococcal isolates from bovine intramammary infections. Veterinary Microbiology 147: 142-148.PubMedCrossRefGoogle Scholar
  6. Piepers, S., De Meulemeester, L., de Kruif, A., Opsomer, G., Barkema, H.W. and De Vliegher, S., 2007. Prevalence and distribution of mastitis pathogens in subclinically infected dairy cows in Flanders, Belgium. Journal of Dairy Research 74: 478-483.PubMedCrossRefGoogle Scholar
  7. Pyörälä, S. and Taponen, S., 2009. Coagulase-negative staphylococci – Emerging mastitis pathogens. Veterinary Microbiology 134: 3-8.PubMedCrossRefGoogle Scholar
  8. Reyher, K.K., Dufour, S., Barkema, H.W., Des Coteaux, L., DeVries, T.J., Dohoo, I.R., Keefe, G.P., Roy, J-P and Scholl, D.T., 2011. The National Cohort of Dairy Farms – A data collection platform for mastitis research in Canada. Journal of Dairy Science 94: 1616-1626.PubMedCrossRefGoogle Scholar
  9. Sampimon, O., Barkema, H.W., Berends, I., Sol, J. and Lam, T., 2009a. Prevalence of intramammary infection in Dutch dairy herds. Journal of Dairy Research 76: 129-136.PubMedCrossRefGoogle Scholar
  10. Sampimon, O.C., Zadoks, R.N., De Vliegher, S., Supré, K., Haesebrouck, F., Barkema, H.W., Sol, J. And Lam, T.J.G.M., 2009b. Performance of API Staph ID 32 and Staph-Zym for identification of coagulase-negative staphylococci isolated from bovine milk samples. Veterinary Microbiology 136: 300-305.PubMedCrossRefGoogle Scholar
  11. Sasser, M., 2001. Identification of bacteria by gas chromatography of cellular fatty acids. Technical note 101. Microbial ID, Inc., Newark, DE, USA.Google Scholar
  12. Zadoks, R.N. and Watts, J.L., 2009. Species identification of coagulase-negative staphylococci: genotyping is superior to phenotyping. Veterinary Microbiology 134: 20-28.PubMedCrossRefGoogle Scholar

Copyright information

© Wageningen Academic Publishers 2011

Authors and Affiliations

  • M-È Paradis
    • 1
    • 3
    Email author
  • D. Haine
    • 1
    • 3
  • S. Messier
    • 1
    • 3
  • J. Middleton
    • 2
  • J. Perry
    • 2
  • A. I. Ramirez
    • 1
  • D. T. Scholl
    • 1
    • 3
    • 4
  1. 1.Faculté de médecine vétérinaireUniversité de MontréalSt-HyacintheCanada
  2. 2.College of Veterinary MedicineUniversity of MissouriColumbiaUSA
  3. 3.Canadian Bovine Mastitis Research NetworkSt-HyacintheCanada
  4. 4.South Dakota State UniversityBrookingsUSA

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