title: 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.
abstract: 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.
authors.affiliation: Microbial Pathogenesis and Toxicology Laboratory, Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0056, USA.
authors.forename: Suresh B
authors.initials: SB
authors.lastname: Selvaraju
authors.forename: Renuka
authors.initials: R
authors.lastname: Kapoor
authors.forename: Jagjit S
authors.initials: JS
authors.lastname: Yadav
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007218:Industrial Microbiology; D008670:Metals; D009161:Mycobacterium / Q000235:genetics*; D009164:Mycobacterium Infections / Q000175:diagnosis / Q000453:epidemiology; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000235:genetics; D013183:Sputum / Q000382:microbiology*
chemical_list: D008670:Metals; D020135:Peptide Nucleic Acids; D004247:DNA