Environmental Isolation of Nontuberculous Mycobacteria and Targeted PCR Detection of Mycobacteroides abscessus (Early View)

Abstract

Nontuberculous mycobacteria (NTM) are increasingly recognised as opportunistic pathogens in humans, with Mycobacteroides abscessus representing one of the most clinically significant species. Rapid and reliable detection of M. abscessus remains challenging due to its slow growth rate and limitations in current diagnostic approaches in Malaysia. In this study, nine species of NTM were successfully isolated from soil, animal faeces, and water samples using a Siew-modified Löwenstein–Jensen agar formulation (SMLJ), with M. abscessus being detected and isolated successfully. We observed that the presence of glycerol in transport or culture media interfered with DNA extraction and subsequent PCR detection, highlighting the importance of glycerol-free conditions when samples are intended for molecular identification. To improve diagnostic resolution, a novel forward primer, MA16S966F (966F), was designed to pair with the universal 1492R primer for specific amplification of the 16S rRNA gene of M. abscessus. The primer demonstrated robust amplification across annealing temperatures of 45–60 °C and exhibited 100% specificity against a panel of bacterial strains, including other mycobacterial species. Sensitivity testing revealed a detection limit of 10 pg of M. abscessus DNA, with very faint amplification still detectable at 1 pg. These findings provide a cost-effective and efficient framework for the environmental isolation and molecular detection of NTM, and establish 966F as a promising tool for rapid screening of M. abscessus in clinical and environmental settings.