DNA-based methodologies for rapid detection, quantification, and species- or strain-level identification of respiratory pathogens (Mycobacteria and Pseudomonads) in metalworking fluids

Abstract

Mycobacteria and pseudomonads occurring in modern metalworking fluids (MWF) have been implicated in occupational health hazards as causal agents for hypersensitivity pneumonitis (HP) and other respiratory illnesses in machine workers exposed to these fluids and their aerosols. Unlike the conventional cultural and biochemical methods, which are often slow and ambiguous and detect only culturable cells, DNA-based methods offer a time-saving alternative for reliable detection and identification of both culturable and nonculturable bacteria in MWF and for selective quantification of individual genera of pathogens of interest in these fluids. This is the first report on DNA-based direct detection of mycobacteria and pseudomonads in MWF without culturing. Genus-specific PCR approach was successfully applied for screening of field MWF samples originating from different industrial users for detection of mycobacteria or pseudomonads including both culturable and nonculturable cells. PCR in combination with amplicon DNA sequencing led to the identification of Mycobacterium chelonae, Pseudomonas nitroreducens, and an undefined Pseudomonas species from these fluids. Genome fingerprinting by pulsed-field gel electrophoresis (PFGE) on Mycobacterium isolates further showed that the isolates represented three strains of M. chelonae although the possibility of one of the strains being clonal with M. immunogenum cannot be excluded. In parallel efforts, a quantitative competitive PCR method developed based on the Pseudomonas-specific PCR was applied to quantify total P. fluorescens cells in contaminated metalworking fluid and MWF aerosol without culturing. The DNA-based protocols developed in this study will allow rapid screening of field MWF samples for the presence of both culturable and nonculturable cells and thus facilitate effective fluid management and timely exposure assessment.