Species diversity of microorganisms previously identified as nontuberculous mycobacteria by DNA hybridization.

Background: Over the past 10 years, the clinical importance of opportunistic bacteria of the order Actinomycetales has increased significantly. While many problems for the Mycobacterium tuberculosis complex have been solved, for nontuberculous mycobacteria, some questions remain open. These pathogens have a number of structural features that allow them to persist in the external environment for a long time.

Methods: The main inclusion criteria were cultural characteristics in assessing the growth of microorganisms on solid egg media. If nontuberculous mycobacteria (NTM) growth was detected, identification signs were carried out using the DNA hybridization method. Subsequently, these cultures were identified using the matrix-assisted laser desorption/ionization-time-of-flight (MALDI-ToF) mass spectrometry method. In case of obtaining unacceptable results of identification from primary inoculations, re-identification to obtain pure cultures was carried out after transferring the material from primary media to agar media: 5% blood agar and universal chromogenic medium. When re-identifying isolated cultures using MALDI-ToF mass spectrometry, all isolated cultures were analyzed, regardless of whether they belonged to the NTM group or not.

Results: DNA hybridization, which accounted for 59.5% of the total number of cultures included in the study, performed species identification of 188 strains. Using MALDI-ToF mass spectrometry, 345 strains were identified.

Conclusion: The use of methods based on DNA hybridization makes it possible to identify quite accurately some of the most common NTM species. MALDI-ToF mass spectrometry is an important technique to allow species identification of most Actinomycetales. However, algorithms to standardize methods for their isolation from clinical material are needed.