Abstract

Chlorhexidine (CHX) is a broad-spectrum antiseptic widely used in community and clinical contexts for many years, recently acquiring higher relevance in nosocomial infections control worldwide. Despite of this, CHX tolerance has been poorly understood among Enterococcus faecium, one of the leading agents causing nosocomial infections. This study provides new phenotypic and molecular data for a better identification of CHX tolerant E. faecium subpopulations in community and clinical contexts. MICCHX distribution of 106 E. faecium suggested the occurrence of tolerant subpopulations in diverse sources (human, animal, food, environment) and phylogenomic backgrounds (clades A1/A2/B), with predominance in clade A1. They carried a specific variant of 2CS-CHXT operon, here identified. It encodes a glucose and an amino-acid-polyamine-organocation family transporters, besides the DNA-binding-response-regulator ChtR with a P102H mutation previously described in only CHX tolerant clade A1 E. faecium, and the ChtS sensor. Combined data from normal MIC distribution and 2CS-CHXT operon characterization supports a tentative epidemiological cut-off (ECOFF) of 8 mg/L to CHX, useful to detect tolerant E. faecium populations in future surveillance studies. The spread of tolerant E. faecium in diverse epidemiological backgrounds alerts for a prudent use of CHX in multiple contexts.\n\nImportanceChlorhexidine is one of the substances included in the World Health Organizations List of Essential Medicines which comprises the safest and most effective medicines needed in global health systems. Although it has been widely applied as a disinfectant and antiseptic since the 1950s in healthcare (skin, hands, mouthwashes, eye drops), its use in hospitals to prevent nosocomial infections has increased worldwide in recent years. Here we provide a comprehensive study on chlorhexidine tolerance among Enterococcus faecium, one of the leading nosocomial agents worldwide, and identified a novel 2CS-CHXT operon as a signature of tolerant strains occurring in diverse phylogenomic groups. Finally, our data allowed to propose a tentative epidemiological cut-off of 8 mg/L, useful to detect tolerant E. faecium populations in future surveillance studies in community and clinical contexts.