Identification of Nontuberculous Mycobacteria (NTM)
The following procedures are used alone or in various combinations for identification of NTM cultures:
High Performance Liquid Chromatography (HPLC)
The HPLC test determines the mycolic acid profile of the organism isolated from a culture. All organisms, either isolated in our lab or received from other laboratories, are verified for acid-fastness and purity by assessment of colonial morphology or evaluation of Ziehl-Neelsen stained smears. A preliminary identification is performed on all isolates by the HPLC method. Differentiation between M. tuberculosis complex and non tuberculous mycobacteria is completely reliable by HPLC. In addition, the interpretation of mycolic acid profiles allows us to identify certain species or groups/complexes of non-tuberculous mycobacteria. M.avium-intracellulare complex is easily identified by HPLC as is M.gordonae, M.marinum, M.xenopi, M.chelonae/abscessus group, M.simiae, M.fortuitum/chelonae group/smegmatis complex, M. kansasii/szulgai, and M. mucogenicum/M.terrae.
M. avium intracellulare complex is considered a final identification for referral isolates. If further species identification is required, we can identify M. avium or M. intracellulare by the GenProbe test. M.chelonae and M. abscessus have nearly identical HPLC patterns and are identified by the ability to grow in the presence of 5% sodium chloride and by use of citrate as a sole carbon source. Species within the M. fortuitum group and species of pigmented rapidly growing mycobacteria are identified by 16S RDNA sequencing. Species that result in a M. kansasii/M. szulgai pattern are identified by AccuProbe or biochemical testing. M.mucogenicum and M. terrae are identified by 16S RDNA sequencing.
The HPLC method can differentiate non-tuberculous mycobacteria from other acid-fast or partially acid fast aerobic Actinomycetes, including Gordonia, Nocardia,Tsukamurella, and Rhodococcus. Species identification for these organisms is performed by sequencing.
We are currently in the process of validating a new HPLC method for identification of mycobacteria and other aerobic Actinomycetes. The SHERLOCK® method, developed by MIDI, uses a software program to match mycolic acid profiles to existing profiles contained in a library database. This method will allow us to identify organisms to the species level that currently require further identification techniques.
Biochemical Tests
Species identification for members within the M. tuberculosis complex is performed by conventional biochemical tests and drug susceptibility testing. These methods include niacin accumulation, nitrate reduction, detection of pyrazinimidase, susceptibility to thiacetazone, tolerance to TCH, and oxygen preference in a semi-solid medium.
16S rDNA Sequencing
Samples unable to be conclusively identified by HPLC, AccuProbe, or biochemical testing are identified to the species level by 16s rDNA sequencing. Sequencing results are always verified by assessment of the subculture.
The objective of this test is to identify Mycobacteria and other aerobic Actinomycetes to the species level by sequencing the first 500 base pairs of the 16S ribosomal gene. The genomic DNA is extracted from bacterial cells grown in liquid or solid media. The 16S rDNA fragment of interest is amplified by PCR and purified. Cycle sequencing is performed on the purified PCR product, labeling the appropriate nucleotide with one of four fluorescent tags (dye terminators). After the extension product is purified using a gel filtration cartridge, electrophoresis is performed on a genetic analyzer. The resulting forward and reverse sequences are aligned and edited to resolve base pair ambiguities in the consensus sequence using the Microseq software from Applied BiosystemsTM. The edited consensus sequence is compared to a sequence database library revealing a list of close matches.
The final species identification is determined based on the percent divergence from the compared sequences in the library, the mycolic acid profile (HPLC), growth characteristics of the organism (including colony morphology, pigmentation and temperature growth preference), and if required, biochemical tests.
GenProbe Technology (AccuProbe)
The AccuProbe test, manufactured by GenProbe, Inc, uses a synthetic, chemiluminescent labeled, DNA probe that hybridizes with the complementary ribosomal RNA of the target organism. Mycobacteria cells are sonicated and the ribosomal RNA is released. Complementary hybrids are formed and the chemiluminescent marker is maintained during the Hybridization Protection Assay. A selection reagent is added to destroy all unbound probe. The hybrids are detected in a luminometer during a chemical reaction that gives off light. The light is measured by the luminometer, and a positive result is obtained when the measurement exceeds the cut-off threshold.
The AccuProbe test is used by the laboratory to confirm identification of M. tuberculosis complex for low-level mycolic acid profiles from the HPLC instrument or for confirmation of species that result in non-definitive HPLC patterns.
The following probes are available from GenProbe: M. tuberculosis complex, M. avium intracellulare complex, M. gordonae, M. kansasii, M. avium, and M. intracellulare.
Genotyping
DNA fingerprinting is performed in this laboratory using the DiversilabTM system from BioMerieux. All bacterial genomes studied to date contain a unique pattern of repetitive sequences that, when amplified, can be used to differentiate between species and strains. Our fingerprinting system amplifies these repetitive elements from a pure culture using a repetitive PCR method and then separates the amplicons by size. This creates a distinct DNA fingerprint that can be used for identification and strain typing. This method is currently used for differentiation between M. chelonae and M. abscessus.
