Evolution of chlorhexidine susceptibility and of the EfrEF operon among Enterococcus faecalis from diverse environments, clones and time spans

Chlorhexidine (CHX) is widely used to control the spread of pathogens (e.g. human/animal clinical settings, ambulatory care, food industry). E. faecalis, a major nosocomial pathogen, is broadly distributed in diverse hosts and environments facilitating its exposure to CHX over the years. Nevertheless, CHX activity against E. faecalis is understudied. Our goal was to assess CHX activity and the variability of ChlR-EfrEF proteins (associated with CHX tolerance) among 673 field isolates and 1784 E. faecalis genomes from PATRIC database from different sources, time spans, clonal lineages and antibiotic resistance profiles. CHX minimum inhibitory concentrations (MICCHX) and minimum bactericidal concentrations (MBCCHX) against E. faecalis presented normal distributions (0.5-64 mg/L). However, more CHX tolerant isolates were detected in the food chain and recent human infections, suggesting an adaptability of E. faecalis populations in settings where CHX is heavily used. Heterogeneity in ChlR-EfrEF sequences was identified, with isolates harboring incomplete ChlR-EfrEF proteins, particularly the EfrE identified in the ST40 clonal lineage, showing low MICCHX ([≤]1mg/L). Distinct ST40-E. faecalis subpopulations carrying truncated and non-truncated EfrE were detected, the former being predominant in human isolates. This study provides a new insight about CHX susceptibility and ChlR-EfrEF variability within diverse E. faecalis populations. The MICCHX/MBCCHX of more tolerant E. faecalis (MICCHX=8mg/L; MBCCHX=64mg/L) remain lower than in-use concentrations of CHX (>500mg/L). However, CHX increasing use combined with concentration gradients occurring in diverse environments potentially selecting multidrug-resistant strains with different CHX susceptibilities, alert to the importance of monitoring the trends of E. faecalis CHX tolerance within a One-Health approach.