Optimized Plasmid Extraction Uncovers Novel and Mobilizable Plasmids in Staphylococcus nepalensis Sharing Antimicrobial Resistance Across Different Bacterial Genera.

Plasmids are key vectors in the dissemination of antimicrobial resistance (AMR), often transcending species and genus boundaries through horizontal gene transfer. Staphylococcus nepalensis, typically regarded as a commensal species, has emerged as a potential reservoir of resistance genes. In this study, we optimized plasmid extraction protocols to enhance the recovery of low-copy plasmids and applied whole-genome sequencing to characterize plasmids from a S. nepalensis strain isolated from the oral microbiota of a healthy cat in Brazil. Plasmid-enriched extraction using the Qiagen miniprep kit, with an additional enzymatic lysis step, significantly improved assembly outcomes, enabling the recovery of four complete plasmids. Three of them carried mobilizable antimicrobial resistance genes (aadK, cat, and tetK), conferring resistance to streptomycin, chloramphenicol, and tetracycline, respectively. Comparative and phylogenetic analyses revealed a high sequence similarity between these plasmids and mobile elements found in diverse pathogenic and environmental bacteria, including Staphylococcus aureus, S. epidermidis, Enterococcus sp., and Pseudomonas aeruginosa, indicating plasmid circulation across bacterial genera. Additionally, one novel plasmid was identified, displaying limited similarity to any known sequence and suggesting the existence of uncharacterized plasmid lineages in commensal staphylococci. These findings highlight the underestimated role of S. nepalensis as a hidden reservoir of mobilizable resistance genes and reinforce the need to surveil non-pathogenic bacteria in AMR monitoring frameworks.