|Year : 2012 | Volume
| Issue : 4 | Page : 453-455
Differences in vancomycin MIC among MRSA isolates by agar dilution and E test method
K Tandel, AK Praharaj, S Kumar
Armed Forces Medical College, Sholapur Road, Pune-40, Maharashtra, India
|Date of Submission||14-Feb-2012|
|Date of Acceptance||28-Apr-2012|
|Date of Web Publication||24-Nov-2012|
Armed Forces Medical College, Sholapur Road, Pune-40, Maharashtra
Source of Support: None, Conflict of Interest: None
In this study, the correlation between vancomycin minimum inhibitory concentration (MIC) obtained by the E test technique and the Clinical And Laboratory Standards Institute (CLSI) agar dilution method was evaluated. A total of 53 Methicillin Resistant Staphylococcus aureus (MRSA) strains were tested by both the methods in the present study. MICs of vancomycin obtained by the E test method were consistently higher (+0.5 to 2 log2 dilutions) than those obtained by the agar dilution method. Out of 53 MRSA isolates, 49 isolates showed higher MIC results by E test than by agar dilution method. Three isolates showed same MIC result by both methods. Since many studies have demonstrated increased clinical failure with MRSA isolates for which vancomycin MICs are increased (>1 μg/ml) but still within the susceptibility range (≤ 2 μg/ml), our findings suggest the requirement to re-look into the breakpoints for vancomycin for determining sensitivity of MRSA isolates. Guidelines should also specify the method to be used for determining the MIC.
Keywords: Methicillin Resistant Staphylococcus aureus, vancomycin, Staphylococcus aureus
|How to cite this article:|
Tandel K, Praharaj A K, Kumar S. Differences in vancomycin MIC among MRSA isolates by agar dilution and E test method. Indian J Med Microbiol 2012;30:453-5
|How to cite this URL:|
Tandel K, Praharaj A K, Kumar S. Differences in vancomycin MIC among MRSA isolates by agar dilution and E test method. Indian J Med Microbiol [serial online] 2012 [cited 2019 Jun 26];30:453-5. Available from: http://www.ijmm.org/text.asp?2012/30/4/453/103768
| ~ Introduction|| |
Vancomycin has been the most reliable therapeutic agent against methicillin-resistant Staphylococcus aureus (MRSA) for the past three decades. However, despite its sustained in vitro microbiologic inhibitory activity, clinicians continue to debate its utility for MRSA infections. , Significant controversy still exists regarding the current and future roles of vancomycin in the treatment of serious MRSA infections. Vancomycin treatment failure is not uncommon, even when the MRSA strains are fully susceptible [breakpoint Minimum Inhibitory Concentration (MIC), ≤ 2 μg/ml] to vancomycin according to the Clinical And Laboratory Standards Institute (CLSI) criterion. 
Among MRSA strains for which vancomycin MICs are elevated (1-2 μg/ml), failure of vancomycin therapy or reduction in efficacy has been widely reported. These reports suggest that modest elevations in MICs may explain some suboptimal clinical outcomes. , In addition, there is increasing number of reports showing discrepancies between in vitro susceptibility test results for vancomycin and clinical outcomes of MRSA infections treated with vancomycin. ,
| ~ Materials and Methods|| |
A total of 53 MRSA isolates collected from May 2009 to June 2010 at a tertiary medical centre were used in this study. The isolates were collected from various clinical specimens (38 from pus, 6 from tracheal aspiration/bronchoalveolar fluid, 5 from blood and 4 from urine). All isolates were processed at the time of collection and stored at -80°C until they were tested. S. aureus isolates were identified by gram staining, catalase test, slide and tube coagulase testing, DNase production and biochemical testing. Cefoxitin (30 μg) disk diffusion testing was performed by the Kirby-Bauer disc diffusion method using CLSI guidelines.  Confirmation of the isolates as S. aureus and methicillin resistance were determined by multiplex PCR for the femB gene and the mecA gene, respectively. Case records of patients with S. aureus bacteraemia demonstrating reduced glycopeptide susceptibility were examined to determine the patient's clinical progress.
Agar dilution method
S. aureus isolates were screened for reduced glycopeptide susceptibility by an agar dilution method. Bacterial suspensions were prepared from overnight cultures on blood agar and their turbidity was adjusted to be equivalent to that of a 0.5 McFarland standard (10 8 cfu/ml). This suspension was inoculated onto Mueller-Hinton agar containing serial dilutions of vancomycin. Inoculation of isolates along with controls was performed with the use of multi-point inoculator. Plates were incubated at 37°C for 24 h.
E test was performed on all isolates according to the manufacturer's instructions (AB bioMe΄rieux). Briefly, a bacterial suspension of a 0.5 McFarland standard inoculum in sterile water was spread on a Mueller-Hinton agar plate. The vancomycin E test strip was placed onto the agar plate, and the plate was incubated at 35°C for 24 h.
Isolates were categorised as susceptible or resistant to vancomycin according to the breakpoints published by the CLSI. 
| ~ Results|| |
All the S. aureus isolates were resistant to cefoxitin disk by the Kirby-Bauer method. All these isolates were having mecA gene and femB gene [Figure 1].
|Figure 1: Gel electrophoresis result showing mec A gene amplicon product at 310 bp and fem B gene amplicon product at 651 bp|
Click here to view
In this study, we evaluated the correlation between vancomycin MICs obtained by the E test technique and the CLSI agar dilution method. A total of 53 MRSA strains were tested by both the methods. Quality control strain S. aureus ATCC 25923 was evaluated concurrently with every set of tests. Results are shown in [Table 1] and [Figure 2].
|Figure 2: Comparison of vancomycin MICs determined by agar dilution method and E test|
Click here to view
| ~ Discussion|| |
MICs of vancomycin obtained by the E test method were consistently higher (+0.5 to 2 log 2 dilutions) than those obtained by the agar dilution method [Figure 2]. Out of 53 MRSA isolates, 49 isolates showed higher MIC results by E test than by agar dilution method. Three isolates showed same MIC result by both methods. Only one isolate showed higher MIC value by agar dilution method than by E test. Among 53 strains for which the E test MIC was >2 μg/ ml [32 strains (60.37%)], only 1 (1.88%) had agar dilution MIC result of>2 μg/ml. All these 32 strains are vancomycin intermediate S. aureus vancomycin intermediate staphylococcus aureus (VISA) strains according to CLSI breakpoints. Out of 53 strains, 52 (98.11%) were having MIC of ≤2 μg/mlby agar dilution method, whereas only 21 (39.62%) strains were having MIC of ≤ 2 μg/mlby E test method.
The CLSI 1 defined resistance breakpoints for MIC and disc diffusion testing of vancomycin against S. aureus over 20 years ago. Subsequently, in 2006, the CLSI redefined vancomycin breakpoints, according to which the current definition for VISA is an S. aureus isolate with a vancomycin broth MIC of 4-8 μg/mlsince they use doubling dilutions of antibiotic (otherwise all isolates with MIC between 2 and 16 μg/mlare called VISA strains; strains with MIC ≤ 2 μg/mlare sensitive and ≥ 16 μg/mlare resistant.) The rationale for changing breakpoints was an increasing association between a vancomycin MIC of 4 μg/ ml and vancomycin treatment failure and also the increased detection of heteroresistant strains. 
Although the emergence of vancomycin-intermediate S. aureus strains and that of vancomycin-resistant S. aureus strains are reasons for concern, these organisms still are extremely rare.  Nevertheless, a number of studies have demonstrated increased clinical failure with MRSA isolates for which vancomycin MICs are increased (>1 μg/ml) but still within the susceptibility range (≤ 2 μg/ml). ,
Automated systems, which used to perform susceptibility testing, do not provide a precise vancomycin MIC. Therefore laboratories are using alternative methods for testing vancomycin in selected cases.  The E test method is an alternative and feasible option for vancomycin testing since it is easy to perform and cost-effective for testing only one drug for one strain. Interpretation of results is also easy. We recommend using E test as a routine test for determining MIC because of the above mentioned reasons. Agar dilution test should be done on all those strains showing higher MIC (intermediate or resistant results) by E test. Routine use of agar dilution is cumbersome and labour intensive. However, the results of the present study clearly shows that the E test provides vancomycin MIC results consistently higher (by 0.5 to 2 log 2 dilutions) than those provided by reference agar dilution tests. If differences of only one dilution are relevant to predicting clinical outcomes of MRSA infections, the MIC method used is a critical part of the equation. Furthermore, the fact that the higher vancomycin MIC results provided by the E test appear to be more reliable in predicting vancomycin treatment responses,  which indicates that the vancomycin susceptibility breakpoint should be revaluated.
| ~ References|| |
|1.||CLSI. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard. CLSI document M7-A8. 7th ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2009. |
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|3.||Neoh HM, Hori S, Komatsu M, Oguri T, Takeuchi F, Cui L, et al. Impact of reduced vancomycin susceptibility on the therapeutic outcome of MRSA bloodstream infections. Ann Clin Microbiol Antimicrob 2007;6:13. |
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[Figure 1], [Figure 2]
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