Laboratory Detection of Vancomycin Nonsusceptible Staphylococcus aureus

Carey‐Ann D. Burnham1, Robin R. Craven1, W. Michael Dunne1

1 Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, Missouri
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 17.6
DOI:  10.1002/9780471729259.mc1706s22
Online Posting Date:  August, 2011
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Abstract

In 2009, the Clinical and Laboratory Standards Institute introduced new guidelines for staphylococcal susceptibility testing, indicating that Kirby Bauer disk diffusion is not a reliable method for detection of reduced susceptibility to vancomycin in this genus. In this unit, we describe a method using a brain‐heart infusion medium containing 3 µg/ml of vancomycin (BHI‐V3) to screen for isolates of S. aureus with reduced susceptibility to vancomycin; this medium represents a sensitive and cost‐effective method for detection of such isolates in the clinical or research laboratory. Curr. Protoc. Microbiol. 22:17.6.1‐17.6.5. © 2011 by John Wiley & Sons, Inc.

Keywords: Staphylococcus aureus; vancomycin; vancomycin‐intermediate Staphylococcus aureus; susceptibility testing; screening agar

     
 
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Table of Contents

  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1:

  Materials
  • Brain‐heart infusion agar with 3 µg/ml vancomycin (BHI‐V3; commercially available from Remel, cat. no. R01173; this is a special order item)
  • 0.5 McFarland standard (a commercially available 0.5 McFarland standard suspension can be purchased; Remel, cat. no. R20140); alternatively, a 0.5 McFarland suspension can be made as described in the Manual of Clinical Microbiology (Chapin and Lauderdale, ), or a colorimeter device, such as the BioMerieux “Densicheck,” can be used to prepare the appropriate inocula
  • Isolates of Staphylococcus aureus to be tested
  • Normal saline
  • Enterococcus faecalis (ATCC #29212)
  • Staphylococcus aureus (ATCC #25925)
  • Sheep blood agar plates
  • 35°C incubator
  • Swabs
  • Micropipettor (able to deliver 10 µl)
  • Sterile pipet tips
  • Additional reagents and equipment for Gram‐stain ( appendix 3B)
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Figures

Videos

Literature Cited

Literature Cited
   Burnham, C.A., Weber, C.J., and Dunne, W.M. Jr. 2010. Novel screening agar for detection of vancomycin‐nonsusceptible Staphylococcus aureus. J. Clin. Microbiol. 48:949‐951.
   Chapin, K.C. and Lauderdale, T. 2007. Reagents, stains and media: Bacteriology. In The Manual of Clinical Microbiology, 9th ed. (P.R. Murray, E.J. Baron, J.H. Jorgense, M.L. Landry, and M.A. Pfaller, eds.), pp 334‐364. ASM Press: Washington, D.C.
   CLSI. Clinical and Laboratory Standards Institute. 2006a. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, 7th ed. CLSI document M7‐A7. Clinical and Laboratory Standards Institute, Wayne, Pa.
   CLSI. Clinical and Laboratory Standards Institute. 2006b. Performance standards for antimicrobial susceptibility testing; 16th informational supplement. CLSI document M100‐S16. Clinical and Laboratory Standards Institute, Wayne, Pa.
   CLSI. Clinical and Laboratory Standards Institute. 2009. Performance standards for antimicrobial susceptibility testing: 19th informational supplement. CLSI document M100‐S19, 19th ed. Clinical and Laboratory Standards Institute, Wayne, Pa.
   Howden, B.P., Davies, J.K., Johnson, P.D., Stinear, T.P., and Grayson, M.L. 2010. Reduced vancomycin susceptibility in Staphylococcus aureus, including vancomycin‐intermediate and heterogeneous vancomycin‐intermediate strains: Resistance mechanisms, laboratory detection, and clinical implications. Clin. Microbiol. Rev. 23:99–139.
   Jorgensen, J.H. and Turnidge, J.D. 2007. Susceptibility test methods: Dilution and disk diffusion methods. In The Manual of Clinical Microbiology, 9th ed. (P.R. Murray, E.J. Baron, J.H. Jorgensen, M.L. Landry, and M.A. Pfaller, eds.), pp 1152‐1172. ASM Press: Washington, D.C.
   Tenover, F.C. and Moellering, R.C. Jr. 2007. The rationale for revising the Clinical and Laboratory Standards Institute vancomycin minimal inhibitory concentration interpretive criteria for Staphylococcus aureus. Clin. Infect. Dis. 44:1208‐1215.
   Walsh, T.R., Bolmstrom, A., Qwarnstrom, A., Ho, P., Wootton, M., Howe, R.A., MacGowan, A.P., and Diekema, D. 2001. Evaluation of current methods for detection of staphylococci with reduced susceptibility to glycopeptides. J. Clin. Microbiol. 39:2439–2444.
   Wootton, M., Howe, R.A., Hillman, R., Walsh, T.R., Bennett, P.M., and MacGowan, A.P. 2001. A modified population analysis profile (PAP) method to detect hetero‐resistance to vancomycin in Staphylococcus aureus in a UK hospital. J. Antimicrob. Chemother. 47:399–403.
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