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.

Proteomic analysis of nontuberculous bacteria protein spectra as the element of subtyping of strains.

Background: For the present, matrix-assisted laser desorption/ionization-time-of-flight (MALDI-ToF) mass spectrometry is the fastest and the most correct method for species identification of microorganisms. Apart from species-level identification, it allows to use a variety of approaches for the analysis and comparison of protein spectra of microorganisms of the same species, which are isolated from a patient at various disease states, that can be used in routine microbiological practice in laboratories fitted with mass analyzers. Methods: Two strains of Mycobacterium fortuitum and two strains of Mycobacterium peregrinum were isolated from sputum samples, which were obtained from patients with different clinical aspects of mycobacteriosis, whereat were reinoculated on the universal chromogenic culture medium "UriSelect 4." Further, the MALDI-ToF mass spectrometry method was used, aiming to obtain protein profiles, which were analyzed using the FlexAnalysis 3.0 software package. Results of the statistical proteomic comparison of mass spectra were visualized using MALDI Biotyper 3.0 Offline Classification software. Results: Presented clinical examples demonstrate that strains of the same species, which are isolated from the same patient at different times of infection, change their cultural properties. Dynamic changes in cultural properties are reflected in changes in protein profiles by comparison spectra of isolates at different stages of colonization, which is reflected in the correlation with the clinical condition of the patient. Conclusion: Thus, the mentioned examples of proteomic analysis, using MALDI-ToF mass spectrometry, demonstrate the possibility of subtyping of strains, that are isolated on a universal chromogenic culture medium, in case of detection in the culture signs of population's heterogeneity, based on cultural properties.

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.

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.

Application of chromogenic media for preliminary identification of acid-resistant bacteria.

Background: The variety of morphological and cultural characteristics of acid-resistant bacteria (ARB) makes it possible to use microscopy and estimate the growth rate and pigment formation when cultivating on solid egg media for preliminary identification only as additional indicative methods. It is necessary to develop new approaches for the cultivation and primary identification of ARB isolated from the biological material. It will allow to obtain data on the prevalence, structure, epidemiological, and clinical features of infectious processes caused by opportunistic ARB. Methods: Three hundred and sixty strains of ARB were isolated from the various biological materials obtained from the patients during the examination for tuberculosis. All biological material samples were negative on Mycobacteria tuberculosis complex. Species identification of all bacteria was performed by matrix-assisted lazer desorption/ion-ization time-of-flight mass spectrometry. The cultural characteristics of ARB were evaluated on a universal chromogenic media. As a selective additive, a mixture of bacitracin and polymyxin sulfate which had no effect on ARB was tested to suppress concomitant Gram-positive and Gram-negative microflora. Results: Cultural characteristics were identified and described for all tested representatives of fast-growing nontuberculous mycobacteria (NTM), as well as for all types of nocardia, gordonia, and streptomycetes. Representatives of other genera of ARB on a universal chromogenic media gave meager growth or did not show it at all. When inoculated on a universal chromogenic media with a selective addition, 100% of the strains from the ARB group showed abundant or moderate growth. Incubation time for fast-growing species was up to 7 days; for slow-growing species, it was up to 28 days. Concomitant control strains of Gram-positive and Gram-negative bacteria on universal chromogenic media with selective growth additive did not show the growth. Conclusions: The use of a universal chromogenic media allows to preliminarily identify NTM and other ARB by cultural characteristics. The addition of bacitracin and polymyxin sulfate does not reduce the growth properties of ARB, which can be used when working with both biological materials and for the isolation of pure ARB cultures from mixtures with other bacteria.