Single point mutations in global regulatory genes restore cephalosporin resistance in a low-MIC <i>Enterococcus faecium</i> natural isolate

<i>Enterococcus faecium</i> exhibits intrinsic resistance to cephalosporins (CPHs), yet the genetic determinants of this phenotype remain incompletely understood. To date, <i>E. faecium</i> strains with low minimum inhibitory concentrations (MICs) of CPH have only been described following genetic manipulation. At Parc Taulí University Hospital, we identified a clinical isolate of ampicillin-susceptible <i>E. faecium</i> (Efm5) that exhibited unusually low MICs to cefotaxime (1 mg/L), ceftriaxone (3 mg/L), and ceftaroline (0.19 mg/L). Upon single exposure to ceftriaxone (100 mg/L), Efm5 rapidly yielded variants with markedly increased MICs of ceftriaxone (>256 mg/L) and cefotaxime (>32 mg/L), while MICs of ampicillin and ceftaroline were unaffected. Whole-genome sequencing revealed that the high-MIC variants carried single-nucleotide polymorphisms leading to non-synonymous mutations in <i>croS</i>, <i>nusG</i>, or <i>rpoB</i> genes. Phenotypic assays confirmed that these mutations were associated with ceftriaxone resistance, and immunoblots revealed increased expression of penicillin-binding protein 5 (PBP5) in all the high-MIC variants. Transcriptional profiling showed upregulation of the <i>pbp5</i> operon, which includes <i>ftsW</i>, <i>psr</i>, and <i>pbp5</i>, in the <i>croS</i> variants. This study provides evidence that <i>E. faecium</i> isolates with low MICs to CPH can arise in clinical settings without laboratory manipulation, enabling further investigation of resistance pathways.