Plate-based dormancy culture system for Mycobacterium smegmatis and isolation of metronidazole-resistant mutants

Mycobacterium smegmatis is an obligate aerobe. However, growth analyses in oxygen-limited liquid cultures have shown that the bacillus is able to survive anoxia with a half-life of 4 days by shifting down to a drug-resistant, dormant state. Metronidazole is the first lead against dormant bacilli and shows selective toxicity for this physiological state. Here, we report a plate-based dormancy culture system employing anoxic jars for M. smegmatis. Its usefulness for the genetic analysis of dormancy was demonstrated by isolating the first metronidazole-resistant mutants. Highly resistant mutants formed slightly yellow (as opposed to creamy) colonies. Furthermore, high-level metronidazole resistance correlated with an increased half-life of 12 days under anoxic conditions. This suggests a link between metronidazole susceptibility and anaerobic survival.