Method for rapid identification and differentiation of the species of the Mycobacterium chelonae complex based on 16S-23S rRNA gene internal transcribed spacer PCR-restriction analysis

Abstract

Members of the Mycobacterium chelonae complex (MCC), including M. immunogenum, M. chelonae, and M. abscessus, have been associated with nosocomial infections and occupational hypersensitivity pneumonitis due to metalworking fluid (MWF) exposures. In order to minimize these health hazards, an effective and rapid assay for detection of MCC species and differentiation of MCC species from other species of rapidly growing mycobacteria (RGM) and from one another is warranted. Here we report such a method, based on the variable 16S-23S rRNA gene internal transcribed spacer (ITS) region. Mycobacterium genus-specific primers derived from highly conserved sequences in the ITS region and the flanking 16S rRNA gene were used. Specificity of the primers was verified using the MCC member species, 11 non-MCC RGM species, 3 slow-growing mycobacterial (SGM) species (two strains each), and 19 field isolates, including 18 MCC isolates (from in-use MWF) and one non-MCC isolate (from reverse osmosis water). The ITS amplicon size of M. immunogenum varied from those of M. chelonae and M. abscessus. Sequencing of the approximately 250-bp-long ITS amplicons of the three MCC member species showed differences in 24 to 34 bases, thereby yielding variable deduced restriction maps. ITS PCR-restriction analysis using the in silico-selected restriction enzyme MaeII or HphI differentiated the three MCC members from one another and from other RGM and SGM species without sequencing. The enzyme MaeII discriminated all three member species; however, HphI could only differentiate M. immunogenum from M. chelonae and M. abscessus. Use of an optimized rapid DNA template preparation step based on direct cell lysis in the PCR tube added to the simplicity and adaptability of the developed assay.

FIG. 1.

Validation of the designed Mycobacterium-specific PCR primers for amplification of the variable ITS regions from MCC reference species, non-MCC reference species (RGM and SGM), and the field isolates. Panel A: ATCC reference strains of RGM species, including M. immunogenum (lane 1), M. chelonae (lane 2), M. abscessus ATCC 19977 (lane 3), M. fortuitum (lane 4), M. mageritense (lane 5), M. peregrinum (lane 6), M. phlei (lane 7), M. mucogenicum (lane 8), M. senegalense (lane 9), M. smegmatis (lane 10), M. vaccae (lane 11), M. wolinskyi (lane 12), Mycobacterium sp. strain RJGII.135 (lane 13), M. abscessus ATCC 23006 (lane 14), and M. septicum (lane 15); 100-bp DNA size marker (lanes M) (Invitrogen, Carlsbad, CA); reference strains of SGM species, including M. avium strain W144 (lane 16), M. avium strain W359 (lane 17), M. avium subsp. paratuberculosis strain 202 (lane 18), M. avium subsp. paratuberculosis strain 1112 (lane 19), M. intracellulare strain W253st (lane 20), and M. intracellulare strain HO3AN5st (lane 21); negative control with no template DNA (lane 22); and 50-bp DNA size marker (lane m) (Promega Biosciences, Inc., Madison, WI). Panel B: MWF isolates M-JY1 through M-JY4 (lanes 1 to 4, respectively), MWF isolates M-JY6 through M-JY14 (lanes 6 to 19, respectively), water isolate M-JY5 (lane 5), negative control with no template DNA (lane 20), and 100-bp DNA size marker (lanes M) (PGC Scientifics, Gaithersburg, MD). The amplicons were resolved on 1% agarose (RGM species and isolates) and 12% polyacrylamide (SGM species).

FIG. 2.

Alignment of the ITS amplicon sequences for reference strains of the three member MCC species (M. abscessus, M. chelonae, and M. immunogenum) and 19 mycobacterial isolates. Based on the sequence homology criterion, 8 isolates matched with M. chelonae and 10 isolates matched with M. immunogenum. The water isolate, M-JY5, with a relatively long sequence, showed significant nonhomology with MCC species. The shaded areas and dashes indicate the differences and the missing nucleotide bases, respectively.

FIG. 3.

Comparison of the ITSPRA patterns for M. chelonae complex member species (M. immunogenum, M. chelonae, and M. abscessus), other RGM, and SGM. The restriction fragments were resolved using 12% polyacrylamide gel. Panels A and B: HphI and MaeII restriction patterns, respectively, of 14 ATCC reference strains of RGM, namely, M. immunogenum (lane 1), M. chelonae (lane 2), M. abscessus ATCC19977 (lane 3), M. fortuitum (lane 4), M. mageritense (lane 5), M. peregrinum (lane 6), M. phlei (lane 7), M. mucogenicum (lane 8), M. senegalense (lane 9), M. smegmatis (lane 10), M. vaccae (lane 11), M. wolinskyi (lane 12), Mycobacterium sp. strain RJGII.135 (lane 13), M. abscessus ATCC 23006 (lane 14), and M. septicum (lane 15); HphI and MaeII restriction patterns, respectively, of mycobacterial isolates M-JY1 through M-JY19 (lanes 16 to 34, respectively); HphI and MaeII restriction patterns, respectively, of 4 strains of SGM species, namely, M. avium strain W144 (lane 35), M. avium strain W359 (lane 36), M. intracellulare strain W253st (lane 37), and M. intracellulare-strain HO3AN5st (lane 38); and 50-bp DNA size marker (lanes m) (Promega Biosciences, Inc., Madison, WI).

FIG. 4.

Work flow for the developed ITSPRA method for identification of an unknown isolate of rapidly growing mycobacteria from metalworking fluids.