Evaluation of a new culture medium for isolation of nontuberculous mycobacteria from environmental water samples

Abstract

Nontuberculous mycobacteria (NTM) are waterborne pathogens commonly found in building water systems where they are a primary concern to vulnerable patient populations and can cause severe disease. The recovery of NTM from environmental samples can be a laborious undertaking and current pre-treatment methods and selective media lack sensitivity. We explored the use of the highly selective Rapidly Growing Mycobacteria (RGM) medium for culturing NTM from environmental water samples compared to existing methods. In total, 223 environmental water samples, including potable and non-potable water, were cultured for NTM using three culture media. In addition to direct culture on RGM medium, each sample was cultured on Middlebrook 7H10 medium and Mitchison 7H11 medium after pre-treatment with 0.2M KCl-HCl. Additionally, 33 distinct species of NTM were inoculated onto RGM medium and 7H10 medium in parallel to directly compare their growth. The use of RGM medium alone without pre-treatment provided a sensitivity (91%) comparable to that offered by culture on both 7H10 and 7H11 with acid pretreatment (combined sensitivity; 86%) with significantly less overgrowth and interference from other organisms on RGM medium. The average concentration of NTM observed on RGM medium alone was comparable to or greater than the NTM concentration on either medium alone or combined. Thirty-three species were examined in parallel and all tested strains of 27 of these species successfully grew on RGM medium, including 19 of 21 from the CDC's healthcare-associated infections species list. RGM medium was successful at recovering environmental NTM without a pre-treatment, greatly reducing labor and materials required to process samples. Simplification of culture processing for environmental NTM will allow for a better assessment of their presence in building water systems and the potential for reduced exposure of susceptible populations.

Fig 1. Visual comparison of two environmental water samples plated on 7H10 and 7H11 after acid treatment and RGM medium directly after six weeks of incubation.

The 7H10 and 7H11 plates grew many and varied microbial species, however RGM medium selected for only NTM A) white, beige colonies and B) white, beige and yellow-orange colonies.

Fig 2. Visual comparison of Purpureocillium lilacinum on 7H10, 7H11, and RGM medium after six weeks of incubation.

A) The 7H10 and 7H11 plates were overgrown with this fungus however the RGM medium partially inhibited the growth allowing recovery of NTM. B) Close up of inhibited growth of fungus from white box, black arrow shows the P. lilacinum colony. This was the only isolate of non-mycobacteria recovered from 223 environmental samples using RGM medium.