Comparison of Decontamination Methods for Isolation of Mycobacterium Species from the Environment

Abstract

The environment harbours many bacterial species, some of which include non-tuberculous mycobacteria which have recently become important in public health. Isolation of mycobacteria from the environment has not been easy because of the presence of other fast growing bacteria and fungi. For isolation of mycobacteria from the environment, decontamination methods that minimize contamination but maximize recovery of mycobacteria are needed. This study sought to compare three decontamination methods for isolation of mycobacteria from the environment. Sixty-five samples were collected from both Buruli ulcer disease endemic and non-endemic villages. Polymerase chain reaction (PCR) was done to detect the biomarker IS2404 as a first screening procedure. Direct microscopy was performed on the IS2404 positive samples and three decontamination methods were evaluated, namely; 4% NaOH/ 5% simplified OA method, 0.3% malachite green/ 0.75 g/50 ml cycloheximide/ 4% NaOH and 3% SDS/ 4% NaOH decontamination methods. Three different media with antibiotic supplementation and one without antibiotic supplementation were used for isolation of mycobacteria from the environment. Thirty-seven out of the 65 samples were positive for IS2404 marker and 5/37(13.5%) were acid-fast positive. Decontamination by NaOH/OA method gave the highest number of total tubes that confirmed mycobacterial growth (42/91, 46.1 %) and the least contamination. The medium containing PANTA-mycobactin-J (PM) was best among the four media used. Isolates obtained from this study were identified by Hain GenoType CM® line probe assay. Forty-four Mycobacterium species were identified and Mycobacterium chelonae was the most frequently isolated species. Decontamination with 4% sodium hydroxide/5% oxalic acid and L-J medium containing PANTA and mycobactin J (PM) were the most efficient in supporting the growth of mycobacteria and may be used as standard for isolating mycobacteria from the environment.