Activity of Drug Combinations against Mycobacterium abscessus Grown in Aerobic and Hypoxic Conditions

Abstract

Infections caused by Mycobacterium abscessus (Mab), an environmental non-tuberculous mycobacterium, are difficult to eradicate from patients with pulmonary diseases such as cystic fibrosis and bronchiectasis even after years of antibiotic treatments. In these people, the low oxygen pressure in mucus and biofilm may restrict Mab growth from actively replicating aerobic (A) to non-replicating hypoxic (H) stages, which are known to be extremely drug-tolerant. After the exposure of Mab A and H cells to drugs, killing was monitored by measuring colony-forming units (CFU) and regrowth in liquid medium (MGIT 960) of 1-day-old A cells (A1) and 5-day-old H cells (H5). Mab killing was defined as a lack of regrowth of drug-exposed cells in MGIT tubes after >50 days of incubation. Out of 18 drugs tested, 14-day treatments with bedaquiline-amikacin (BDQ-AMK)-containing three-drug combinations were very active against A1 + H5 cells. However, drug-tolerant cells (persisters) were not killed, as shown by CFU curves with typical bimodal trends. Instead, 56-day treatments with the nitrocompounds containing combinations BDQ-AMK-rifabutin-clarithromycin-nimorazole and BDQ-AMK-rifabutin-clarithromycin-metronidazole-colistin killed all A1 + H5 Mab cells in 42 and 56 days, respectively, as shown by lack of regrowth in agar and MGIT medium. Overall, these data indicated that Mab persisters may be killed by appropriate drug combinations.

Keywords: Mycobacterium abscessus; aerobiosis; anaerobiosis; colistin; cystic fibrosis; drug combinations; drug tolerance; nimorazole; nitrocompounds; persisters.

Figure 1

Growth of Mab-10 in aerobic (A), and hypoxic (H) conditions (Wayne dormancy culture model). Means ± standard deviations of colony forming units (CFU)/ml and optical density (OD) values are shown.

Figure 2

Survival of A1 and H5 cells of Mab-10 after exposure to bedaquiline (BDQ)-amikacin (AMK)-rifabutin (RFB), 2-drug components, and single drugs, as estimated by CFU counts. A representative experiment out of two is shown.

Figure 3

Activity against A1 and H5 cells of 3-drug combinations containing bedaquiline-amikacin (BDQ-AMK). (A,E) BDQ-AMK and its components BDQ and AMK; (B,F) BDQ-AMK ± rifabutin (RFB); (C,G) BDQ-AMK ± clarithromycin (CLR); (D,H) BDQ-AMK ± moxifloxacin (MXF). Increases in the activities of (BDQ-AMK + third drug) versus BDQ-AMK are highlighted in grey.

Figure 4

Activity against A1 and H5 cells of 3-drug combinations containing bedaquiline-amikacin (BDQ-AMK). (A,E) BDQ-AMK ± clofazimine (CLF); (B,F) BDQ-AMK ± linezolid (LNZ); (C,G) BDQ-AMK ± tigecycline (TGC); (D,H) BDQ-AMK ± metronidazole (MTR). Increases in the activities of (BDQ-AMK + third drug) versus BDQ-AMK are highlighted in grey.

Figure 5

Activity against A1 and H5 cells of 3-drug combinations containing bedaquiline-amikacin (BDQ-AMK). (A,E) BDQ-AMK ± tinidazole (TND); (B,F) BDQ-AMK ± benznidazole (BNZ); (C,G) BDQ-AMK ± nimorazole (NMR); (D,H) BDQ-AMK ± secnidazole (SCN). Increases in the activities of (BDQ-AMK + third drug) versus BDQ-AMK are highlighted in grey.

Figure 6

Activity against A1 and H5 cells of 3-drug combinations containing bedaquiline-amikacin (BDQ-AMK). (A,E) BDQ-AMK ± niclosamide (NCL); (B,F) BDQ-AMK ± nitazoxanide (NTZ); (C,G) BDQ-AMK ± ornidazole (ORN); (D,H) BDQ-AMK ± nitroxoline (NTR). Increases in the activities of (BDQ-AMK + third drug) versus BDQ-AMK are highlighted in grey.

Figure 7

Activity against A1 and H5 cells of 5-drug combinations containing BDQ-AMK added with RFB (or CLF or MXF, both potentiating A1 cells), and with CLR-MTR (potentiating H5 cells). Dashed lines indicate the limit of detection (5 CFU/mL). A representative experiment out of two is shown.

Figure 8

Activity against A1 and H5 cells of BDQ-AMK-RFB-CLR-MTR, without or with substitution of MTR with TND, SCN or NMR. Dashed lines indicate the limit of detection (5 CFU/mL). A representative experiment out of two is shown.

Figure 9

Activity against A1 and H5 cells of 3-drug combinations containing bedaquiline-amikacin (BDQ-AMK) ± 25 µg/mL or 100 µg/mL of colistin (CLS). Increases in the activities of BDQ-AMK-CLS versus BDQ-AMK are highlighted in grey. Dashed lines indicate the limit of detection (5 CFU/mL).

Figure 10

Activity against A1 and H5 cells of BDQ-AMK-RFB-CLR-MTR ±25 µg/mL or 100 µg/mL of CLS. Dashed lines indicate the limit of detection (5 CFU/mL). A representative experiment out of two is shown.