Genetic Characterization of a Linezolid‐ and Penicillin‐Resistant <i>Enterococcus hirae</i> Isolate Co‐Harboring <i>poxtA</i> and <i>pbp5fm</i>

Linezolid and penicillin are critical for treating multidrug resistant (MDR) Gram-positive infections, but the emergence of resistance to both seriously threatens public health. Here, we first report the cocarrying <i>poxtA</i> (oxazolidinone resistance) and <i>pbp5fm</i> (β-lactam resistance) genes by the plasmid in a strain of <i>Enterococcus hirae</i> HDC14-2 derived from porcine. The isolate also exhibits MDR phenotypes to phenicols, oxazolidinones, tetracyclines, β-lactams, aminoglycosides, macrolides, and lincosamides. Whole-genome sequencing (WGS) revealed these resistance genes, along with <i>tet</i>(<i>L</i>), <i>tet</i>(<i>M</i>), <i>catA</i>, <i>erm</i>(B), <i>aac</i>(<i>6</i>)-<i>aph</i>(<i>2"</i>), <i>aadE</i>, <i>spw</i>, <i>lsa</i>(E), <i>lnu</i>(B), <i>sat4</i>, and <i>aphA3</i>, were clustered in a novel MDR region flanked by IS<i>1216</i> elements on plasmid pHDC14-2.133K. This IS<i>1216</i>-bounded MDR region formed translocatable units (TUs), including an IS<i>1216</i>-<i>poxtA</i> TU that was also identified on a secondary plasmid, pHDC14-2.27K. Functional assays demonstrated the excisability and mobility of these TUs, indicating its potential ability integration into other plasmids or chromosomes. Critically, electrotransformation confirmed the transfer of pHDC14-2.27K (<i>poxtA</i>-carrying) to <i>Enterococcus faecalis</i> JH2-2, with retained TU activity and minimal fitness cost. This study provides the evidence of colocalized <i>poxtA</i> and <i>pbp5fm</i> on plasmids in enterococci, highlighting their role in disseminating pan-resistance among bacteria. Although <i>E. hirae</i> is not an important pathogenic bacterium to humans and animals, but its potential risk to horizontally spread of these resistance genes important in medicine still cannot be ignored.