Volume 3, Issue 1 (January 2006)
Enhanced Recovery and Real-Time PCR Based Quantification of Mycobacteria from Metalworking Fluids
Non-tuberculous mycobacteria, especially Mycobacterium immunogenum and M. chelonae, that are associated with metalworking fluids (MWF) have been implicated in occupational hypersensitivity pneumonitis (HP), asthma, and other respiratory illnesses in the MWF exposed metal workers. Cultural recovery and quantification of mycobacterial population from the contaminated MWF is often cumbersome, especially due to the presence of other microbial contaminants and due to the frequently coexisting culturable and non-culturable fractions of the mycobacterial population in these fluids triggered by improper fluid management and storage conditions. Here we report on role of sample storage conditions in reducing cultural recovery, optimization of methods for enhanced recovery, and culture-independent DNA-based quantification of mycobacteria (culturable and non-culturable) from MWF matrices. The recovery optimization included strategies for selective suppression of gram-positive and gram-negative co-contaminants using antibiotic mixture and revival of the injured cells (viable-but-non-culturable, VBNC). A real-time PCR method designed based on Mycobacterium-specific PCR primers  was further developed to attain a minimum quantification limit of 1 CFU/ml, using pristine or used MWF simulated with M. immunogenum in the background of microbial co-contaminants. The method was validated by comparing its quantification efficiency with conventional agar plating and evaluated for direct quantification of total mycobacteria, including culturable and non-culturable, using contaminated field MWF. The state-of-the art methods developed in this study for selective recovery and real-time PCR based quantification would allow rapid and efficient analysis of field MWF samples for mycobacteria including both culturable and non-culturable sub-populations.