Laboratory Testing for Vancomycin-resistant Staphylococcus aureus

Key points

  • Vancomycin resistance in Staphylococcus aureus (VRSA) is exceptionally rare. Most isolates of Staphylococcus aureus (S. aureus) are susceptible to vancomycin.
  • Not all susceptibility testing methods detect vancomycin-resistant isolates.
  • It is critical to report suspected VRSA to health departments and CDC.

Resistance mechanisms

To survive, germs can develop defense strategies, called resistance mechanisms against treatment drugs, like antibiotics and antifungals. Germs can share their resistance mechanisms with other germs and can carry genes for many types of resistance.

All VRSA have contained the vancomycin resistance gene vanA, commonly found in vancomycin-resistant enterococci (VRE). The VanA gene typically confers (provides) high-level vancomycin resistance (MICs= 512-1024 µg/ml).

All VRSA isolates detected and confirmed in the U.S. to date have also been methicillin (oxacillin)-resistant (MRSA) due to the mecA gene.

Some VRSA-positive patients have a history of infections caused by both VRE containing vanA and MRSA. The vanA determinant likely transferred from the VRE to the MRSA strain via plasmids or transposons, resulting in the VRSA.

For vancomycin-intermediate S. aureus (VISA), vancomycin must bind to the growing cell wall complex and reach the cell membrane to inhibit cell growth. Most VISA isolates are methicillin (oxacillin)-resistant.

Types of tests

Both automated and non-automated susceptibility testing methods can detect VISA/VRSA. It should be noted that disk diffusion is not a recommended method for testing vancomycin susceptibility in S. aureus.

Information about susceptibility testing methods and their limitations should be made available to healthcare providers. Providers can request additional testing when they suspect reduced susceptibility to vancomycin.


Tests typically used to detect VRSA

  • Non-automated MIC methods including reference broth microdilution, agar dilution and gradient diffusion strips.
  • Automated methods.
  • Vancomycin screen agar plate [brain heart infusion (BHI) agar containing 6 µg/ml of vancomycin].


Tests typically used to detect VISA

  • Non-automated MIC methods including reference broth microdilution, agar dilution and gradient diffusion strips.
  • Automated methods.
  • Vancomycin screen agar plate [brain heart infusion (BHI) agar containing 6 µg/ml of vancomycin] usually detects VISA for which the vancomycin MIC = 8 µg/ml); some strains for which the vancomycin MICs are 4 µg/ml will fail to grow.

Laboratory guidelines

Dilution methods

Laboratorians should perform broth microdilution and agar dilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines, which include the following recommendations:

  • Inoculum: Use direct colony suspension.
  • Incubation: 35 ± 2°C, ambient air, for 24 hr.
  • Endpoint: Examine very closely for any indication of growth.

Vancomycin agar screen

Laboratorians should perform vancomycin agar screening for S. aureus according to CLSI guidelines.

The vancomycin agar screen test uses commercially prepared agar plates to screen pure cultures of bacteria for vancomycin resistance. These plates contain BHI agar and 6 µg/ml of vancomycin. Spot 10µl inoculum of a 0.5 McFarland suspension on the agar using a micropipette (final concentration=106 CFU/ml). Alternatively, dip a swab in the McFarland suspension, express the excess liquid, and spot an area of 10-15 mm in diameter or streak a portion of the plate. For quality control, laboratories should use Enterococcus faecalis ATCC 29212 as the susceptible control and E. faecalis ATCC 51299 as the resistant control.

You can test up to eight isolates per plate but should always perform quality control each day of testing.

Plates prepared in-house using various lots of media performed inconsistently and were inferior to those obtained commercially (CDC unpublished data). Performance of commercially prepared plates varies by individual manufacturer.

Testing for additional agents

There are several antimicrobial agents that usually retain activity against VISA and VRSA isolates (e.g., daptomycin, linezolid and ceftaroline). Laboratories should perform AST for these agents and any others considered for clinical treatment. The laboratory of the Division of Healthcare Quality Promotion at CDC can perform this testing.

It is essential to send probable isolates of VRSA to health departments and CDC as quickly as possible, even if the laboratory has the capability to test additional agents in-house; to facilitate organism confirmation and enhance infection control efforts. Laboratorians should send isolates to CDC by their local or state health department. Keep repeated subcultures to a minimum before storage and shipping. See CDC Test Directory for specific shipping guidance.

Interpreting results

Dilution methods:

• Vancomycin-intermediate S. aureus: MIC = 4-8 μg/mL
• Vancomycin-resistant S. aureus: MIC = ≥16 μg/mL1

The vast majority of suspected VRSA are due to a mixed culture of VRE and MRSA. Verify any vancomycin-intermediate or vancomycin-resistant result for S. aureus by restreaking the isolate for purity and repeating a validated MIC method and organism identification. Send any S. aureus for which the vancomycin MIC is >=8 µg/ml to a referral laboratory.

Vancomycin agar screen

  • Growth of more than one colony means a positive result. Inspect all staphylococci that grow on these plates for purity.
  • Perform a vancomycin MIC test using a validated MIC method to determine vancomycin MICs on S. aureus that grow on the vancomycin screening agar.

Reporting results

When laboratorians suspect VISA/VRSA, it's critical they promptly:

  • Notify the submitting facility's infection control team, local and/or state health department(s) and CDC.
  • Save and send any S. aureus for which the vancomycin MIC is >=8 µg/ml to the health department and CDC for confirmatory testing.

VISA and VRSA should be reported to CDC through the National Notifiable Disease Surveillance System (NNDSS). Suspected VRSA (i.e., isolates that have been confirmed to be pure and are vancomycin-resistant on repeat AST) should be promptly reported to state/local health authorities and subsequently to CDC for public health investigation.


CLSI is a global, interdisciplinary, nonprofit, standards-developing and educational organization that promotes the development and use of voluntary consensus standards and guidelines within the healthcare community.

Definition of term: glycopeptide

The term glycopeptide refers to a group of antimicrobial agents that includes vancomycin and teicoplanin. Since the first two VISA isolates in the United States were also resistant to teicoplanin, the term glycopeptide-intermediate S. aureus (GISA) indicates this broader resistance profile. While GISA may be a more specific term for strains intermediate to both vancomycin and teicoplanin, not all VISA strains are intermediate to teicoplanin; therefore, VISA is a more accurate and widely used term.

  1. Clinical and Laboratory Standards Institute/NCCLS. Performance Standards for Antimicrobial Susceptibility Testing. Sixteenth informational supplement. M100-S16. Wayne, PA: CLSI, 2006.