The Two-Component System ChtRS Contributes to Chlorhexidine Tolerance in Enterococcus faecium

Abstract

Enterococcus faecium is one of the primary causes of nosocomial infections. Disinfectants are commonly used to prevent infections with multidrugresistant E. faecium in hospitals. Worryingly, E. faecium strains that exhibit tolerance to disinfectants have already been described. We aimed to identify and characterize E. faecium genes that contribute to tolerance to the disinfectant chlorhexidine (CHX). We used a transposon mutant library, constructed in a multidrug-resistant E. faecium bloodstream isolate, to perform a genome-wide screen to identify genetic determinants involved in tolerance to CHX. We identified a putative two-component system (2CS), composed of a putative sensor histidine kinase (ChtS) and a cognate DNAbinding response regulator (ChtR), which contributed to CHX tolerance in E. faecium. Targeted chtR and chtS deletion mutants exhibited compromised growth in the presence of CHX. Growth of the chtR and chtS mutants was also affected in the presence of the antibiotic bacitracin. The CHX-and bacitracin-tolerant phenotype of E. faecium E1162 was linked to a unique, nonsynonymous single nucleotide polymorphism in chtR. Transmission electron microscopy showed that upon challenge with CHX, the ΔchtR and ΔchtS mutants failed to divide properly and formed long chains. Normal growth and cell morphology were restored when the mutations were complemented in trans. Morphological abnormalities were also observed upon exposure of the ΔchtR and ΔchtS mutants to bacitracin. The tolerance to both chlorhexidine and bacitracin provided by ChtRS in E. faecium highlights the overlap between responses to disinfectants and antibiotics and the potential for the development of cross-tolerance for these classes of antimicrobials.

Spontaneously Arising Streptococcus mutans Variants with Reduced Susceptibility to Chlorhexidine Display Genetic Defects and Diminished Fitness

“…Earlier studies with other oral streptococci, including Streptococcus sanguinis and Streptococcus mitis, showed that repeated exposure to CHX resulted in increased MICs to CHX, but these findings were not deemed to be of clinical significance (13)(14)(15)(16)(17). In enterococci, increased tolerance to antiseptics such as CHX in hospital settings has inspired research into the mechanisms for increased CHX tolerance, including studies of CHX effects on the transcriptome (18) and analysis of the impact of CHX on a transposon mutant library (19). More specifically, Bhardwaj and colleagues (18) used transcriptome sequencing (RNA-Seq) to show that VanA-type vancomycin resistance genes are upregulated in Enterococcus faecium following exposure to CHX.…”

“…More specifically, Bhardwaj and colleagues (18) used transcriptome sequencing (RNA-Seq) to show that VanA-type vancomycin resistance genes are upregulated in Enterococcus faecium following exposure to CHX. In E. faecium, two genes encoding a two-component regulatory system were found to contribute to CHX tolerance (19). Thus, it is clear that Firmicutes have the capacity to develop increased tolerance to CHX, but the mechanisms by which tolerance may develop, especially in streptococci, are only beginning to be explored.…”

Reduced chlorhexidine and daptomycin susceptibility arises in vancomycin-resistantEnterococcus faeciumafter serial chlorhexidine exposure

“…The two main mechanisms include CHX efflux (23, 42-44, 64-67) and changes in outer membrane content (68, 69). The recently identified two-component system ChtRS contributes to CHX tolerance in E. faecium , presumably via regulation of expression of genes in its regulon, which is currently undefined (70). In this study, we have confirmed a role for the heterodimeric ABC transporter in CHX susceptibility in VREfm.…”

Characterization of Spontaneously Arising Chlorhexidine-Tolerant Variants inStreptococcus mutans

“…However one recent study (6) reported that when CHX was continuously flowed on biofilms formed on polystyrene blocks in vitro, detached cells displayed a higher minimal inhibitory concentration (MIC) to CHX (1.25 μg mL −1 ), whereas most isolates of S. mutans evaluated had MICs < 1μg mL 1 . In enterococci, increased tolerance to antiseptics such as CHX in the hospital setting has inspired research into methods of increased CHX tolerance, including studies of CHX effects on the transcriptome (11) and on a transposon mutant library (12). More specifically, Bhardwaj and colleagues (11) used RNA-Seq to show that VanA-type vancomycin resistance genes are upregulated in Enterococcus faecium following CHX exposure.…”

“…More specifically, Bhardwaj and colleagues (11) used RNA-Seq to show that VanA-type vancomycin resistance genes are upregulated in Enterococcus faecium following CHX exposure. In E. faecium, two genes encoding for a two-component regulatory system were found to contribute to CHX tolerance (12). Thus, it is clear that Firmicutes have the capacity to develop increased tolerance to CHX, however the mechanisms by which tolerance may develop, especially in streptococci, is only beginning to be explored.…”