Molecular Investigations of Linezolid Resistance in Enterococci OptrA Variants from a Hospital in Shanghai

PURPOSE: The OptrA protein is a member of ATP-binding cassette (ABC) transporters, a transporter family which can confer resistance to oxazolidinone antibiotics by transferring plasmid. We aim to describe the distribution of optrA-harbored Enterococcus in Huashan hospital in 2017 and to address the effects of optrA mutations on the susceptibility of linezolid antibiotic drug. METHODS: Linezolid-resistance-related genes were tested for Enterococcus by polymerase chain reaction (PCR) and then sequenced for amino acid substitution site analysis. Broth microdilution and agar dilution test were applied to determine the minimal inhibitory concentration (MIC) of linezolid for Enterococcus containing optrA. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to evaluate the genotypes of optrA-positive isolates. To verify the functions of two main optrA variants, optrA over-expressing strains were constructed. RESULTS: Among 20 optrA-positive strains, only two were resistant to linezolid. No amino acid substitution existed in 23S rRNA V domain among Enterococcus faecalis. None had cfr, cfr(B) or cfr(C) genes. F101L and G4D/K/R or T150A were the main substitutions of ribosomal protein L4, L3, respectively. We found one Enterococcus faecium isolate co-contained optrA and poxtA and another E. faecalis isolate co-contained optrA and cfr(D), but they were not resistant to linezolid. Among 20 optrA-positive strains, ST-16 was the main type. Two main optrA variants KD (T112K, Y176D) and RDK (I104R, Y176D, E256K) slightly raised enterococci’s MIC of linezolid. CONCLUSION: OptrA exists in linezolid non-resistant enterococci with diverse amino acid substitutions. The variants play different roles in changing the MIC of linezolid.