Table 1.
Bacterial strains and plasmids used in the study.
Table 2.
Median vancomycin MICs in E. faecium 1,231,410.
Fig 1.
A ΔddcP mutant has an altered vancomycin/H-CHG synergy phenotype.
Optical density (OD600nm) of (A) E. faecium 1,231,410 wild-type (E. faecium 410), (B) the ddcP deletion mutant, and (C) the ddcP complemented strain with and without vancomycin and H-CHG treatment. E. faecium was cultured in BHI broth until the OD600 reached 0.6, as described in materials and methods. Equal volumes of cultures were split into BHI (control; red circles) or BHI containing vancomycin (50 μg/ml) and H-CHG (4.9 μg/ml) (blue squares). OD600 values were monitored for 6 h. Error bars indicate standard deviations from n = 3 independent experiments. Significance was assessed using the one-tailed Student’s t-test. * denotes P-value < 0.05. Stars indicate significant differences between vancomycin- and H-CHG-treated cultures in panel B versus A, and in panel C versus B. Note that growth curve of E. faecium 410 wild-type in the presence of vancomycin and chlorhexidine from Fig 1A has been shown again in S1A Fig for comparison with the pbp deletion mutants.
Fig 2.
A ΔddcP mutant has reduced susceptibility to vancomycin/glycine synergy.
(A) E. faecium 410 wild-type and (B) ddcP deletion mutant cultures were grown at 37°C in BHI until OD600 reached 0.6 as described in materials and methods. Equal volumes of cultures were split into BHI (control; red circles) or BHI containing vancomycin (50 μg/ml) and glycine (0.2 M) (green squares). OD600 values were monitored for 6 h and a reading at 24 h was recorded. Error bars indicate standard deviations from n = 3 independent experiments. Significance was assessed using the one-tailed Student’s t-test. * denotes P-value < 0.05. Stars indicate significant differences between vancomycin- and glycine-treated cultures in panel B versus A.
Fig 3.
E. faecium 1,231,410 can adapt to vancomycin/H-CHG synergy.
The growth kinetics of E. faecium 410 in the presence of vancomycin and H-CHG were observed over a two-day (40 h) growth curve. Panel (A) Representative OD600 of E. faecium 410 after treatment with 0X (control; red circles), vancomycin (orange squares), H-CHG (green triangles) or vancomycin and H-CHG (inverted blue triangles). E. faecium culture was grown at 37°C in BHI until OD600 reached 0.6 and equal volumes of cultures were split into BHI with different antimicrobials (shown by arrow) as described in materials and methods. OD600 values were monitored for 6 h and after 24 h, the vancomycin and H-CHG-treated recovered culture (circled and indicated with dashed arrow) was used as an inoculum to repeat the growth curve (shown in panel B).
Fig 4.
Mutations in the phosphate-specific transport (pst) operon result in escape from vancomycin and H-CHG synergy.
Growth of (A) E. faecium 410 wild-type, (B) SE101, (C) E. faecium 410Δpst, and (D) SE101Δpst. E. faecium was cultured in BHI until the OD600 reached 0.6. Equal volumes of cultures were split into BHI (control; red circles) or BHI containing vancomycin (50 μg/ml) and H-CHG (4.9 μg/ml) (blue squares). OD600 values were monitored for 6 h and the 24 h time point was recorded. Error bars indicate standard deviations from n = 3 independent experiments. Significance was assessed using the one-tailed Student’s t-test. * denotes P-value < 0.05. Stars indicate significant differences between vancomycin- and H-CHG-treated cultures in panel B versus A, in panel C versus A, and in panel D versus B.