Chlorhexidine Induces VanA-Type Vancomycin Resistance Genes in Enterococci

Abstract

Chlorhexidine is a bisbiguanide antiseptic used for infection control. Vancomycin-resistantE. faecium(VREfm) is among the leading causes of hospital-acquired infections. VREfm may be exposed to chlorhexidine at supra- and subinhibitory concentrations as a result of chlorhexidine bathing and chlorhexidine-impregnated central venous catheter use. We used RNA sequencing to investigate how VREfm responds to chlorhexidine gluconate exposure. Among the 35 genes upregulated ≥10-fold after 15 min of exposure to the MIC of chlorhexidine gluconate were those encoding VanA-type vancomycin resistance (vanHAX) and those associated with reduced daptomycin susceptibility (liaXYZ). We confirmed thatvanAupregulation was not strain or species specific by querying other VanA-type VRE. VanB-type genes were not induced. ThevanHpromoter was found to be responsive to subinhibitory chlorhexidine gluconate in VREfm, as was production of the VanX protein. UsingvanHreporter experiments withBacillus subtilisand deletion analysis in VREfm, we found that this phenomenon is VanR dependent. Deletion ofvanRdid not result in increased chlorhexidine susceptibility, demonstrating thatvanHAXinduction is not protective against chlorhexidine. As expected, VanA-type VRE is more susceptible to ceftriaxone in the presence of sub-MIC chlorhexidine. Unexpectedly, VREfm is also more susceptible to vancomycin in the presence of subinhibitory chlorhexidine, suggesting that chlorhexidine-induced gene expression changes lead to additional alterations in cell wall synthesis. We conclude that chlorhexidine induces expression of VanA-type vancomycin resistance genes and genes associated with daptomycin nonsusceptibility. Overall, our results indicate that the impacts of subinhibitory chlorhexidine exposure on hospital-associated pathogens should be further investigated in laboratory studies.

FIG 1

Growth kinetics of E. faecium 1,231,410 after H-CHG treatment. (A) Representative optical density curve. E. faecium was grown at 37°C in BHI with shaking at 100 rpm until the optical density at 600 nm (OD₆₀₀) reached 0.3 to 0.4. Twenty-five milliliters of culture was added to an equal volume of prewarmed BHI containing different concentrations of H-CHG such that final concentrations of 0× (control; green circles), 1/2× (red squares), 1× (blue crosses), or 2× MIC (orange hexagons) were attained. The cultures were incubated at 37°C with shaking, and OD₆₀₀ was monitored. (B) Viability curve. Viable cell count (CFU per milliliter) for 1× MIC-treated cultures (blue crosses) and control cultures (green circles) was assessed. Error bars indicate standard deviations from 3 independent experiments. *, P < 0.05 by the Student one-tailed t test; significance was assessed only for the 15-min time point. For transcriptomic analyses, RNA was harvested from cultures exposed to 0× (control) and 1× MIC of H-CHG for 15 min.

FIG 2

H-CHG induces VanA-type vancomycin resistance genes in E. faecium and E. faecalis. RT-qPCR was used to quantify the expression of vanA or vanB upon exposure to H-CHG for 15 min compared to control conditions. Expression of vanA and -B was internally normalized to clpX as described in the text. Expression of vanA or -B in control cultures was set to 1 (not shown). The fold change in vanA or -B expression in cultures treated with 1× MIC of H-CHG relative to the control was quantified for two independent experiments for all strains queried. E. faecium 1,231,410 vanA expression upon exposure to 1/4× or 1/2× MIC of H-CHG for 15 min was additionally queried. Efm, E. faecium; Efs, E. faecalis; HIP, HIP11704; TUH, TUH4-64.

FIG 3

vanH promoter activity in E. faecium 1,231,410. (A) Average growth curve of E. faecium PB104. Aliquots of an exponentially growing E. faecium PB104 culture at an OD₆₀₀ of 0.4 to 0.5 were added to 25 ml of BHI-chloramphenicol (control; red circles) or BHI-chloramphenicol containing H-CHG or vancomycin such that 1/4× MIC of H-CHG (green triangles), 1/2× MIC of H-CHG (blue triangles), 1× MIC of H-CHG (orange diamonds), or 2 μg/ml of vancomycin (pink squares) were attained. Cultures were incubated with shaking at 100 rpm at 37°C, and the OD₆₀₀ was monitored. Error bars indicate standard deviations from 4 independent experiments. (B) β-Galactosidase activity assays. Enzyme activity assays were performed as described in the text. Samples were harvested from E. faecium PB104 cultures at multiple time points after exposure to H-CHG, vancomycin, or control conditions. Error bars indicate standard deviations from 4 independent experiments. The one-tailed Student t test was used to assess significance. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

FIG 4

Detection and quantification of VanX protein levels in E. faecium cultures. (A) Representative Western blot. A total of 250 μg of protein extracted from E. faecium PB221 cultures was analyzed by Western blotting with anti-polyhistidine antibody as described in the text. A 23-kDa protein (indicated by arrow) was detected in cultures treated with H-CHG and vancomycin. Lanes: 1, control cells after 1.5 h of incubation; 2, control cells after 2 h of incubation; 3, cells after 1.5 h of incubation with 1/4× MIC of H-CHG; 4, cells after 2 h of incubation with 1/4× MIC of H-CHG; 5, cells after 1.5 h of incubation with 1/2× MIC of H-CHG; 6, cells after 2 h of incubation with 1/2× MIC of H-CHG; 7, cells after 1.5 h of incubation with 1× MIC of H-CHG; 8, cells after 2 h of incubation with 1× MIC of H-CHG; 9, cells after 2 h of incubation with 20 μg/ml of vancomycin. (B) Quantification of VanX protein levels. The amount of VanX protein was quantified by calculating the IDV (integrated density value) of each of the protein band by using Alphaimager spot density tool. Average values are shown. Error bar represent the standard deviations from 3 experiments. The one-tailed Student t test was used to assess significance. *, P < 0.05.

FIG 5

RT-qPCR analysis of vanA gene expression in response to vancomycin and H-CHG. RT-qPCR was used to quantify the expression of vanA ligase upon exposure to vancomycin (50 μg/ml) for 2 h (A) and 1× MIC H-CHG for 15 min (B) versus unexposed cultures for the E. faecium 410 wild type and ΔvanR and ΔvanRS deletion mutants. The expression of the vanA gene was internally normalized to clpX. The expression of vanA in control cultures was set to 1 (not shown), and relative fold change expression in vancomycin and H-CHG treated cultures from two independent experiments was quantified (trial 1, red bars; trial 2, green bars).