Insights towards the impact of subinhibitory chlorhexidine on antimicrobial susceptibility and horizontal gene transfer in Enterococcus faecium

Enterococcus faecium, a human and animal commensal broadly distributed in the environment, is currently one of the most challenging multidrug-resistant (MDR) healthcare-associated pathogens worldwide. It is often exposed to chlorhexidine (CHX), a broad-spectrum antiseptic, extensively used in healthcare, domestic, and food production settings, and a diffused polluter. However, the impact of gradients of CHX concentrations, including at subinhibitory levels, on E. faecium adaptation to various antimicrobials remains unclear. Our study aimed to explore the effects of subinhibitory CHX concentrations on biocides and antibiotics susceptibility as well as in the transfer of clinically relevant antibiotic resistance genes among E. faecium (n = 11) from diverse sources and clonal backgrounds. Serial exposure to increasing CHX concentrations resulted in strain-specific MIC_CHX and MBC_CHX changes among six E. faecium studied. These strains presented different CHX genotypes, namely the P102H mutation in DNA-binding response regulator ChtR in two strains showing twofold increased MIC_CHX and/or MBC_CHX, and an absent EfrEF transporter in a strain exhibiting increased CHX susceptibility after exposure. Whole-genome comparison between parental and CHX-adapted strains found no alterations in genes with a recognized role in CHX reduced susceptibility. Additionally, in a different assay, subinhibitory CHX exposure enhanced the transfer (up to 12.5-fold) of vancomycin or linezolid resistance genes among most E. faecium strains tested, except one lacking a functional EfrEF transporter. Our data suggest that subinhibitory CHX concentrations could have a role in Enterococcus adaptation to CHX and in the spread of antibiotic resistance through horizontal transfer events. Further investigation is warranted to elucidate the underlying mechanisms driving these phenomena in E. faecium, ensuring the continued effectiveness of both CHX and antibiotics, and safeguarding Public Health.