Peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) assay for specific detection of mycobacterium immunogenum and DNA-FISH assay for analysis of pseudomonads in metalworking fluids and sputum.
Selvaraju-SB; Kapoor-R; Yadav-JS
Mol Cell Probes 2008 Oct-Dec; 22(5-6):273-280
Specific and rapid detection and quantification of mycobacteria in contaminated metalworking fluid (MWF) are problematic due to complexity of the matrix and heavy background co-occurring microflora. Furthermore, cross-reactivity among neighboring species of Mycobacterium makes species differentiation difficult for this genus. Here, we report for the first time a species-specific peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method for Mycobacterium immunogenum, a non-tuberculous Mycobacterium species prevalent in MWF and implicated in occupational lung disease hypersensitivity pneumonitis and pseudo-outbreaks. A novel species-specific 14-bp PNA probe was designed for M. immunogenum based on its 16S rRNA gene sequence and was validated for specificity, by testing against a panel of other phylogenetically closely related rapidly growing mycobacteria and representative species of gram-positive, gram-negative, and acid fast organisms. In addition, a DNA-FISH protocol was optimized for co-detection of Pseudomonas, the most predominantly co-occurring genus in contaminated MWF. Reliable quantification for both the test organisms was achieved at or above a cell density of 10(3)cellsml(-1), a recognized minimum limit for microscopic quantification. The mycobacterial PNA-FISH assay was successfully adapted to human sputum demonstrating its potential for clinical diagnostic applications in addition to industrial MWF monitoring, to assess MWF-associated exposures and pseudo-outbreaks.
Bacteria; Bacterial-disease; Bacterial-infections; Bacteriology; Cell-function; Cell-metabolism; Cellular-function; Cellular-structures; Hypersensitivity; Infection-control; Infectious-diseases; Lung-cells; Lung-disorders; Lung-irritants; Metalworking-fluids; Microbial-test-systems; Microbiology; Microorganisms; Microscopic-analysis; Occupational-health; Occupational-diseases; Occupational-exposure; Pulmonary-system-disorders; Quantitative-analysis; Respiratory-system-disorders; Ribonucleic-acids;
Author Keywords: Mycobacterium immunogenum; PNA-FISH; Pseudomonads; Metalworking fluids; Sputum; Hypersensitivity pneumonitis
Jagjit S. Yadav, Microbial Pathogenesis and Toxicology Laboratory, Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0056
Molecular and Cellular Probes
University of Cincinnati