Comment on: Identification of antimicrobial activity among FDA-approved drugs for combating Mycobacterium abscessus and Mycobacterium chelonae

Abstract

Sir, In the article titled ‘Identification of antimicrobial activity among FDA-approved drugs for combating Mycobacterium abscessus and Mycobacterium chelonae’ published in the July 2011 issue of JAC, it was reported that the FDA-approved drug metronidazole has potent activity (<0.015 mg/L; ∼87 nM) against the rapid-growing mycobacteria (RGM) Mycobacterium abscessus.1 This finding intrigued us and our clinical colleagues who treat M. abscessus-infected patients here at NIH. We therefore tried to repeat their experiment with M. abscessus ATCC 19977 and some recent patient isolates of M. abscessus to evaluate their susceptibility to metronidazole. We regret to report that we did not observe any MIC99, even at the highest concentration of the drug we tested (2 mM) after 48, 72 and 96 h. In accordance with the method published in the paper, we grew the M. abscessus ATCC 19977 (and four clinical isolates) from glycerol stock in 7H9 Middlebrook medium, passaged them once and let them grow to an OD650 of 0.2–0.4. The cell cultures were then diluted to an OD650 of 0.0002 in 7H9 medium and 50 μL of the cells was added to 50 μL of drug at various concentrations in a 96-well plate. They were incubated at 37°C for 48, 72 and 96 h and no killing of this RGM was observed (Figure 1a). Figure 1. (a) MICs of drugs for M. abscessus ATCC 19977 after 72 h. Each of the drugs was assayed in duplicate. MTZ, metronidazole. Each drug was serially diluted (50% each time) from well 1 to well 11, and well 12 does not have any drug. The concentration (in ... It is known that M. abscessus is susceptible to clarithromycin,2 and we therefore included this as a positive control in the experiment. PA-824, another nitroimidazole that is currently in Phase II clinical trials for the treatment of tuberculosis, has shown aerobic as well as anaerobic activity against the slow-growing mycobacteria Mycobacterium tuberculosis.3 It was also tested for activity against the RGM M. abscessus. The susceptibility of M. abscessus to clarithromycin clearly shows that there was no flaw in the experimental design, yet M. abscessus grew in the presence of 100 μM metronidazole and 50 μM PA-824 (Figure 1a). To investigate the susceptibility of M. abscessus ATCC 19977 and four clinical isolates to a higher concentration of metronidazole we determined the MIC99 starting with a 2 mM concentration of metronidazole, but we still did not observe any cidal effect of metronidazole even at such a high concentration (Figure 1b). In addition, no killing was observed with PA-824 at a concentration of 2 mM (data not shown). Strains tested have been identified as belonging to the M. abscessus group by partial sequencing of the secA gene4 and/or MALDI-TOF (matrix-assisted laser desorption ionization–time of flight) mass spectrometry.5 Metronidazole is known for its activity and has been used in the treatment of diseases caused by anaerobic microorganisms like Trichomonas vaginalis,6 Entamoeba histolytica7 and Clostridium difficile6 and microaerophilic bacteria like Helicobacter pylori.8 Though metronidazole is active against hypoxically adapted non-replicating M. tuberculosis, it is inactive against rapidly replicating M. tuberculosis under aerobic conditions.9 Metronidazole, which belongs to the class of 5-nitroimidazoles, has a single electron reduction potential of −0.4 to −0.5 V, which is out of the range normally accessible to the mammalian redox system. Under a limiting oxygen concentration in microbes the redox potential range is −0.42 V and lower and is suitable for the bioreductive activation of metronidazole. Upon single electron reduction of nitroimidazole, a nitro radical anion is produced,10 which could reduce oxygen in a microaerophilic organism to form superoxide.11 In the presence of transition elements like iron or copper, the hydrogen peroxide and superoxide react to form a hydroxyl radical by the Haber–Weiss reaction,10 causing inhibition of DNA synthesis and DNA strand breakage by this futile cycle. Metronidazole induces the expression of SOS response genes, which are responsible for DNA repair by the RecA pathway,10 as a result of which recA mutants in Escherichia coli and H. pylori are more susceptible to metronidazole treatment.12,13 Under normoxic conditions reduction of molecular oxygen by reduced metronidazole would be expected to render it harmless, as superoxide formed would be rapidly decomposed by the microbial superoxide dismutase.10 Our finding clearly demonstrates that metronidazole is inactive against M. abscessus under aerobic conditions, as has been reported. Metronidazole is potent against anaerobic organisms, but cannot be used in the treatment of infection with M. abscessus. Metronidazole does not kill RGM M. abscessus at a concentration of 2 mM (342.3 mg/L), which is more than 20 000-fold the concentration of what was reported by Chopra et al.1