<i>In vivo</i> development of elevated linezolid resistance mediated by a deletion in ribosomal protein L3 in clinical <i>Enterococcus faecium</i>

Gram-positive bacteria can develop resistance to linezolid through mutations in the 50S large-subunit ribosomal proteins, such as L3. However, this mechanism is rarely reported in clinical enterococcal strains. Here, we describe a pair of <i>Enterococcus faecium</i> strains, 1505efm and 1583efm, isolated from a meningioma patient at different stages of linezolid treatment. Strain 1583efm, which was obtained after 17 days of linezolid treatment, exhibited an eightfold higher linezolid MIC (64 mg/L) than the initially isolated strain 1505efm (8 mg/L). Meanwhile, 1583efm became susceptible to erythromycin, tetracycline, and high-level streptomycin due to the loss of plasmid-borne resistance genes <i>erm(A)</i>, <i>erm(B)</i>, <i>tet(M)</i>, <i>ant(6)-Ia</i>, and <i>aph(3')-III</i>. Whole-genome comparison revealed a deletion in the <i>rplC</i> gene of 1583efm, resulting in the loss of 21 amino acids (∆Tyr139-Arg159) in ribosomal protein L3. Structural modeling showed that this deletion disrupts the linezolid hydrogen bond network near the ribosomal peptidyl transferase center, reducing its binding affinity. The mutation, which remained stable over 50 generations without antibiotics, imposed a significant fitness cost on the bacterial cells and led to an increase in the expression of the truncated <i>rplC</i> gene. Our study uncovers a novel linezolid resistance mechanism involving the deletion of ribosomal protein L3 in clinical <i>E. faecium</i> strains within the host.