Comparison of protein profiles obtained by matrix-assisted laser desorption/ionization-Time-of-flight mass spectrometry in representatives of the family Mycobacteriaceae grown on various nutrient media.

Background: The nutrient medium effects on the quality of the matrix-assisted laser desorption/ionization-time-of-flight (MALDI-ToF) mass spectra. The standard library includes spectra of microorganisms of the family Mycobacteriaceae grown on the Lowenstein-Jensen and Middlebrook Media. There are new methods for culturing microorganisms from this group, including inoculation on chromogenic media. Methods: The study included 240 strains of NTM isolated from patients during tuberculosis examination. The inoculation of the biological material was carried out on solid culture media of Lowenstein-Jensen and universal chromogenic media. Identification of bacteria from both types of media was performed by MALDI-ToF mass spectrometry (Bruker Daltonik GmbH, Germany). Analysis of protein spectra was performed. Results: For all strains, the spectra revealed both coinciding peaks (regardless of the cultivation medium) and significant differences, including the complete absence of some peaks depending on the medium. The results of a greater divergence of peaks in mass and intensity were obtained for slow-growing species than for fast-growing species. For all analyzed cultures, the number of peaks in the mass spectra was significantly higher when cultivating on a universal chromogenic medium than on a Lowenstein-Jensen medium. Conclusions: The use for NTM cultivation of a universal chromogenic medium makes it possible to obtain acceptable identification results by MALDI-ToF mass spectrometry using a standard library.

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.

Evaluation of the influence of the cultivation medium on the result of identification of microorganisms from the group of acid-resistant bacteria of the order actinomycetales by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Background: Problem with mass spectrometers is the difficulty in identifying some genera of acid-fast bacteria (AFB). Due to the peculiarities of the colony architectonics (the formation of dry colonies with a complex structure) and the structure of the cell wall, the probability of obtaining a sufficient amount of ribosomal proteins is significantly reduced. This problem is partially solved using an extended method of direct application and extraction with formic acid, which can significantly improve the quality of the identification. Methods: The study analyzed strains of microorganisms obtained during the examination of patients with suspected tuberculosis. In total, 287 strains of nontuberculous mycobacteria (NTM) were obtained. In addition, 63 strains of the most common bacteria from the AFB group were analyzed. Matrix-assisted laser desorption/ionization (MALDI) was used. Three main methods of sample preparation of microorganisms according to the manufacturer's recommendations for use with the MALDI-time-of-flight (ToF) mass spectrometry method were used in the work: Direct coating method, extended direct coating method, and formic acid extraction method. Results: When evaluating the influence of the cultivation medium on the result of NTM identification by MALDI-ToF mass spectrometry, statistically significant results of the influence of the medium on the result of NTM identification were revealed for all compared parameters. Conclusions: Optimization of sample preparation protocols and assessment of the impact on the identification of new methods of cultivating microorganisms can significantly improve the quality of the identification of both clinically significant microorganisms from the AFB group and saprophytic microflora, the clinical significance of which has not been proven at the moment.