Expeditious identification and quantification of mycobacteria species in metalworking fluids using peptide nucleic acid probes

Abstract

Microbial contamination of metalworking fluids (MWFs), particularly by mycobacteria species, poses a potential health and safety risk for machine tool workers. This hazard can be magnified by undiagnosed or long-term exposure to mycobacteria that is created by the extensive time required to detect mycobacteria in MWF systems (10 to 26 days). This research investigates the use of molecular technology to reduce the mycobacteria detection time in MWFs to within one day. Novel fluorescent peptide nucleic acid (PNA) probes, designed to target the specific 16S rRNA of Mycobacterium parafortuitum, are applied as a means to demonstrate the effectiveness of the approach in MWF The specific hybridization of the PNA probes is detected using epi-fluorescence microscopy and flow cytometry (FCM), and the performance of two PNA probe classes (traditional and beacon) is compared in this application. It is shown that both epi-fluorescence microscopy and FCM can be utilized in conjunction with the PNA probes to specifically identify M. parafortuitum among a mixed microbial community in MWF It is also shown that the M. parafortuitum labeling methods are insensitive to the synthetic, semi-synthetic, and soluble oil MWF chemistries investigated. In addition, the use of FCM for quantification of M. parafortuitum is demonstrated, and important considerations for applying FCM in the field are described.