Activities of antibiotic combinations against resistant strains of Pseudomonas aeruginosa in a model of infected THP-1 monocytes

Abstract

Antibiotic combinations are often used for treating Pseudomonas aeruginosa infections but their efficacy toward intracellular bacteria has not been investigated so far. We have studied combinations of representatives of the main antipseudomonal classes (ciprofloxacin, meropenem, tobramycin, and colistin) against intracellular P. aeruginosa in a model of THP-1 monocytes in comparison with bacteria growing in broth, using the reference strain PAO1 and two clinical isolates (resistant to ciprofloxacin and meropenem, respectively). Interaction between drugs was assessed by checkerboard titration (extracellular model only), by kill curves, and by using the fractional maximal effect (FME) method, which allows studying the effects of combinations when dose-effect relationships are not linear. For drugs used alone, simple sigmoidal functions could be fitted to all concentration-effect relationships (extracellular and intracellular bacteria), with static concentrations close to (ciprofloxacin, colistin, and meropenem) or slightly higher than (tobramycin) the MIC and with maximal efficacy reaching the limit of detection in broth but only a 1 to 1.5 (colistin, meropenem, and tobramycin) to 2 to 3 (ciprofloxacin) log10 CFU decrease intracellularly. Extracellularly, all combinations proved additive by checkerboard titration but synergistic using the FME method and more bactericidal in kill curve assays. Intracellularly, all combinations proved additive only based on both FME and kill curve assays. Thus, although combinations appeared to modestly improve antibiotic activity against intracellular P. aeruginosa, they do not allow eradication of these persistent forms of infections. Combinations including ciprofloxacin were the most active (even against the ciprofloxacin-resistant strain), which is probably related to the fact this drug was the most effective alone intracellularly.

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 C_max (solid lines) (human C_max 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 C_max (solid lines) (human C_max 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 FME_obs for each antibiotic alone or for the combination. FME_obs 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 FME_obs 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).