Respiratory
Pathogens In Metalworking Fluids: DNA-Based Methodologies for Rapid Detection,
Quantification, and Species- or Strain-Level Identification of Mycobacteria
and Pseudomonas
Jagjit S. Yadav,
PhD
Microbiology
Laboratory, Department of Environmental Health
University of
Cincinnati Medical Center
Cincinnati, Ohio
Mycobacteria and Pseudomonas
species occurring in the synthetic metalworking fluids (MWF) have been
implicated as causal agents for hypersensitivity pneumonitis (HP) and other
respiratory illnesses in machine workers exposed to these fluids and their
aerosols. Their rapid detection/identification and specific quantification in
MWF samples is warranted for early detection and elimination of these
occupational hazards. The conventional culture-based methods for detection and
quantification are largely based on agar plating using general or specific
microbiological media, which makes them time-consuming and ambiguous yielding
only the viable (culturable) counts. Conventional biochemical and physical
methods used for identification of Mycobacterium species from MWFs are
slow and often ambiguous besides the major disadvantage that generally they
fail to identify isolates particularly to the species and strain-level.
Objectives of this NIOSH-funded study
are to optimize and apply DNA-based methods (PCR and genome fingerprinting) to
provide a time-saving alternative for detection and identification of
Mycobacteria in field samples of MWF and for real-time quantification of
potential pathogens (Pseudomonas and Mycobacteriium) in the
fluids-in-use. We have developed a rapid genus-specific PCR-based method for
detection of Mycobacteria in MWF. The method was successfully applied
for screening of field MWF samples for detection of culturable as well as non-culturable
mycobacteria. Two of the four samples of commercial MWF yielded positive PCR
signals for presence of Mycobacteria. One of these samples yielded colonies on
Middlebrook agar whereas the other showed no culturable isolates, indicating
that the developed genus-specific PCR can detect even the non-culturable cells
as present in the second positive sample. Subsequently, this method in
combination with DNA sequencing was applied to identify several colony isolates
from MWF samples, originating from different industrial users. We have so far
identified two strains of Mycobacterium chelonae from these isolates.
Genome fingerprinting on these isolates is in progress to distinguish their
strains.
In parallel efforts, Pseudomonas-specific PCR method was developed
to detect and quantify total Pseudomonads in contaminated metalworking fluids.
Initial efforts included development of a competitive quantitative method using
Ps. fluorescens as a reference strain. The method was evaluated and used
to estimate total Pseudomonas cell number in both fluid and aerosol samples of
MWF. Experiments are in progress to improve this method into a real-time PCR
based quantification method and extension of the protocol for quantification of
total (both culturable and non-culturable) mycobacteria in MWF.