In vitro and intracellular inhibitory activities of nosiheptide against Mycobacterium abscessus

Abstract

The high level of inherent drug resistance of Mycobacterium abscessus makes the infection caused by it very difficult to be treated. The objective of this study was to evaluate the potential of nosiheptide (NOS) as a new drug candidate for treating M. abscessus infections. The microplate AlamarBlue assay was performed to determine the minimum inhibitory concentrations (MICs) of NOS for 28 reference strains of rapidly growing mycobacteria (RGM) and 77 clinical isolates of M. abscessus. Time-kill kinetic and post-antibiotic effect (PAE) of NOS against M. abscessus was evaluated. Its bactericidal activity against M. abscessus in macrophages was determined by an intracellular colony numerating assay. NOS manifested good activity against the reference strains of RGM and M. abscessus clinical isolates in vitro. The MICs of NOS against M. abscessus clinical isolates ranged from 0.0078 to 1 μg/ml, and the MIC₅₀ and MIC₉₀ were 0.125 μg/ml and 0.25 μg/ml, respectively. The pattern of growth and kill by NOS against M. abscessus was moderate with apparent concentration-dependent characteristics, and the PAE value of NOS was found to be ~6 h. Furthermore, NOS had low cell toxicity against the THP-1 cell line after 48 h of exposure (IC₅₀ = 106.9 μM). At 4 μg/ml, NOS exhibited high intracellular bactericidal activity against M. abscessus reference strains with an inhibitory rate of 66.52% ± 1.51%, comparable with that of clarithromycin at 2 μg/ml. NOS showed suitable inhibitory activities against M. abscessus in vitro and in macrophages and could be a potential drug candidate to treat M. abscessus infection.

Keywords: Mycobacterium abscessus; cytotoxicity; intracellular bactericidal activity; nosiheptide; susceptibility.

Figure 1

The minimum inhibitory concentration (MIC) distribution of nosiheptide (NOS) against Mycobacterium abscessus isolates. The MIC was determined by microtiter plate AlamarBlue assay. The MIC outcome of NOS against M. abscessus ATCC19977 was on the top. The blank group: only bacteria without drugs; NC group: negative control group, this group was free of bacteria and drugs to prove that the experiment was not contaminated.

Figure 2

Determination of membrane-permeabilizing properties of NOS by FCM of M. abscessus ATCC 19977. Bacterial cells are treated with NOS and clarithromycin (CLA) in a time-dependent manner. The percentage of bacterial cells shows the increased uptake of propidium iodide with increasing time intervals. (A) Bacterial cells without treatment serve as a negative control. (B–D) The results after 1, 3, and 6 h of treatment with NOS (4 μg/ml). (E–G) The results after 1, 3, and 6 h of treatment with CLA (4 μg/ml). Samples were prepared in phosphate-buffered saline (PBS).

Figure 3

Time-kill kinetics of (A) NOS and (B) CLA against M. abscessus ATCC 19977. Antibiotic concentrations are presented as different symbols. Dimethyl sulfoxide (DMSO) was used as a negative control, and CLA was used as a positive control. *P < 0.05; **P < 0.01.

Figure 4

Post-antibiotic effect (PAE) in the growth of M. abscessus ATCC19977 after pulse dosing with NOS and CLA. CLA was used as a positive control and DMSO as a negative control. All data are shown as the means ± SD (n = 3). NOS vs. DMSO. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5

Cytotoxicity assay of NOS in the differentiated (A) and undifferentiated (B) THP-1 cell lines. DMSO group: the negative control; NSC 228155 group: the positive control (at a final concentration of 2.5 μM). All data are shown as the means ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 6

Intracellular bactericidal activities of different concentrations of nosiheptide (NOS) and CLA (the positive control) against M. abscessus ATCC 19977 in macrophages. (A) NOS at an MOI of 10:1. (B) clarithromycin at an MOI of 10:1. (C) NOS at an MOI of 1:1. (D) clarithromycin at an MOI of 1:1. DMSO group: the negative control. All data are shown as the means ± SD (n = 3). ****P < 0.0001.

Figure 7

The intracellular bacterial survival rate of M. abscessus ATCC 19977 with NOS exposure. (A) At an MOI of 1:1. (B) at an MOI of 10:1. NOS group: infected macrophage treated with NOS (2 μg/ml); CLA group: the positive control, infected macrophage treated with CLA (2 μg/ml); NOS + CLA group: infected macrophage treated with NOS (1 μg/ml) and CLA (1 μg/ml); DMSO group: the negative control. All data are shown as the means ± SD (n = 3). ****P < 0.0001.