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

Enterococcus faecium exhibits intrinsic resistance to cephalosporins (CPH), yet the genetic determinants of this phenotype remain incompletely understood. To date, E. faecium strains with low minimum inhibitory concentrations (MICs) to CPH have only been described following genetic manipulation. At Parc Taulí University Hospital, we identified a clinical isolate of ampicillin-susceptible E. faecium (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 to ceftriaxone (>256 mg/L) and cefotaxime (>32 mg/L), while MICs to ampicillin and ceftaroline were unaffected. Whole-genome sequencing revealed that the high-MIC variants carried single nucleotide polymorphisms (SNPs) leading to non-synonymous mutations in croS , nusG or rpoB 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 pbp5 operon, which includes ftsW , psr and pbp5 , in the croS variants. This study provides the first evidence that E. faecium isolates with low-MICs to CPH can arise in clinical settings without laboratory manipulation, challenging the prevailing notion of absolute intrinsic CPH resistance in this species and offering a novel framework to explore previously unrecognized resistance pathways.