1. What is the difference between vancomycin-susceptible S. aureus, VISA and VRSA?

Most isolates of S. aureus are susceptible to vancomycin. The concentration of vancomycin required to inhibit these strains (called the minimal inhibitory concentration or MIC) is typically between 0.5 and 2 micrograms/mL (μg/mL). In contrast, S. aureus isolates for which vancomycin MICs are 8-16 μg/mL are classified as vancomycin intermediate, and isolates for which vancomycin MICs are ≥32 μg/mL are classified as vancomycin resistant. The definitions for classifying isolates of S. aureus are based on the interpretive criteria published by Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS).

CLSI lists only susceptible disk diffusion interpretive criteria (in mm) for vancomycin and Staphylococcus spp. There has not been a sufficient number of non-susceptible isolates to develop resistant and intermediate breakpoints. Organisms for which the vancomycin zone diameters are ≥15mm are considered susceptible, although several studies show that this breakpoint is unreliable for detecting VISA strains.

2. What is CLSI (formerly, NCCLS)?

CLSI (www.nccls.org) 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.

3. What are glycopeptide-intermediate S. aureus (GISA)?

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) was used to indicate 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 more widely used term.

4. Why are VISA and VRSA isolates important?

Vancomycin continues to be a critical antimicrobial agent for treating infections caused by S. aureus strains that are resistant to oxacillin (MRSA; www.cdc.gov/ncidod/hip/aresist/mrsa.htm) and other antimicrobial agents. The decreased susceptibility of VISA and VRSA strains to vancomycin leaves clinicians with relatively few therapeutic options for treating these infections.

5. Can routine susceptibility tests detect VISA and VRSA?

Not all susceptibility testing methods detect VISA and VRSA isolates. Two out of three confirmed VRSA isolates were not reliably detected by automated testing systems. In addition, VISA isolates are not detected by disk diffusion. Methods that do detect VISA and VRSA are the vancomycin screen agar plate brain heart infusion (BHI) agar with 6 µg/ml of vancomycin) and non-automated MIC methods [reference broth microdilution, agar dilution, and Etest® using a 0.5 McFarland standard to prepare inoculum (AB Biodisk, Piscatway, NJ)]. Laboratories that use automated methods or disk diffusion should also include a vancomycin screen plate for enhanced detection of VISA and VRSA. If possible, laboratories should incorporate the vancomycin agar screen plate for testing all S. aureus. Alternatively, the screening may be limited to MRSA isolates, since nearly all VISA and all VRSA were also MRSA. S. aureus isolates that grow on the vancomycin agar screen plate should be checked for purity and the species identification of the organism confirmed. Vancomycin susceptibility should be retested by a validated MIC method incubated for a full 24 hours.

6. What is the vancomycin agar screen test?

The vancomycin agar screen test uses commercially prepared plates to screen pure cultures of bacteria for vancomycin resistance. These plates contain BHI agar and 6 µg/ml of vancomycin. A 10µl inoculum of a 0.5 McFarland suspension should be spotted on the agar using a micropipette (final concentration=10⁶ CFU/ml). Alternatively, a swab may be dipped in the McFarland suspension, the excess liquid expressed, and used to inoculate the vancomycin agar plate. For quality control, laboratories should use Enterococcus faecalis ATCC 29212 as the susceptible control and E. faecalis ATCC 51299 as the resistant control. Up to eight isolates can be tested per plate; quality control should be performed each day of testing. Growth of more than one colony is considered a positive result. All staphylococci that grow on these plates should be inspected for pure culture, and the original clinical isolates should be tested by a validated MIC method for confirmation. Plates prepared in-house using various lots of media performed inconsistently and were inferior to those obtained commercially. Performance of commercially prepared plates varies by individual manufacturer (CDC unpublished data).

7. Are any modifications required of routine disk diffusion and MIC methods when testing vancomycin and staphylococci?

Testing should incorporate the following CLSI (formerly NCCLS) recommendations:

Inoculum: Use direct colony suspension

Incubation: 35°C, ambient air, for a full 24 hr.

Endpoint: Examine very closely for any indication of growth

According to the newest CLSI (formerly NCCLS) standards, a vancomycin-intermediate or –resistant result for staphylococci isolate should be verified by repeating a validated MIC method and the organism identification.

8. What special steps should be taken when an S. aureus is suspected of being a VISA or VRSA?

Please refer to the VISA/VRSA testing algorithm (www.cdc.gov/ncidod/hip/lab/visa_vrsa_algo.htm), which presents a strategy of detecting and confirming VISA and VRSA using appropriate test methods.

9. Should repeat testing include any specific antimicrobial agents that might not be included in the routine panel?

Yes. The following additional antimicrobial agents should be testing against VISA and VRSA isolates. Clindamycin, daptomycin, linezolid, quinupristin/dalfopristin, rifampin, and trimethoprim-sulfamethoxazole. The laboratories of the Division of Healthcare Quality Promotion of the Centers for Disease Control and Prevention will perform tests for these additional antimicrobial agents. It is essential to send probable VISA and VRSA as quickly as possible, even if the laboratory has the capability to test additional agents in-house to facilitate confirmation and enhance infection control efforts. Isolates should be sent to CDC via your local or state health department. CDC may be notified of presumptive VISA/VRSA by sending an email to SEARCH@cdc.gov. [S.E.A.R.C.H.](www.cdc.gov/ncidod/hip/ncidod/aresist/search.htm)

10. How should local clinical laboratories save presumptive VISA or VRSA isolates?

It is best to freeze isolates at -60°C or lower in standard stock culture medium, such as a broth containing 15% glycerol, or in defibrinated sheep blood. However, if a laboratory cannot do this, the isolate should be subcultured to a non-selective agar slant (e.g., trypticase soy agar) and incubated overnight at 35°C. The following day, the caps should be tightened and the slants store at 2-8°C. Repeated subcultures should be kept to a minimum prior to storage.

11. Are VISA and VRSA isolates resistant to oxacillin?

All VRSA isolates to date were oxacillin-resistant (i.e., MRSA) and contained mecA. Most VISA isolates were also oxacillin-resistant. However, two VISA isolates became oxacillin-susceptible upon repeat isolation from the patient and one was oxacillin-susceptible but contained mecA.

12. What are the mechanisms of resistance for VRSA and VISA?

All VRSA isolates to date contained the vanA vancomycin resistance gene. The vanA gene is usually found in enterococci and typically confers high-level vancomycin resistance (MICs= 512-1024µg/ml) to these organisms. Vancomycin-resistant enterococci containing vanA were isolated from all patients in addition to MRSA. It is likely that the vanA determinant was transferred via plasmids or transposons from enterococci to a resident MRSA strain, resulting in the VRSA.

The mechanism of decreased vancomycin susceptibility in VISA strains is not fully understood. VISA cells have thicker cell walls that contain many cell wall subunits capable of binding vancomycin extracellularly. Vancomycin must reach the cell membrane and bind to the growing cell wall complex to inhibit cell growth.

13. Should VISA and VRSA be reported to the infection control team?

Yes. There is significant concern about the spread of VISA and VRSA among patients because of limited treatment options. If a VISA or VRSA is suspected, specific infection control precautions need to be initiated by infection control personnel to decrease the risk of transmission to others. It is critical for laboratory workers to contact the infection control team immediately when a VISA or VRSA is suspected. Additionally, laboratories should notify the local and/or state health department and the Division of Healthcare Quality Promotion, National Center for Infectious Diseases, CDC, by telephone 800-893-0485 or by sending an email to SEARCH@cdc.gov. The isolate should be saved and sent to the health department and CDC for confirmatory testing.

14. Where can I learn more about antimicrobial susceptibility testing?

CDC developed a training tool for laboratorians to enhance their understanding and improve their proficiency in performing antimicrobial susceptibility testing
(M.A.S.T.E.R.; www.phppo.cdc.gov/dls/master/default.asp).

15. How do I report a VISA or VRSA to the CDC?

To report or request testing of suspected VISA or VRSA isolates, send an email to SEARCH@cdc.gov with your contact information (i.e., name, title, telephone number, laboratory or facility name, and a description of your testing methods and results facility and/or laboratory name, telephone number).[S.E.A.R.C.H.](www.cdc.gov/ncidod/hip/ncidod/aresist/search.htm)

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