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.

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.

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.

General characteristics, features of cultivation and antibiotic resistance representatives of mycobacterium fortuitum group representatives (review of literature).

Recently, more and more scientific works have been devoted to non-tuberculous mycobacteria, both by domestic and foreign researchers. One of the main reasons for this is the increase in patients with immunosuppression of various origins, improvement of the quality of laboratory and instrumental diagnostics of mycobacteriosis. This article focuses on the representatives of the M. fortuitum group, as the main pathogens among the group of fast-growing mycobacteria. The data on the modern classification based on the use of molecular genetic studies are indicated. The M. fortuitum group includes: Mycobacterium fortuitum, M. peregrinum, M. senegalense, M. porcinum, M. houstonense, M. neworleansense, M. boenickei, M. conceptionense, M. septicum, M. alvei. According to the new data, mycobacteria were divided into 5 clades (Abscessus-Chelonae, Fortuitum-Vaccae, Terrae, Triviale, Tuberculosis-Simiae), and based on molecular genetic studies, new genera in the Mycobacteriaceae family were isolated: Mycolicibacter spp., Mycolicibacillus spp., Mycolicibacillus spp., Mycobacteroides spp., Mycolicibacterium spp. In accordance with the new classification, representatives of the Mycobacterium fortuitum group belong to the genus Mycolicibacterium. The main epidemiological features of the main sources of the spread of mycobacteria, factors and ways of their transmission are indicated. Due to their wide distribution in the environment, representatives of the M. fortuitum group are capable of causing diseases of the pulmonary and extrapulmonary localization. The distinctive features of pathogenicity factors, due to which the course of the disease is determined, are noted. The article also indicates the main difficulties and features of determining the sensitivity to antimicrobial chemotherapy drugs, provides data on the main features of antibiotic resistance of M.fortuitum group. In preparing the review, literature sources obtained from international and domestic databases were used: Scopus, Web of Science, Springer, RSCI.

[Structure of microorganisms isolated from bronhoalveolar lavage from patients in the department of reanimation and intensive therapy.] / Структура микроорганизмов, Ð²Ñ‹Ð´ÐµÐ»ÐµÐ½Ð½Ñ‹Ñ Ð¸Ð· Ð±Ñ€Ð¾Ð½Ñ Ð¾Ð°Ð»ÑŒÐ²ÐµÐ¾Ð»ÑÑ€Ð½Ð¾Ð³Ð¾ лаважа от пациентов отделения реанимации и интенсивной терапии.

The aim of the work was to determine and compare the structure of microorganisms isolated from bronchoalveolar lavage from patients in the ICU of Clinics in 2016 and 2019. This work presents the results of a bacteriological examination of 229 samples from 139 patients for 2016 and 387 samples from 218 patients for 2019. The predominant microorganism in 2016 was Acinetobacter baumanii - 75 (26.2%). Less common were Klebsiella pneumoniae - 55 (19.2%), Pseudomonas aeruginosa - 35 (12.2%), Escherichia coli - 19 (6.6%). In 2019, the prevailing microorganism was K.pneumoniae - 158 (19.1%). As in 2016, A.baumanii - 115 (13.9%) and P. aeruginosa - 57 (6.9%) were most often found, but unlike 2016, in 2019 there was a high incidence of such pathogens as Enterococcus faecalis - 52 (6.3%), Candida albicans - 43 (5.2%), Staphylococcus aureus and Stenotrophomonas maltophilia - 40 (4.8%). One of the features is the presence of polymicrobial associations. In 2016, microorganisms isolated in monoculture predominated (53.4%), while in 2019 the frequency of occurrence of monocultures decreased and amounted to 24.7%. At the same time, a two-component association prevailed (31.5%). Thus, in ICU it is necessary to regularly monitor nosocomial pathogens not only to make the right decision when choosing antimicrobial therapy, but also to identify new potential nosocomial pathogens.