Development, optimization, standardization, and validation of a simple in-house agar gradient method to determine minimum inhibitory concentration of vancomycin for Staphylococcus aureus

Abstract

Background: The Clinical and Laboratory Standards Institute recommends reporting minimum inhibitory concentration (MIC) values of vancomycin for Staphylococcus aureus. Commercial MIC strips are expensive, and the traditional broth microdilution method is cumbersome. With this background, we attempted to develop and standardize an in-house agar gradient method to determine MIC values of vancomycin for S. aureus.

Objectives: To develop and validate an in-house vancomycin MIC strip, based on simple agar gradient method for S. aureus as per bioassay development guidelines.

Materials and methods: Filter paper gradient strips were made in house and impregnated with varying concentrations of vancomycin to create an antibiotic gradient. During standardization, MICs of ninety clinical strains of S. aureus and ATCC 29213 were tested by the broth microdilution and commercial strip followed by the in-house strip. During the validation stage, MICs of ninety different clinical strains of S. aureus and ATCC 29213 were determined by the in-house strip followed by MIC detection by broth microdilution and commercial strips. A reading of more than ± 1log₂ dilution compared with broth microdilution was considered as an outlier.

Results: During the initial stage, there were 7/90 outliers in the clinical strains, and no outliers were seen with the ATCC 29213 control strain. Corrective action included increasing precaution during the antibiotic impregnation on the strip. During validation stage, only 4/90 outliers were observed in the clinical strains. The commercial strips had 29/90 among clinical and 15/30 outliers in the control strain during the prevalidation phase. Despite maintaining cold chain during the validation phase, the outliers for commercial strip were 18/90 and 4/30 for clinical and control strains, respectively.

Conclusion: Reporting vancomycin MIC for S. aureus may be attempted using the in-house method after validating it with a gold standard broth microdilution method and quality control as per protocol.

Keywords: AMR; Epsilometer test; antimicrobial testing; in-house agar gradient method; minimum inhibitory concentration; vancomycin.

Figure 1

The dimensions of the in-house strip and its relative size compared to the commercially available strip placed together on the same plate to avoid inoculum variation

Figure 2

How the 8-channel micropipette was used to aspirate 5 μL of antibiotic solution to create a gradient along the strip as in (a) and visual confirmation was done to check equal volume being impregnated on every spot of the strip (arrows) as in (b)

Figure 3

How the impregnation was done exactly at the center (arrows) of each point on the strip

Figure 4

(a and b) How to take the reading of the strip. If there was growth in hole separating the two breakpoints, the higher breakpoint was considered as the breakpoint minimum inhibitory concentration as in a, there is growth adjacent to 0.5 μg/mL; therefore, the minimum inhibitory concentration is 1 μg/mL. Similar is the case with b

Figure 5

(a and b) Gross discrepancy among minimum inhibitory concentration values from the commercial strip (higher) against the in-house strip

Figure 6

(a and b) When the impregnation technique was faulty, there were gross errors in the zones meeting on both sides