Identification of the Optimal Cultivation Period Required to Isolate Representatives of Mycobacterium abscessus Complex Isolated from Patients with Cystic Fibrosis.

BACKGROUND: In patients with cystic fibrosis (CF), representatives of the fast-growing Mycobacterium abscessus complex (MABSc) are often distinguished, but the culture of the material taken from such patients increases the growth time. We analyzed the terms of cultivation of MABSc representatives on dense nutrient media and also evaluated the productivity of a modified nutrient medium based on agar for the isolation of Burkholderia cepacia complex (BCC). METHODS: Sixty-four strains of MABSc isolated from patients with CF and suspected tuberculosis were analyzed. The material from the patients was cultured on a universal chromogenic medium, 5% blood agar, yolk-salt agar, selective medium for isolation of BCC, and Löwenstein-Jensen medium. The cultures were incubated for 5 days (37°C, aerobic conditions), after for 23 days (28°C, aerobic conditions). The productivity of the developed nutrient medium was evaluated by the number of cells that gave visible growth after culturing 0.1 mL of a bacterial suspension of 103 CFU/mL. RESULTS: 76.8% of the strains grew in a 2-week period, and 23.2% of the strains were obtained at a later date from 18 to 28 days (average: 21.23 days). The modified medium with a concentration of 240 mg of iron (III) polymaltose hydroxide proved to be the most optimal for the isolation of MABSc. CONCLUSION: When using a chromogenic medium for culture material from patients with CF, it is necessary to extend incubation up to 28 days to increase the probability of MABSc isolation. The modified BCC medium showed a good selectivity result but required further investigation.

Comparison of laboratory methods for identifying members of the family Mycobacteriaceae.

Background: The introduction of a method based on matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF mass spectrometry) into the practice of laboratories significantly increased the identification of acid-resistant bacteria (ARB). Methods: Seventy-four nontuberculous mycobacteria (NTM) cultures identified by deoxyribonucleic acid (DNA) hybridization, polymerase chain reaction, Sanger sequencing, and MALDI-ToF mass spectrometry. Results: Analysis of the identification results obtained by the methods of DNA hybridization and Sanger sequencing showed a complete match only for 67.6% of samples of the total number of cultures included in the study. The partial match of the identification results was 68.9%. When comparing the results of the identification of 74 samples obtained by MALDI-ToF mass spectrometry to the results obtained by sequencing, full match of identification of Mycobacterium chimaera/Mycobacterium intracelullare, Mycobacterium porcinum/Mycobacterium peregrinum and Mycobacterium tuberculosis complex was found for 90.5% of the samples; the partial match of the results - for 4.1%.. DNA hybridization as a method for identifying NTM showed acceptable sensitivity and specificity; however, for mass spectrometry, a significantly higher sensitivity with comparable specificity was determined. Conclusions: Mass spectrometry is an important element in the modern system of species identification of microorganisms. The optimization of sample preparation protocols and assessment of the impact on the identification of new techniques of cultivation of microorganisms can significantly improve the quality of identification of microorganisms from the ARB group. In this case, accurate species identification and the development of algorithms for its application will improve the diagnosis of diseases caused by ARB.