Loss of LafB activity reverses daptomycin resistance in <i>E. faecium</i>

Infections caused by multidrug-resistant enterococci, particularly vancomycin-resistant <i>Enterococcus</i> (VRE), present significant therapeutic challenges. Daptomycin, a last-line treatment for VRE, often loses efficacy due to the emergence of resistance. In this study, we revealed the critical role of the <i>lafB</i> gene as a key determinant of daptomycin susceptibility and resistance in <i>E. faecium</i>. We showed that in the absence of a functional <i>lafB</i>, daptomycin-resistant mutants did not emerge <i>in vitro</i>, and derivatives of clinical daptomycin-resistant strains engineered to lack functional <i>lafB</i> were rendered even more sensitive to daptomycin than wild-type daptomycin-susceptible strains. These findings indicated that functional <i>lafB</i> is critical for key known mechanisms of daptomycin resistance, and mutations in <i>lafB</i> have phenotypic dominance to those that otherwise confer resistance. Therefore, inhibiting the activity of the <i>lafB</i> gene product is predicted to prevent or reverse resistance, offering a promising new strategy for extending the efficacy of daptomycin for treating enterococcal infections.IMPORTANCEDaptomycin is one of the few remaining effective antibiotics for treating vancomycin-resistant enterococcal infections but is limited by the emergence of resistance during protracted therapy. Here, we show that without a functional <i>lafB</i> gene, daptomycin-resistant mutants do not arise under conditions where wild-type strains readily generate daptomycin-resistant mutants. Furthermore, we show that loss of function mutation of the <i>lafB</i> gene in daptomycin-resistant clinical isolates renders them more susceptible to daptomycin than wild-type <i>Enterococcus faecium</i>. This indicates that an effective small molecule inhibitor of LafB activity or <i>lafB</i> gene expression would be a useful adjunctive for extending and restoring the therapeutic utility of daptomycin.