Article Figures & Data
Figures
- FIG 1
Concentration-response curves of selected antibiotics against extracellular (left panels) and intracellular (THP-1 cells; right panels) P. aeruginosa PAO1, PA50, and PA291. The graphs show the change in the number of CFU (Δlog10 CFU from the initial inoculum) per ml of broth (extracellular, open symbols, dotted lines) or per mg of cell protein (intracellular, closed symbols, solid plain lines) after 24 h of incubation at increasing extracellular concentrations expressed in mg/liter (total drug). The solid horizontal line corresponds to a bacteriostatic effect (no change from the initial inoculum), and the dotted horizontal line shows the limit of detection (4.5 log CFU decrease). Values are means ± standard errors of the means (SEM) (2 experiments performed in triplicate); when not visible, the error bars are smaller than the symbols. CIP, ciprofloxacin; CST, colistin; MEM, meropenem; TOB, tobramycin.
- FIG 2
Influence of time on the rate and extent of killing of P. aeruginosa PAO1, PA50, and PA291 in broth by antibiotics alone or in combination used at concentrations corresponding to their respective MIC (dotted lines) or their human Cmax (solid lines) (human Cmax values are as follows: CIP, 4.6 mg/liter; CST, 5 mg/liter; MER, 57 mg/liter; and TOB, 6 mg/liter [see reference in reference 17]). The ordinate shows the change in the number of CFU (log10 scale) per ml of broth. The solid horizontal line corresponds to a bacteriostatic effect (no change from the initial inoculum), and the dotted horizontal line shows the limit of detection (−4.5 log CFU decrease).Values are means ± SEM (2 experiments performed in triplicate); when not visible, error bars are smaller than the symbols. CIP, ciprofloxacin; CST, colistin; MEM, meropenem; TOB, tobramycin.
- FIG 3
Influence of time on the rate and extent of killing of intracellular P. aeruginosa PAO1, PA50, and PA291 by antibiotics alone or in combination used at concentrations corresponding to their respective MIC (dotted lines) or their human Cmax (solid lines) (human Cmax values are as follows: CIP, 4.6 mg/liter; CST, 5 mg/liter; MER, 57 mg/liter; TOB, 6 mg/liter [see reference in reference 17]). The ordinate shows the change in the number of CFU (log10 scale) per mg of cell protein. The solid horizontal line corresponds to a bacteriostatic effect (no change from the initial inoculum), and the dotted horizontal line shows the limit of detection (−4.5 log CFU decrease). Values are means ± SEM (2 experiments performed in triplicate); when not visible, error bars are smaller than the symbols. CIP, ciprofloxacin; CST, colistin; MEM, meropenem; TOB, tobramycin.
- FIG 4
FME plots of antibiotics against extracellular (open symbols, dashed lines) and intracellular (closed symbols, solid lines) P. aeruginosa strains. In each graph, the abscissa shows the FMEs value calculated for antibiotics A and B based on concentration effects shown in Fig. 1 (see Table 3 for the corresponding concentrations), and the ordinate shows the value of the FMEobs for each antibiotic alone or for the combination. FMEobs values for the combination that are >1 denote a synergistic effect, values equal to 1 an additive effect, values <1 but higher an indifferent effect, and values <1 and lower than FMEobs for each individual antibiotic in the combination an antagonistic effect.
- FIG 5
Comparative activities of antibiotics at a fixed concentration giving rise to an expected FME of 0.5 and tested alone (open and hatched bars) (see Table 3 for the corresponding concentrations) or combined (gray bars) against P. aeruginosa strains in broth (left) or infecting THP-1 cells (right). Data are expressed as the decrease in CFU per ml of broth (extracellular) or per mg cell protein (intracellular) compared to the initial inoculum. Values are the means ± SEM (2 experiments performed in triplicate). The horizontal dotted line corresponds to the limit of detection. Statistical analysis: P < 0.05 (*) or P < 0.01 (**) for the combination compared to each of the two antibiotics alone (t test).
Tables
- TABLE 1
Pertinent regression parameters of concentration-response curves for extracellular (broth) and intracellular (THP-1 cells) activity of antibiotics against P. aeruginosa strainsa
Antibiotic and strain MICb (mg/liter) Extracellular activity Intracellular activity Eminc Emaxd Cse EC50f Emin Emax Cs EC50 CIP PAO1 0.25 3.9 (2.8 to 4.9) >−4.5 0.6 (0.4 to 0.9) 0.2 (0.1 to 0.4) 2.8 (2.0 to 3.5) −2.7*# (−3.1 to −2.3) 1.5 (0.9 to 2.4) 0.4 (0.2 to 0.8) PA50 16 4.0 (3.3 to 4.7) >−4.5 1.3 (0.9 to 1.7) 38.8*# (21.7 to 69.1) 3.3 (2.89 to 3.7) −2.3*# (−3.1 to −1.5) 1.9 (1.4 to 2.5) 21.0*# (12.1 to 36.4) PA291 0.5 3.3 (2.7 to 3.9) >−4.5 1.9 (1.5 to 2.5) 2.4 (1.7 to 3.4) 2.0* (1.5 to 2.6) −3.1# (−3.5 to −2.8) 1.2 (0.8 to 1.6) 0.9 (0.5 to 1.4) CST PAO1 1 3.6 (2.5 to 4.7) >−4.5 1.2 (0.8 to 1.8) 1.8 (1.0 to 3.4) 2.9 (2.5 to 3.3) −0.9 (−1.1 to −0.7) 2.1 (1.5 to 3.0) 0.7 (0.4 to 1.2) PA50 4 3.9 (3.3 to 4.5) >−4.5 1.4 (1.2 to 1.6) 6.7 (4.8 to 9.5) 3.5* (2.9 to 4.0) −0.35# (−0.6 to −0.1) 0.7 (0.3 to 1.7) 0.2 (0.1 to 0.5) PA291 1 3.1 (2.7 to 3.6) >−4.5 2.3 (1.8 to 3.0) 3.2 (2.0 to 5.1) 2.7 (2.1 to 3.2) −1.7* (−2.0 to −1.5) 0.8 (0.5 to 1.3) 0.6 (0.3 to 1.3) MEM PAO1 1 3.8 (3.0 to 4.3) >−4.5 0.8 (0.7 to 1.0) 1.2 (0.9 to 1.6) 2.7 (2.2 to 3.2) −1.7# (−2.0 to −1.4) 1.4 (1.0 to 2.1) 0.5 (0.3 to 0.8) PA50 2 4.0 (3.3 to 4.7) >−4.5 1.4 (1.1 to 1.7) 3.8 (2.6 to 5.5) 2.9 (2.5 to 3.4) −1.4 (−1.7 to −1.1) 1.2 (0.8 to 1.7) 1.1 (0.7 to 1.9) PA291 64 3.3 (2.9 to 3.8) >−4.5 0.6 (0.5 to 0.8) 119.9*# (59.6 to 241.6) 2.1* (1.9 to 2.4) ND 1.2 (1.0 to 1.6) 222.1*# (62.7 to 786.8) TOB PAO1 0.5 3.9 (3.1 to 4.8) >−4.5 0.6 (0.4 to 1.0) 0.4 (0.2 to 0.7) 3.2 (2.9 to 3.6) −0.9 (−1.2 to −0.6) 10.3# (7.2 to 14.7) 2.8# (1.8 to 4.4) PA50 1 4.1 (2.8 to 5.4) >−4.5 1.2 (0.9 to 1.6) 1.8 (1.2 to 2.9) 3.4 (2.9 to 3.8) −0.9# (−1.3 to −0.6) 11.5# (7.6 to 17.3) 3.0 (1.8 to 5.0) PA291 1 3.5 (3.3 to 3.8) >−4.5 0.9 (0.8 to 1.0) 1.2 (1.0 to 1.4) 2.3* (1.8 to 2.9) −2.0* (−2.4 to −1.5) 5.4*# (4.3 to 6.7) 2.5 (1.3 to 4.7) ↵a After 24 h of incubation with extracellular concentrations ranging from 0.01 to 100 to 200 mg/liter. CIP, ciprofloxacin; CST, colistin; MEM, meropenem; TOB, tobramycin. r2 > 0.91; values are given as means with 95% confidence intervals in parentheses. Statistical analysis was done by one-way analysis of variance with Tukey's test for multiple comparisons of each parameter. *, denotes significant difference among strains for the same antibiotic; #, significant difference between antibiotics for the same strain (P ≤ 0.05). ND, not determined (plateau not reached at the highest concentration tested).
↵b EUCAST/CLSI susceptibility breakpoints (susceptible): CIP, ≤0.5/1; CST, 4/2; MEM, 2/2; TOB, 4/4.
↵c CFU increase (in log10 units) from the corresponding initial inoculum at 24 h as extrapolated for an infinitely low antibiotic concentration.
↵d CFU decrease (in log10 units) from the corresponding initial inoculum at 24 h as extrapolated from an infinitely large antibiotic concentration.
↵e Static concentration, i.e., the extracellular concentration (total drug, in multiples of the MIC) resulting in no apparent bacterial growth (number of CFU identical to the initial inoculum), as calculated from the Hill equation of the concentration-response curve.
↵f Extracellular antibiotic concentration (mg/liter) causing a reduction of the inoculum halfway between the number of CFU extrapolated for Emin and Emax.
- TABLE 2
FIC indices from checkerboard titration synergy testing
Strain Range of calculated FIC indices for drug combinationa (interpretation) CIP-CST CIP-TOB CIP-MEM CST-TOB CST-MEM TOB-MEM PAO1 0.56–1 (additivity) 0.75–1 (additivity) 0.63–1 (additivity) 0.63–1 (additivity) 0.26–1 (synergy/additivity) 0.56–1 (additivity) PA50 0.56–1 (additivity) 1 (additivity) 1 (additivity) 0.26–1 (synergy/additivity) 0.56–1 (additivity) 0.63–1 (additivity) PA291 1 (additivity) 0.53–1 (additivity) 0.63–1 (additivity) 0.75–1 (additivity) 1 (additivity) 0.75–1 (additivity) ↵a CIP, ciprofloxacin; CST, colistin; MEM, meropenem; TOB, tobramycin. FICs were determined by checkerboard titration; the values correspond the minimal and maximal FIC values calculated for the different combinations of concentrations tested.
- TABLE 3
Concentrations of antibiotics (Cxp) used for the FME experiments illustrated in Fig. 4 and for the comparative activities of the most effective combinations in Fig. 5
Antibiotic Target FME Cxp (mg/liter)a for strain and model: PAO1 PA50 PA291 Extracellular Intracellular Extracellular Intracellular Extracellular Intracellular Ciprofloxacin 0.1 0.20 0.45 26.52 35.43 1.16 0.71 0.3 0.32 0.69 45.22 51.52 1.84 1.15 0.5 0.53 1.11 78.86 80.48 3.06 1.96 0.7 1.02 2.08 157.38 148.07 5.89 3.83 0.9 3.47 6.98 549.93 485.99 20.09 13.18 Colistin 0.1 1.66 2.39 6.50 2.98 2.97 0.98 0.3 2.65 3.32 9.86 3.91 4.73 1.45 0.5 4.44 5.00 15.91 5.59 7.88 2.29 0.7 8.61 8.91 30.01 9.71 15.25 4.24 0.9 29.48 28.48 100.52 29.09 52.06 14.01 Meropenem 0.1 1.13 0.86 3.75 6.82 57.84 113.80 0.3 1.79 1.23 5.9 9.44 108.72 210.28 0.5 2.99 1.91 9.78 14.15 200.30 383.28 0.7 5.79 3.48 18.84 25.14 413.98 789.15 0.9 10.77 11.36 64.12 80.12 1482.39 2815.20 Tobramycin 0.1 0.39 11.31 1.75 12.42 1.15 3.50 0.3 0.61 15.34 2.77 16.83 1.82 5.20 0.5 1.00 22.61 4.6 24.78 3.03 8.26 0.7 1.91 39.56 8.89 43.31 5.85 15.40 0.9 6.50 124.32 30.30 135.96 19.97 51.10 ↵a Concentration of each individual antibiotic in the combination calculated to reach an FME ranging from 0.1 to 0.9, using the EC50s from the sigmoid equation of the concentration-effect experiments presented in Fig. 1 (see values in Table 1) and based on the following equation: Cxp = (FME × EC50)/(1 − FME). Concentration values in bold are those used in Fig. 5, corresponding to a FME of 0.5 for each drug in the combination.
