Synergistic Interactions of Indole-2-Carboxamides and β-Lactam Antibiotics against Mycobacterium abscessus

Abstract

New drugs or therapeutic combinations are urgently needed against Mycobacterium abscessus Previously, we demonstrated the potent activity of indole-2-carboxamides 6 and 12 against M. abscessus We show here that these compounds act synergistically with imipenem and cefoxitin in vitro and increase the bactericidal activity of the β-lactams against M. abscessus In addition, compound 12 also displays synergism with imipenem and cefoxitin within infected macrophages. The clinical potential of these new drug combinations requires further evaluation.

Keywords: MmpL3; Mycobacterium abscessus; drug synergism; indole-2-carboxamide; macrophage; therapeutic activity; β-lactam.

FIG 1

Structures of imipenem, cefoxitin, and the lead indole carboxamides 6 and 12 used in this study.

FIG 2

Synergistic activity of indole-2-carboxamide derivatives with IPM and FOX in vitro. CFU counts of Cpd12 (A) and Cpd6 (B) given alone and in combination with imipenem (IPM) or cefoxitin (FOX). M. abscessus cultures were incubated at 30°C in CaMHB for 5 days in the presence of the indicated compounds (μg/ml) and plated on LB agar prior to CFU enumeration. (C) For CFU determination, the M. abscessus mutant A309P (spontaneous resistant strain to Cpd12 carrying the A309P mutation in MmpL3) was exposed to the indicated antibiotics (μg/ml) at 30°C in CaMHB for 5 days. Graphs represent the mean of three independent experiments completed in triplicate. Data are expressed as the mean ± standard deviation (SD). The statistical test used is a nonparametric Mann-Whitney t test in which the combinations were compared to the drugs alone. ns, nonsignificant; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 3

CFU determination of clinical isolates exposed to Cpd12 given alone or in combination with imipenem (IPM) or cefoxitin (FOX). M. abscessus cultures were incubated at 30°C in CaMHB for 5 days in the presence of the indicated compounds (μg/ml) and plated on LB agar prior to CFU enumeration. Data are expressed as the mean ± SD from three independent experiments completed in triplicate. The statistical test used is a nonparametric Mann-Whitney t test in which the combinations were compared to the drugs alone. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P < 0.0001.

FIG 4

Impact of Cpd12 alone or in combination on intracellular-residing M. abscessus. THP-1 macrophages were infected with M. abscessus S expressing TdTomato (multiplicity of infection [MOI] of 2:1) and treated with the indicated compounds (μg/ml). (A) CFU were determined at day 0 and day 2 postinfection. Data represents the mean ± SD of three independent experiments completed in triplicate. For statistical analysis, a nonparametric Mann-Whitney t test was performed. ***, P ≤ 0.001; ****, P < 0.0001. (B) Percentage of infected THP-1 macrophages at day 0 and day 2 postinfection. Data shown are mean values ± SD for one representative experiment completed in triplicate. One-way analysis of variance (ANOVA) Kruskal-Wallis was used as a statistical test. ****, P < 0.0001. (C) Immunofluorescent fields were taken at day 2 postinfection at magnification 40× (using confocal microscopy) showing the nuclei of macrophages (DAPI in blue) infected with red-fluorescent M. abscessus in the absence or in the presence of the drugs used alone or in combination. Yellow arrows emphasize red-fluorescent M. abscessus (tdTomato) within macrophages. Only intracellular bacteria that were individually observed under the microscope were recorded.