Antimicrobial Resistance Profiling of Enterococcus

Minimum inhibitory concentrations (MICs) to ampicillin, gentamicin, vancomycin, teicoplanin, ciprofloxacin, tigecycline, linezolid, daptomycin and quinupristin/dalfopristin were examined by E-test (Liofilchem, Italy). MICs results were interpreted according to the recommendations of The European Committee on Antimicrobial Susceptibility Testing (EUCAST Breakpoint tables for interpretation of MICs and zone diameters, version 11.0, 2021, http://www.eucast.org/clinical_breakpoints/). The Clinical and Laboratory Standards Institute (CLSI) guidelines, 2021, https://clsi.org/standards/ were used to interpret the MICs for daptomycin. The presence of vanABCDMN genes was investigated by colony multiplex PCR assay using the primer sequences and PCR protocol described by Nomura et al. [22 (link)]. Briefly, a modified PCR mix for detection of the investigated genes was applied containing 0.4 µM (each) primer, 200 µM (each) dNTP, 1 U of Taq (Canvax, Spain), 1X reaction buffer, 2.5 mM MgCl₂, ultrapure PCR H₂O and 10 ng DNA template to a final volume of 20 µL. The PCR thermal conditions consisted of initial denaturation (94 °C for 4 min), followed by 30 cycles of denaturation (94 °C for 30 s), annealing (62 °C for 35 s) and extension (68 °C for 1 min), with a single final extension of 7 min at 68 °C. The amplified PCR products were analyzed by capillary electrophoresis.