A new method for species identification and differentiation of Mycobacterium chelonae complex based on amplified hsp65 restriction analysis (AHSPRA)

Members of the Mycobacterium chelonae complex (MCC), namely M. chelonae, Mycobacterium abscessus and Mycobacterium immunogenum, have been implicated in nosocomial infections and occupational respiratory illnesses like hypersensitivity pneumonitis (HP) associated with contaminated metalworking fluid (MWF) exposures. Close relationship among these member species makes their differentiation cumbersome using the existing methods. Here we report a simple and rapid method for unambiguous identification and differentiation of the three-member species of the MCC group with PCR-restriction analysis targeting a 667-bp segment of a variable region of the 65-kDa-heat shock protein (hsp65) gene. This assay, described as Amplified hsp65 Restriction Analysis (AHSPRA), can discriminate all the three individual species using a one-step restriction digestion using either BbvI or Eco0109I. The enzyme NarI can differentiate M. immunogenum from the other two MCC species (M. chelonae and M. abscessus). The developed method was validated using several non-MCC reference species of other rapidly growing mycobacteria (RGM) and MCC field isolates from MWF samples. Direct cell-lysis was used instead of the conventional DNA template preparation, which improved the rapidity, simplicity and adaptability of the developed method. The results suggest that the developed method can unambiguously differentiate species of the M. chelonae complex from other RGM species and from one another.