Standard e tests

Automated strain identification with Gram-positive (GP) Vitek 2 identification card (bioMerieux, France) according to the manufacturer’s instructions was performed. Susceptibility to methicillin was determined with cefoxitin discs (FOX 30 μg, Oxoid), according to the current EUCAST recommendations; mecA gene was detected with the PCR technique [15 (link)].
The first-line screening test to determine sensitivity to vancomycin and teicoplanin with AST-P580 cassettes in Vitek 2 system (bioMerieux, France) was performed, MIC values for VAN and TEI were determined with standard E-tests (bioMerieux, France). All strains which occurred less susceptible (even in the range of sensitivity, MIC VAN/TEI values at least 1 mg/L or higher), were classified to the further investigations. In the next step E-test GRD (glycopeptide resistance detection), (bioMerieux, France) was performed according to manufacturer’s guidelines, as well as agar dilution method, according to EUCAST ISO 20776–1. Forty seven MRSA strains that were both: positive in the GRD gradient test (MIC value equal to 8 mg/L or higher for VAN or TEI), and with MIC value for VAN ≥3 mg/L (in diagnostic manner at least 4 mg/L) in agar dilution method, were classified as an appropriate materials for analysis in this study.

Standard Etests (bioMérieux) were performed according to manufacturer’s instructions. Macro Etests were performed as previously described (4 (link)). Vancomycin population analysis profiles (PAPs) were performed as previously described (50 (link)) and by allowing 48 h of growth in order to isolate slow-growing VISA subpopulations. Broth cultures were performed in the wells of 96-well plates in 200 µl of HI broth alone and supplemented with 0.5, 1, 1.5, 2, 2.5, or 3 mg/liter of vancomycin. To avoid batch effects, all mutant strains and wild-type strain were tested with the same batch of HI broth and HI broth plus vancomycin solutions, and growth was monitored in the same 96-well plate. Overnight HI broth cultures of the strains to be tested were adjusted to an OD₆₀₀ of 5, and 2 µl was inoculated into each well. Growth was then immediately monitored at OD₆₀₀ for 36 h using a PerkinElmer EnSight plate reader.