Allele-specific effects of mutations in the rifampin resistance-determining region (RRDR) of RpoB on physiology and antibiotic resistance in <i>Enterococcus faecium</i>

<i>Enterococcus faecium</i> is a member of the human gut microbiota that has evolved into a problematic nosocomial pathogen and a leading cause of infections in hospitalized patients. Treatment of <i>E. faecium</i> infections is complicated by antibiotic resistance, making it important to understand resistance mechanisms and their broader consequences in this pathogen. Here, we explored the collateral effects of rifampin resistance-associated mutations in the <i>E. faecium</i> RNA polymerase β-subunit (RpoB). Of 14,384 publicly available <i>E. faecium</i> genomes, nearly one-third carried a mutation in the rifampin resistance-determining region (RRDR) of RpoB. In a local population of 710 <i>E. faecium</i> clinical isolates collected from patients at a single hospital, we found significant associations between the presence of RRDR mutations and prior exposure to rifamycin antibiotics, as well as associations between RRDR mutations and altered daptomycin susceptibility. To investigate the phenotypic impacts of RRDR mutations, we generated and studied four isogenic strains with distinct RRDR mutations (Q473K, G482D, H486Y, and S491L) that overlapped with clinical isolate variants. Transcriptomic and phenotypic analyses revealed allele-specific effects on <i>E. faecium</i> gene expression, growth dynamics, antibiotic susceptibility, isopropanol tolerance, and cell wall physiology. One frequently observed mutation, H486Y, caused minimal transcriptional changes and enhanced bacterial fitness under antibiotic stress. In contrast, the S491L mutation induced extensive transcriptional changes and slowed bacterial growth but also conferred increased isopropanol tolerance, potentially enhancing bacterial survival on hospital surfaces. Overall, our findings highlight the multifaceted impacts of RRDR mutations in shaping <i>E. faecium</i> physiology and antibiotic resistance, two important features of this hospital-associated pathogen.IMPORTANCEUnderstanding how antimicrobial resistance affects bacterial physiology is critical for developing effective therapeutics against bacterial infections. In this study, we found that rifampin resistance-associated mutations in RpoB are widespread in <i>Enterococcus faecium,</i> a leading multidrug-resistant pathogen. By studying isogenic wild-type and RpoB mutant strains, we discovered that RpoB mutations, although conferring resistance to rifampin, have distinct allele-specific effects on other bacterial phenotypes. Some of these collateral effects appear to promote <i>E. faecium</i> resistance to antibiotics and survival in the hospital environment, raising questions about the selective pressures driving their emergence. Overall, our study underscores the importance of examining the collateral effects of resistance-associated mutations in multidrug-resistant pathogens, which could help mitigate their persistence and spread among vulnerable patients.