Molecular Mechanisms of Linezolid Resistance

Mechanism of resistance to linezolid was determined by PCR (cfr, cfrB, oprA, and poxtA) and Sanger sequencing (23S rRNA, rplC, rplD, and rplV). DNA extraction was obtained from a fresh culture (24 hours) according to the manufacturer’s protocol (GenElute TM Bacterial Genomic DNA Kits, Sigma Aldrich, St. Louis, MO, USA). The list of enterococcal specific primers and settings of PCR used in the study are given in the Supplementary Table S2. PCR products of cfr, oprA were resolved in 1.5% agarose (TopVision Agarose, Thermo Scientific, St. Louis, MO, USA) in electrophoresis (5 V/cm) for 45 minutes. NCTC13923 was used as control strain for optrA detection in this study. The detection of cfr, poxtA, and cfrB was performed according to procedures as described previously [8 (link),12 (link),35 (link)]. The amplified fragments of the 23S rRNA, rplC, rplD, and rplV were sequenced by analyzer Applied Biosystems 3130xL. Point mutation/s associated with linezolid resistance were analyzed using software Bionumerics 7.6.2 (Applied Maths, Ghent, East Flanders, Belgium).