Vol 98, No 6 (2021)

Introduction. Lactoferrin is a cationic monomeric glycoprotein produced by acinar cells and glands, present in different places of the mucous membrane in different concentrations. In connection with the development of various variants of hygienic and medicinal products for the treatment of inflammatory diseases of the oral cavity based on lactoferrin, there was a need for an objective assessment of its antibacterial and antibiofilms properties, followed by an analysis of the preservation of activity in various variants of the isolation of this protein from the substrate and storage.
Aim - to improve the effectiveness of evaluating the antibacterial activity of lactoferrin and the duration of its preservation in various biological substrates containing the active substance and individual experimental batches of the manufactured drug using automatic cultivation.
Materials and methods. As part of the experiment, a microbiological diagnostic technique employing a system for the automatic cultivation of microbial populations was used. A pre-prepared bacterial suspension was inoculated into the nutrient broth and the studied lactoferrin samples were added, followed by cultivation and analysis of the possible antibacterial effects of transferrin protein. To determine the sensitivity of the isolated strains, we used our own modification of the serial dilution method developed at the Department of microbiology, virology, immunology of the A.I. Yevdokimov Moscow State University of Medicine and Dentistry. The experiment was based on the programmed automatic cultivation using the RTS-1 bioreactor. The interpretation of the results was carried out by changing the optical density at a wavelength of λ = 850 nm. The study of the growth dynamics of microorganisms was carried out in several repetitions, which was reflected in the graphs of the development of bacterial populations. The assessment of the growth control of the corresponding bacterial species was reflected in the change in the optical density values, on the basis of which the curve was built. Results and discussion. According to the results of an experimental study of the growth curves of bacterial populations, statistically significant differences in the number of viable cells in different phases of the growth curves were noted, when using different lactoferrin samples. Higher activity of human recombinant lactoferrin samples was established. An analysis of growth dynamics revealed differences in the onset of the maximum reproduction and its inhibition under the influence of various aggravating factors during cultivation. The bacteriostatic effect of lactoferrin is realized through the binding of iron ions, depriving the bacteria of this microelement, causing inhibition of their development. Along with this, lactoferrin is active against certain virulence factors of microorganisms, splitting them like serine proteases, and thus prevents their penetration into human cells.
Conclusion. The method used for automatic cultivation of microorganisms in the bioreactor used allows one to obtain reproducible results, is available for wide use, and can be recommended for obtaining objective, comparable, reliable information about the antimicrobial properties of various samples of the bactericidal protein lactoferrin produced by the domestic pharmaceutical industry. The studied substrate containing recombinant human lactoferrin of Russian production is characterized by high antibacterial activity that persists for 3 years as minimum.

Introduction. About 1,000,000 cases of infections caused by Acinetobacter spp. per year are registered globally, making up 1.8% of all the cases of hospital-acquired infections. In compliance with long-term studies carried out in in this country and abroad, Acinetobacter baumannii is a clinically important representative of the Acinetobacter genus. Intraspecific typing of microorganisms is an integral part of a clinical microbiologist's contribution to scoring the outbreaks of purulent-septic infections within the sphere of HAI surveillance. Most of the practicing microbiological laboratories cannot use genotypic typing methods because of their high costs.
Objective. Developing a test panel for intraspecific identification of A. baumannii sequence types (ST 1167, ST 944, ST 208) based on their phenotypic properties.
Materials and methods. Intraspecific membership of 74 A. baumannii strains obtained from four multipurpose health settings of a large industrial centre was studied using a genetic method (multilocus sequence typing) and a suite of phenotypic methods (biochemical tests, biofilmogenous capacity, growth inhibition zones to antibacterial drugs, sensitivity to aniline dyes, disinfectants and Acinetobacter bacteriophage) was studied.
Results. Phenotypic features of three predominant A. baumannii sequence types (ST 1167, 944, 208) were determined.
Discussion. An efficacious economy set of differentiating tests allowing identification of intraspecific features of A. baumannii multiresistant strains was сreated.
Conclusion. The test panel will enable the laboratories that cannot use sequencing methods to conduct intraspecific differentiation of common A. baumannii sequence types as part of microbiological monitoring.

Introduction. Currently, the participation of women in space flights is increasing. In this regard, questions about the influence of space factors on the state of the female body arise inevitably. Model experiments, in particular, "dry" immersion, are most convenient for studying the influence of individual factors of space flight on the organism. The aim of this work is a comparative assessment of the state of the vaginal microbiota of 6 female volunteers before and after three-day "dry" immersion.
Materials and methods. Microbial samples of all volunteers were stained according to Gram with a sequential culture study in accordance with the medical technology. The species identification of microorganisms was performed by MALDI-TOF-MS analysis using an Autoflex III time-of-flight mass spectrometer with Maldi BioTyper software.
To assess changes in the state of the vaginal microflora and microflora of the cervical canal, eubiotic index was used. It reflects the number of positive states of microbiota to the number of negative ones.
Results. After 3 days of "dry" immersion volunteers, who had high titer of aerobic microorganisms before isolation, had significant increase of the amount of aerobic microorganisms, while the number of lactobacilli decreased. The other group of volunteers showed activation of colonization resistance of the vaginal microflora. Volunteers, who had a significant contamination with anaerobic opportunistic microflora before isolation, had reduction of the number of all anaerobes, including lactobacilli. The eubiotic index, calculated for the cervical canal, decreased after 3 days of immersion. The data obtained indicate that after 3 days of isolation, the state of the microflora has deteriorated.

Objective. Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) affect 150 million people annually.
Purpose: Characteristics of non-hospital strains of UPEC isolated from patients with UTI in Yaroslavl in 2016– 2017.
Materials and methods. Susceptibility of UPEC strains (n = 20) to antibacterials was measured by the serial dilution method; the antibiotic resistance and virulence genes, phylogroups, O-serogroups and sequence types were identified by PCR and whole genome sequencing. The virulence of the strains was studied using the model of Galleria mellonella larvae.
Results. UPEC strains were classified as resistant (n = 11) and multi-drug resistant (n = 9) pathogens. Betalactamase genes bla_TEM (n = 10), bla_CTX-M (n = 6), class 1 integrons (n = 8), and gene cassettes dfrA17-aadA5 (n = 2), dfrA1 (n = 1) and aacA4-cmlA1 (n = 1) were identified. UPEC-virulence genetic determinants coding adhesins fimH, papG, sfaS, focG, afa/draBC, csgA, siderophores iroN, fyuA, iutA, counteracting factors of host immunity ompT, traT, toxins hlyA, cnf1, usp, capsule transporter kpsMTII, colicin cvaC, and pathogenicity islands I₅₃₆, II₅₃₆, III₅₃₆, IV₅₃₆, II_J96 и II_CFT073 were detected. Highly virulent and slightly virulent for G. mellonella larvae UPEC strains were obtained with LD₅₀ 10⁴–10⁵ and 10⁶–10⁷ CFU, respectively. The phylogroups A, B1, B2, E and F, serogroups О2, О4, О6, O9, O11, О15, О18, О25, О75 and O89, known sequence types ST14, ST58, ST69, ST73, ST93, ST127, ST131, ST-141, ST165, ST297, ST457, ST537, ST744, ST1434 and novel ST9239 and ST10102 were revealed.
Conclusions. The identified genetic diversity of non-hospital UPEC strains is consistent with the observed global trend in the spread of human pathogens, which are characterized with both high virulence and multiple drug resistance. This makes possible to assess prospectively the current epidemiological situation, give a forecast for its development in the future, as well as determine the optimal therapeutic options.

Introduction. Mycobacterium tuberculosis is the causative agent of tuberculosis. Drug susceptibility testing is performed by phenotypic and molecular tests. Commonly used for phenotypic drug susceptibility testing is the automated BACTEC system in a liquid culture medium. Drug susceptibility by line probe molecular tests was introduced almost 15 years ago. Recently whole genome sequencing (WGS) analysis of M. tuberculosis strains demonstrated that genotyping of drug-resistance could be accurately performed. Several software tools were developed.
Our study aimed to perform whole-genome sequencing on phenotypically confirmed multi-drug resistant (MDR) M. tuberculosis strains, to identify drug-resistant mutations and to compare whole-genome sequencing profiles with line probe assay and phenotypic results.
Materials and methods. We performed analysis on 34 MDR M. tuberculosis Bulgarian strains. Phenotypic drug susceptibility testing was performed on the BACTEC system. For molecular testing of drug susceptibility to first- and second-line tuberculostatics, we applied line probe assay Geno Type MTBDR plus v.1.0 и Geno Type MTBDR sl v.1.0. Sequencing was performed on MiSeq. Generated FASTQ files were analyzed for known drugresistant mutations with the software platform Mykrobe v.0.8.1.
Results. All three methods — phenotypic analysis using the BACTEC system, genetic analysis of strains applying the Geno Type test and Mykrobe software gave comparable sensitivity/resistance results for the studied strains. All phenotypically proven rifampicin and isoniazid-resistant strains were 100% confirmed using Mykrobe software. The C-15T mutation is a marker for isoniazid resistance in strains of the SIT41 spoligotype. We observed a 75% (21/28) agreement between BACTEC and Mykrobe for ethambutol resistance. Phenotypically, 87% (n = 27) of the strains are resistant to streptomycin, but only 59% (n = 19) are proven by Mykrobe software. Comparing phenotypic and genotypic resistance to ofloxacin, amikacin and kanamycin, we observed 100% coincidence of results.
Conclusions. Whole-genome sequencing approach is relatively expensive and laborious but useful for detailed analysis such as epidemiological genotyping and molecular drug susceptibility testing.

Nontyphoid strains of Salmonella enterica pose a great threat to human health. The problem of salmonellosis is aggravated compounded by the progressive spread of antibiotic resistance among clinical and agricultural strains of S. enterica. This literature review summarizes the current knowledge of the mechanisms of antibiotic resistance in S. enterica and illustrates the diversity and complexity of molecular systems providing antibiotic resistance. The spectrum of natural resistance is described and the adaptive (acquired) mechanisms of resistance to representatives of the main classes of antibiotics, including fluoroquinolones, aminoglycosides, tetracyclines, nitrofurans, sulfonamides, fosfomycin and chloramphenicol, are thoroughly characterized. Particular emphasis is placed on the analysis of the molecular genetic mechanisms of S. enterica resistance to representatives of the most important classes of antibiotics — β-lactams, and to reserve antibiotics — polymyxins (colistin). Genetic determinants of resistance, transmitted by a horizontal path route are also described. The review analyzes only those variants of the molecular mechanisms of antibiotic resistance where the clinical significance has been proven by a set of correct genetic (sequencing) and biochemical (confirmation of the spectrum of hydrolyzed β-lactams) studies. The main ways of regulating the expression of antibiotic resistance are also described. Many S. enterica strains exhibit a combination of different mechanisms of antibiotic resistance and have a multiple resistance. The question was raised about the heterogeneity of the distribution of resistance among different groups/serotypes within the S. enterica species. In particular, some clonal complexes with signs of resistance are more successful pathogens in humans and animals. Salmonella, like most other bacteria, exhibit a non-canonical type of antibiotic resistance — biofilm resistance, which is realized through several mechanisms, the main of which are the filtering/sorption capacity of the biofilm matrix and the transformation of biofilm cells into dormant and persistent forms.
Despite the fact that the functional significance of the molecular assemblies that determine antibiotic resistance is the same for all enterobacteria, the specification of the mechanisms of resistance in Salmonella is a necessary link for the development of molecular diagnostic systems for assessing the sensitivity to antimicrobial drugs.