Journal: bioRxiv

Article Title: Enterococcus faecalis and pathogenic streptococci inactivate daptomycin by releasing phospholipids

doi: 10.1101/149971

Figure Lengend Snippet: Streptococci and enterococci release phospholipid and inactivate daptomycin. (a-h), percentage survival of bacteria after 8 h incubation in broth containing the indicated concentrations of daptomycin. (i-p) the concentration of phospholipid in culture supernatants of bacteria exposed to daptomycin as determined by reactivity with a fluorescent dye (RFU: relative fluorescence units). Note the different Y-axis scale for staphylococci vs other bacteria. (q-x) relative percentage of daptomycin activity remaining in culture supernatants of bacteria exposed to daptomycin for 8 h. The activity of daptomycin incubated in culture medium only for 8 h was taken to be 100 %. For all data, the mean of 4 independent experiments are shown, and error bars represent the standard deviation of the mean.

Article Snippet: We initially determined the daptomycin minimum inhibitory concentration (MIC) for a representative panel of Gram-positive pathogens: S. aureus SH1000 [ ], S. epidermidis ATCC 12228 [ ], GAS strain A40 [ ]; GBS strains 515 [ ] and COH1 [ ]; S. gordonii strain Challis [ ]; and E. faecalis strains JH2-2 [ ] and OG1X [ ].

Techniques: Bacteria, Incubation, Concentration Assay, Fluorescence, Activity Assay, Standard Deviation

Journal: bioRxiv

Article Title: Enterococcus faecalis and pathogenic streptococci inactivate daptomycin by releasing phospholipids

doi: 10.1101/149971

Figure Lengend Snippet: De novo lipid biosynthesis is required for enterococcal inactivation of daptomycin. Phospholipid concentration (RFU) in culture supernatants from S. aureus (a) or E. faecalis OG1X (b) incubated for 8 h in media containing daptomycin (10 µg ml -1 ) only (dap) or both daptomycin and 0.5 X MIC platensimicin (dap + pla). (c,d) relative % daptomycin activity in supernatants from cultures described in (a) and (b), respectively. Data in (a) and (b) were analysed using a one-way ANOVA with Tukey’s post-hoc test. Data in (c) and (d) were analysed by a Student’s t -test. *P=<0.05.

Article Snippet: We initially determined the daptomycin minimum inhibitory concentration (MIC) for a representative panel of Gram-positive pathogens: S. aureus SH1000 [ ], S. epidermidis ATCC 12228 [ ], GAS strain A40 [ ]; GBS strains 515 [ ] and COH1 [ ]; S. gordonii strain Challis [ ]; and E. faecalis strains JH2-2 [ ] and OG1X [ ].

Techniques: Concentration Assay, Incubation, Activity Assay

Journal: bioRxiv

Article Title: Enterococcus faecalis and pathogenic streptococci inactivate daptomycin by releasing phospholipids

doi: 10.1101/149971

Figure Lengend Snippet: Loss of daptomycin activity in supernatant is not due to antibiotic binding to bacteria. (a) percentage survival of E. faecalis OG1X incubated with various concentrations of daptomycin (dapt) or Bodipy-daptomycin (Bodipy-dapt) for 8 h. (b) relative percentage daptomycin activity in culture supernatants described in (a). (c) binding of Bodipy-daptomycin to S. aureus or E. faecalis OG1X after 8 h incubation in media containing the indicated concentration of the labelled antibiotic. (d) quantification of Bodipy-daptomycin (RFU) remaining in culture supernatants from S. aureus or E. faecalis OG1X, after 8 h incubation with Bodipy-daptomycin as described in (c) * indicates significantly different from 0 h time point. (e) relative percentage activity of daptomycin (5 μg ml -1 ) activity in TSB only (TSB + dapt), in the supernatant from E. faecalis incubated with daptomycin for 8 h (s/n) and after the addition of 5 μg ml -1 daptomycin to the supernatant from E. faecalis incubated with daptomycin for 8 h (s/n + dapt). Data in (d) and (e) were analysed by a two-way ANOVA with Tukey’s post-hoc test. Graphs show the mean average and, where shown, error bars represent the standard deviation of the mean. For each panel *P=<0.05.

Article Snippet: We initially determined the daptomycin minimum inhibitory concentration (MIC) for a representative panel of Gram-positive pathogens: S. aureus SH1000 [ ], S. epidermidis ATCC 12228 [ ], GAS strain A40 [ ]; GBS strains 515 [ ] and COH1 [ ]; S. gordonii strain Challis [ ]; and E. faecalis strains JH2-2 [ ] and OG1X [ ].

Techniques: Activity Assay, Binding Assay, Bacteria, Incubation, Concentration Assay, Standard Deviation