Transferable optrA-mediated linezolid resistance in enterococci from high-altitude river sediments of Gilgit-Baltistan, Pakistan

Unlabelled: Environmental dissemination of antimicrobial resistance is a growing global concern. This study investigated the occurrence, genetic context, and mobility of the oxazolidinone resistance genes in enterococci isolated from high-altitude river sediments in the Gilgit-Baltistan region of Pakistan. A total of 17 florfenicol-resistant enterococci were recovered from 3 of the 12 sampling sites analyzed. Four multidrug-resistant strains (two Enterococcus faecium, one E. faecalis, and one E. hirae) harboring the optrA gene were selected for whole-genome sequencing using Illumina and Nanopore platforms. Genomic analyses showed that optrA was plasmid-borne in all isolates and co-localized with additional resistance genes, including macrolide, phenicol, and tetracycline resistance determinants. The optrA genetic context was frequently associated with insertion sequences, suggesting their role in the mobilization of multidrug-resistance elements. Mini-translocatable units of the optrA genetic context were detected as circular forms in two of four strains, further supporting their mobility potential. Conjugation assays demonstrated successful horizontal transfer of the optrA gene mediating linezolid resistance, with transfer frequencies ranging from 2.03 × 10⁻⁶ to 6.36 × 10⁻⁴ transconjugants per recipient cell. optrA-carrying plasmids remained stable after serial passages without antibiotic pressure. Phylogenetic analysis showed that isolates belonged to distinct sequence types and clustered with strains of diverse geographic and ecological origins, suggesting dissemination of optrA across different genetic backgrounds rather than clonal expansion. The occurrence of linezolid-resistant enterococci in high-altitude river sediments suggests their widespread distribution and emphasizes the need for broader environmental surveillance programs to monitor clinically relevant resistances even in remote ecosystems. Importance: This study provides important insights into the environmental dissemination of antimicrobial resistance by demonstrating the occurrence and genetic diversity of clinically relevant resistance determinants in enterococci isolated from remote high-altitude river sediments. The findings emphasize that natural environments could serve as reservoirs and exchange hubs for antimicrobial resistance, even in ecosystems with limited direct anthropogenic impact. Therefore, integrated One Health surveillance strategies that include environmental compartments alongside human and animal populations are required to better monitor and mitigate the emergence of clinically significant resistant bacteria.