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). Enterococcus 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 1,784 E. faecalis genomes from the PATRIC database from different sources, time spans, clonal lineages, and antibiotic-resistance profiles. The CHX MIC (MIC(CHX)) and minimum bactericidal concentration (MBC(CHX)) against E. faecalis presented normal distributions (0.5 to 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 MIC(CHX) (≤1mg/L). Distinct ST40-E. faecalis subpopulations carrying truncated and nontruncated EfrE were detected, with 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 MIC(CHX)/MBC(CHX) of more tolerant E. faecalis (MIC(CHX) = 8 mg/L; MBC(CHX) = 64 mg/L) remain lower than in-use concentrations of CHX (≥500 mg/L). However, increased CHX use, combined with concentration gradients occurring in diverse environments, potentially selecting multidrug-resistant strains with different CHX susceptibilities, signals the importance of monitoring the trends of E. faecalis CHX tolerance within a One Health approach. IMPORTANCE Chlorhexidine (CHX) is a disinfectant and antiseptic used since the 1950s and included in the World Health Organization’s list of essential medicines. It has been widely applied in hospitals, the community, the food industry, animal husbandry and pets. CHX tolerance in Enterococcus faecalis, a ubiquitous bacterium and one of the leading causes of human hospital-acquired infections, remains underexplored. Our study provides novel and comprehensive insights about CHX susceptibility within the E. faecalis population structure context, revealing more CHX-tolerant subpopulations from the food chain and recent human infections. We further show a detailed analysis of the genetic diversity of the efrEF operon (previously associated with E. faecalis CHX tolerance) and its correlation with CHX phenotypes. The recent strains with a higher tolerance to CHX and the multiple sources where bacteria are exposed to this biocide alert us to the need for the continuous monitoring of E. faecalis adaptation toward CHX tolerance within a One Health approach.