Whole-Genome Sequencing of Linezolid-Resistant and Linezolid-Intermediate Susceptibility Enterococcus faecalis Clinical Isolates in a Mexican Tertiary Care University Hospital

Background: Linezolid-non-susceptible Enterococcus faecalis (LNSEf) has emerged as a critical clinical concern worldwide; however, data from Latin American settings remain scarce. Methods: We investigated 392 non-duplicated E. faecalis isolates collected over 12 months in a Mexican tertiary care hospital. Antimicrobial susceptibility was assessed phenotypically, and 24 isolates with linezolid minimum inhibitory concentrations ≥4 µg/mL underwent whole-genome sequencing. Bioinformatic analyses included multilocus sequence typing (MLST), detection of linezolid resistance mechanisms (optrA, cfrA, 23S rRNA mutations), and phylogenetic reconstruction. Results: Of the 392 isolates, 6.12% demonstrated linezolid non-susceptibility, predominantly associated with plasmid- or chromosomally encoded optrA, with only two isolates harboring cfrA. No mutations were detected in 23S rRNA domain V or ribosomal proteins L3/L4. Clinically, LNSEf isolates were linked to immunosuppression, prior surgical procedures, and extended hospital stays. MLST revealed broad genetic diversity, indicating both clonal expansion and independent acquisitions of resistance. Despite retaining susceptibility to ampicillin, vancomycin, and daptomycin, most LNSEf isolates possessed extensive resistance to other classes of antibiotics, particularly aminoglycosides and fluoroquinolones. Conclusions: LNSEf poses a growing threat in this region, driven largely by optrA. Strengthened genomic surveillance, stringent infection control, and optimized antimicrobial stewardship are vital to contain its spread.