Real-time PCR assays for genus-specific detection and quantification of culturable and non-culturable mycobacteria and pseudomonads in metalworking fluids

Abstract

Genus-specific real-time PCR assays were developed and optimized for the direct culture-independent detection and quantification of Mycobacteria and Pseudomonads in contaminated metalworking fluids (MWF) and the results were compared with conventional culturing using selective media. It included optimization of the direct DNA isolation from the fluid matrix and the amplification conditions using genus-specific primers. Mycobacterium-specific primers based on 65-kDa heat shock protein (hsp) gene, and Pseudomonas-specific primers based on 16S rRNA gene were used. A standard curve was developed each for the two model bacterial species Mycobacterium immunogenum and Pseudomonas fluorescens, representing two important genera frequently isolated from MWF. A minimum quantification limit of 10 cells/ml was achieved although as low as 1 cell/ml yielded a detectable amplicon signal. Of the twenty MWF field samples contaminated with mixed microflora, only two samples yielded putative colonies of Mycobacteria and Pseudomonads by culturing method, while seven samples responded to the genus-specific real-time PCR detection and quantification for each genus. In contrast to the low culturable counts, the real-time PCR based cell counts ranged from 1.3 x 10(2) to 5.5 x 10(5)cells/ml and 5.2 x 10(2) to 7.0 x 10(5)cells/ml for Mycobacteria and Pseudomonads, respectively, indicating a significant non-culturable fraction in the fluids, for the two genera. This is the first application of real-time PCR protocol to MWF samples for detection and quantification of total (culturable and non-culturable) Mycobacteria and Pseudomonads without culturing.