Molecular Identification

The genus Mycobacterium consists of a diverse group of acid-fast bacilli. As previously mentioned, conventional methods for identification of these organisms are well established and inexpensive, but these methods are time-consuming and labor-intensive, and they provide inconclusive results, often leading to species identifications based on a "best-fit" approach. The introduction of molecular methods has revolutionized the identification of mycobacteria by reducing time to identification as well as providing more accurate speciation.

DNA Hybridization. DNA hybridization is used to identify some of the more common mycobacterial species isolated on solid culture media or from broth culture. Of importance, these tests can be performed with sufficient growth from primary cultures. Non-isotopically labeled (i.e., acridinium ester-labeled) DNA probes specific for mycobacterial ribosomal RNA (rRNA)

BOX 45-5 Mycobacterial Species Identified bythe INNO LiPA Mycobacteria Version 2 (Innogenetics. Ghent, Belgium)

  1. tuberculosis complex
  2. avium complex and members (M. avium, M. intracellular, and
  3. scrofulaceum) M. kansasii (groups I, II, and III)
  4. xenopi M. gordonae
  5. chelonae (groups I, II, and III)

sequences are commercially available (GenProbe, San Diego, Calif) and are listed in Box 45-4. rRNA is released from the mycobacteria by means of a lysing agent, sonication, and heat. The specific DNA probe is allowed to react with the extracted rRNA to form a stable DNA-RNA hybrid. Any unhybridized DNA-aaidinium ester probes are chemically degraded. When an alkaline hydrogen peroxide solution is added to elicit chemi-luminescence, only the hybrid-bound acridinium ester is available to emit light; the amount of light emitted is direcdy related to the amount of hybridized probe. The light produced is measured on a chemilurninometer. Numerous laboratories have incorporated these tests into their routine procedures.

Amplification and Amplification with Reverse Hybridization. Subsequent to the introduction of commercially available hybridization assays, commercially available and in-house developed nucleic acid amplification tests were successfully used for the early identification of M. tuberculosis complex grown in liquid cultures such as the BACTEC MGIT cultures. In addition, a commercially available system in which the 16S-23S rRNA spacer region of mycobacterial species (INNO* LiPA Mycobacteria; Innogenetics, Ghent, Belgium) ha* been successfully used to directly detect and identify eight of the most clinically relevant mycobacterial spffl des in aliquots of positive liquid cultures (Box 45-5) $ second commercial system, Genotype Mycobacterium (Hain Lifescience GmbH, Nehrin, Germany), using j similar format, has additional probes for M. celatum, Mj malmoense, M. peregrinum, M. phlei, and two subgroups OP

frl formitum as well as a supplemental kit that allows for 16 additional mycobacterial species.

Other Molecular Methods of Identification. Amplification and Restriction Enzyme Analysis or UNA Sequencing. As previously mentioned, it has become increasingly important that identification of mycobacteria be accurate because organisms that are' responsible for disease have important consequences for the proper selection of therapy. Thus, molecular methods have become increasingly important due to their rapidity and, in most cases, production of unequivocal results. Most of these methods are mainly based on 16S rRNA, 16S rDNA, the internal transcribed spacer (ITS) between 16S-23S rDNA and 16S rDNA, and the heat shock protein hsp65 gene of mycobacteria. PCR-based sequencing for mycobacterial identification consists of PCR amplification of mycobacterial DNA with genus-specific primers and sequencing of the amplicons. The organism is identified by comparison of the nucleotide sequence with reference sequences. The target most commonly used for this approach is the gene coding for the 16S ribosomal RNA that is present in all bacterial species. This region contains both conserved and variable regions, thus making it an ideal target for identification purposes. Despite its accuracy, problems remain in that the sequences in some databases are not accurate, there is no present consensus as to the quantitative definition of a genus or species based on 16S rRNA gene sequence data, and procedures are not standardized.

Another common noncommercial approach-for identification is to amplify by PCR a highly conserved gene, such as the hsp65 gene, and perform restriction enzyme analysis on the PCR product. Following restriction enzyme digestion, usually using two different enzymes, digested products are separated by gel electrophoresis with subsequent sizing of resulting fragments The restriction fragment patterns are species-specific and are able to differentiate many species of NTM.

DNA Microarrays. DNA microarrays are also attractive lor the rapid examination of large numbers of DNA sequences by a single hybridization step. This approach has been used to simultaneously identify mycobacterial species and detect mutations that confer rifampin resistance in mycobacteria. Fluorescent-labeled PCR arnplicons generated from bacterial colonies are hybridized to a DNA array containing nucleotide probes. The bound amplicons emit a fluorescent signal that is detected with a scanner. Using this approach, 82 unique 16S ribosomal RNA sequences allow for the discrimination of 54 mycobacterial species and 51 sequences [hat contain unique rpoB gene mutations (mutations that confer resistance to rifampin).31

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