Antimicrobial susceptibility testing, minimum inhibitory concentrations determination, and detection of heterogeneous vancomycin-intermediate Staphylococcus aureus

Susceptibility testing of S. aureus to the primary antimicrobial agents was performed by disk diffusion testing on Mueller-Hinton agar (MHA) as per the latest CLSI guidelines.[21] All the disks were procured from HiMedia, Mumbai, Maharashtra, India. MRSA were identified by cefoxitin (30 μg) disk testing and cefoxitin MIC by Etest (HiMedia, Mumbai, Maharashtra, India) as per the CLSI 2015 (zone diameter, sensitive ≥22 mm and resistant ≤21 mm; MIC, sensitive ≤4 μg/ml and resistant ≥8 μg/ml).[21] Inducible clindamycin resistance in isolates displaying erythromycin resistance was detected by the D-test.[21]

Susceptibility testing to vancomycin was performed to determine the MIC by both Etest (HiMedia, Mumbai, Maharashtra, India) and agar dilution with incorporation of intermediate dilutions in the agar dilution test for better correlation between the two methods of testing. In case of discrepancies between Etest and agar dilution, the values of Etest MICs were deemed to be final because Etest has been found to be a more sensitive method for MIC determination and considered the best technique to assess exact MIC as it allows for visualization of small colonies around the zones of inhibition.[22] With Etest as the gold standard for susceptibility testing, detection of hVISA was done by GRD Etest on MHA with a 5% blood agar plate according to the manufacturer's instructions (HiMedia, Mumbai, Maharashtra, India) in strains with vancomycin MIC 1–2 μg/ml.[11,22] The test isolate was considered positive for hVISA if the Etest GRD strip result was ≥8 μg/ml for vancomycin or teicoplanin.[11,22] Isolates displaying either VISA (vancomycin MIC >2–< 16 μg/ml) or hVISA (detected by GRD Etest) phenotype were designated as strains with RVS.[4]

Minimum inhibitory concentration was also determined for alternative therapeutic options such as linezolid, daptomycin, and tigecycline by Etest (HiMedia, Mumbai, Maharashtra, India) and interpreted as per the CLSI guidelines,[21] except tigecycline (for which CLSI currently does not provide interpretative zone diameters). S. aureus strains ATCC 25923, ATCC 51299, and ATCC 43300 were used as quality control strains for disk diffusion, MIC testing, and MRSA testing, respectively. Tigecycline susceptibility was interpreted as ≥19 mm zone diameter and ≤0.5 μg/ml MIC breakpoint as per the US Food and Drug Administration guidelines, respectively.[23] Strains with intermediate resistance were included in the percentage of resistant isolates. Multidrug resistance was defined as nonsusceptibility to at least one agent in three or more antimicrobial categories.[24]