2.2.4. Antimicrobial susceptibility profiling

The antimicrobial susceptibility testing (AST) was performed according to the standards recommended by the Clinical and Laboratory Standards Institute (CLSI) (2012) on Performance Standards for Antimicrobial Susceptibility Testing for Enterococcus spp. The assay was performed by microbroth dilution using the antibiotics ampicillin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamycin, kanamycin, streptomycin, tetracycline (all from Sigma-Aldrich), and vancomycin (CheilJedang Pharma Co., Republic of Korea) on cation-adjusted Mueller-Hinton broth supplemented with MRS (5.0 g/L). The assay was performed in a 96-well microplate (SPL Life sciences, Pocheon-si, Gyeonggi-do, Republic of Korea) and comprised 10 antibiotic dilutions in two-fold and controls (growth and sterility controls). The inocula were adjusted to 0.5 McFarland units (approximately 10⁷ CFU/mL) and distributed accordingly to obtain a final concentration of 10⁵ CFU/mL. The plates were incubated at 35 ± 1 °C for 18 h. The lowest concentration with complete bacterial inhibition was recorded as the MIC and analyzed according to the standards set for Enterococcus spp. (Rychen et al., 2017; Wiegand et al., 2008).