Abstract
Environmental exposure is considered to be responsible for nontuberculous mycobacterial infections in humans. To facilitate the isolation of mycobacteria from soil, Middlebrook 7H10 agar was optimized as an enhanced selective medium by increasing the concentration of malachite green. A series of modified Middlebrook 7H10 agar media with malachite green concentrations ranging from 2.5 to 2500 mg/L was evaluated using 20 soil samples decontaminated with 3% sodium dodecyl sulfate plus 2% NaOH for 30 min. Among these modified Middlebrook 7H10 media, the medium with malachite green at a concentration of 250 mg/L, i.e., at the same concentration as in Löwenstein–Jensen medium, was the most effective in terms of the number of plates with mycobacterial growth. This medium was further evaluated with 116 soil samples. The results showed that 87.1% (101/116) of the samples produced mycobacterial growth, and 15 samples (12.9%) produced no mycobacterial growth. Of the plates inoculated with the soil samples, each in duplicate, 5.2% (12/232) showed late contamination. In total, 19 mycobacterial species were isolated, including seven (36.8%) rapidly growing mycobacteria and 12 (63.2%) slowly growing mycobacteria. Our results demonstrate that the modified Middlebrook 7H10 agar with 250 mg/L malachite green is useful for the primary isolation of nontuberculous mycobacteria from soil.
Highlights
Nontuberculous mycobacteria (NTM) are members of the genus Mycobacterium, excluding Mycobacterium tuberculosis complex and Mycobacterium leprae
Performance of selective media inoculated with decontaminated soil samples To determine optimal malachite green (MG) concentration in modified Middlebrook 7H10 agar for mycobacteria culture from soil, we first tested MG concentration ranging from 2.5 to 2500 mg/L
65% (13/20) of soils inoculated onto Modified Middlebrook 7H10 agar with 250 mL/L MG produced mycobacterial growth
Summary
Nontuberculous mycobacteria (NTM) are members of the genus Mycobacterium, excluding Mycobacterium tuberculosis complex and Mycobacterium leprae. M. tuberculosis is one of the most important pathogens from this genus. It has been estimated by the World Health Organization that over one-third of the world population is infected with M. tuberculosis (Glaziou et al 2013). The rates of tuberculosis (TB) have gradually declined (Glaziou et al 2013), whereas those of NTM infections are on the rise in many areas (Brode et al 2014). Soil is the likely source of NTM responsible for human infections, especially in soil-related occupations (e.g., farmers) (Reed et al 2006; Gopinath and Singh 2010; Hamada et al 2016). Several studies have focused on the comparison and optimization of soil decontamination methods (Portaels et al 1988; Kamala et al 1994a; Livanainen 1995; Parashar et al 2004) and the development of media with enhanced selectivity (Ichiyama et al 1988; Chilima et al 2006; Narang et al 2009; Aboagye et al 2016) for NTM; no robust and standardized method for the primary isolation of NTM from soil is yet available
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