Vol 98, No 3 (2021)

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Adaptation of the MTT assay for detection of neutralizing antibodies against the SARS-CoV-2 virus

Gracheva A.V., Korchevaya E.R., Kudryashova A.M., Borisova O.V., Petrusha O.A., Smirnova D.I., Chernyshova I.N., Svitich O.A., Zverev V.V., Faizuloev E.B.


Introduction. The ability of SARS-CoV-2 antibodies to neutralize the virus is the primary indicator of their specific activity. The test for virus neutralizing antibodies (NAbs) is much needed in different biomedical studies.

The aim of the study is to find optimum conditions for microscopic and spectrophotometric detection of SARSCoV-2 NAbs by inhibition of cytopathic effect (CPE) in cell cultures.

Materials and methods. Blood sera collected from COVID-19 convalescent patients and healthy individuals (n = 96) were tested using the ELISA method. The SARS-CoV-2 coronavirus, Dubrovka strain (GenBank accession no. MW514307.1) was grown in culture medium of Vero cell line CCL-81 (ATCC). Real-time RT-PCR, ELISA, and Sanger sequencing were used for detection of the virus. The results of the neutralization test (NT) were assessed through the microscopic examination for CPE and by the methyl thiazolyl tetrazolium (MTT) assay.

Results. SARS-CoV-2 was isolated from a COVID-19 patient and adapted to grow in cell culture. At a low dose of infection (MOI = 0.00001), the virus caused a pronounced CPE with the cell viability less than 3%, thus making it possible to assess NT results by CPE inhibition. The NT and ELISA-based comparative study of sera showed positive correlation between virus NAb titers and Nab titers to S-protein RBD (Spearman’s r = 0.714; p < 0.001). The results of NAbs microscopic and spectrophotometric detection (the MTT assay) also demonstrated positive correlation (Spearman’s r = 0.963; p < 0.05).

Conclusion. The SARS-CoV-2 virus adapted to Vero cell culture served to develop a NAb titer assessment system, which can be used both in microscopic studies and for an MTT assay in spectrophotometric studies. The MTT assay provides automated reading of NT results, optimizes the statistical analysis of the obtained data, and minimizes subjectivity in assessment of results. Being a vital dye, MTT penetrates only viable cells, thus contributing to the reliability of the obtained results compared to other dyes.

Journal of microbiology, epidemiology and immunobiology. 2021;98(3):253-265
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A novel source of hospital microorganisms in healthcare settings

Chezganova E.A., Efimova O.S., Sakharova V.M., Efimova A.R., Sozinov S.A., Ismagilov Z.R., Brusina E.B.


Background. Most healthcare-associated infections (HAI) develop due to a colonization of patients and healthcare workers by hospital strains of pathogens. The aim to study was to assess whether the dust within the health facilities can harbor microorganisms acting as a reservoir of HAIs.

Materials and methods. Dust samples collected in the air ducts and ventilation grilles of health facilities underwent a detailed physicochemical analysis by means of scanning electron microscopy, dynamic light scattering, energy-dispersive X-ray spectroscopy, and high-temperature catalytic oxidation. Bacterial and viral diversity was investigated using an automated biochemical analyzer and polymerase chain reaction, respectively. Investigation of the microenvironment included detection of biofilms using a catalase indicator and quantification of viable microorganisms per 1 m3 air.

Results. Dust from the hospital ventilation grilles and air ducts was contaminated with microorganisms in 71.13% of cases. Strikingly, multidrug-resistant and biofilm-forming strains have been found in 69.4% and 48.0% of samples, respectively. The total viable count before and after opening doors and windows was 276 and 462 colony-forming units/m3 respectively (p = 0.046). Biodiversity was represented by 21 genera of microorganisms which were consistently detected upon 6 months of follow-up. All samples contained a nanosized particulate matter. Chemical elements comprising dust were carbon (16.26–50.69%), oxygen (20.02–37.50%), nitrogen (1.59–25.03%), hydrogen (2.03–6.67%), sulfur (0.15–2.38%), calcium (0.19–7.49%), silicon (0.21–4.64%), chlorine (0.05–2.83%), sodium (0.07–1.86%), aluminum (0.36–1.78%), iron (0.08–1.61%), magnesium (0.11–1.40%), potassium (0.04–0.85%), and phosphorus (0.04–0.81%).

Discussion. A wide range of multidrug-resistant strains of bacteria, detected in a hospital particulate matter with a diverse chemical composition, indicates the persistence of HAI-causing pathogens in the hospital environment.

Conclusion. Dust from the ventilation grilles and adjacent air ducts should be considered as an additional reservoir of multidrug-resistant strains of bacteria in the healthcare settings.

Journal of microbiology, epidemiology and immunobiology. 2021;98(3):266-275
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Mechanism of persistence of indigenous bifidobacteria under the impact of acetate in the human colon biotope

Bukharin O.V., Andryuschenko S.V., Perunova N.B., Ivanova E.V.


Aim. To determine the role of the acetate in the persistence of indigenous bifidobacteria in the colon biotope through the lysozyme resistance in model conditions of the acetylation–deacetylation of peptidoglycan.

Materials and methods. The study was performed on 16 strains of the two indigenous bifidobacteria speсies: Bifidobacterium bifidum и Bifidobacterium longum subsp. longum. Bifidobacteria was cultivated in the 0.6% O2 and 9% CO2 atmosphere at the temperature 37ºС in CO2 incubator for 48 hours. The production of the acetate by the bifidobacteria was determined by gas chromatography. The effect of the acetate on the lysozyme resistance of non-indigenous gram-positive bacteria was determined on the Listeria monocytogenes ICIS-280 model strain by the cultivation in LB-Lennox broth with ammonium acetate added in the concentration range matching the concentrations produced by the studied bifidobacteria, in lysozyme serial dilutions at final concentrations 5 μg/ml to 40 μg/ml within 24 hours.

Results. It was found that the acetate release of Bifidobacterium longum subsp. longum was on average two times higher that of Bifidobacterium bifidum (27.0 and 14.7 mmol/liter, respectively) and was quite consistent with the concentrations of acetic acid determined in the intestinal contents (up to 50 mmol/liter). Cultivation of bifidobacteria in a medium with lysozyme, ammonium acetate and their combination did not have a significant impact on their growth parameters at the maximum used concentrations of these substances. In the test strain, the addition of ammonium acetate in the range created by bifidobacteria caused a decrease in the minimum inhibitory concentration of lysozyme by more than two times — from 40 μg/ml to less than 20 μg/ml. In the control medium without lysozyme, no inhibition of the growth of the indicator culture was observed up to the maximum concentrations of ammonium acetate.

Conclusion. The mechanism of persistence (survival) of indigenous bifidobacteria in the human intestinal biotope has been identified, which is associated with the production of acetic acid at a level that selectively suppresses lysozyme resistance of non-indigenous gram-positive microbiota viareversible deacetylation of peptidoglycan. This allows indigenous bifidobacteria to maintain a stable dominant position in the biotope.

Journal of microbiology, epidemiology and immunobiology. 2021;98(3):276-282
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Effect of antibodies to agglutinogens 1 and 2, filamentous hemagglutinin and pertussis toxin on formation of Bordetella pertussis biofilms on abiotic substrate

Zaytsev E.M., Britsina M.V., Ozeretskovskaya M.N., Mertsalova N.U., Bazhanova I.G.


Aim. Study of the effect of antibodies to agglutinogens 1 and 2, filamentous hemagglutinin (FHA) and pertussis toxin (PT) on the formation of biofilms by Bordetella pertussis strains on the abiotic substrate.

Materials and methods. Vaccine-derived and freshly isolated strains of B. pertussis were used. Cultures of strains grown on a dense nutrient medium were used as an inoculum for obtaining biofilms. The intensity of biofilm formation in round-bottomed polystyrene 96-well plates in the presence of antisera to agglutinogens 1 and 2, antiserum to FHA, and monoclonal antibodies (MСА) to the S1, S2, and S3 subunits of PT was evaluated by staining with 0.1% gentian-violet solution.

Results. Most of the studied strains were sensitive to antibodies, which was manifested in complete suppression of biofilm formation. All strains were sensitive to antiserum to agglutinogen 1, antiserum to FHA, and MCA to the S2 subunit of KT. Three out of 4 studied strains with this agglutinogen in their composition were sensitive to antiserum to agglutinogen 2: No. 475 (serotype 1.2.3), No. 317 (serotype 1.2.3) and No. 178 (serotype 1.2.0). Relative resistance to antiserum was detected only in serotype 1.2.0 strain No. 305, but with minimal dilution, the intensity of biofilm formation was 1.8 times lower than in the control group. Strains No. 703 (serotype 1.0.3) and No. 287 (serotype 1.0.3) that did not have agglutinogen 2 were resistant to antiserum. Four and 5 out of the 6 strains used were sensitive to the S1 and S3 subunits of PT, respectively. Strain No. 305 was resistant to MCA to the S1 and S3 subunits, and strain No. 287 to MCA to the S1 subunit. At the same time, the intensity of biofilm formation was 2 and 1.8 times lower than in the control at the minimum MCA dilution.

Conclusion. These data indicate that the growth of biofilms of B. pertussis strains is suppressed by antibodies both to the surface structures of the microbial cell (agglutinogens 1 and 2, FHA) and to the S1, S2 and S3 subunits of PT.

Journal of microbiology, epidemiology and immunobiology. 2021;98(3):283-289
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Biological properties and genetic characteristics of experimental diagnostic Vibrio cholerae bacteriophages

Pogozhova M.P., Gayevskaya N.E., Vodopyanov A.S., Pisanov R.V., Anoprienko A.O., Romanova L.V., Tyurina A.V.


Background. Currently, the researches focused on the design of new diagnostic and preventive preparations based on bacteriophages are underway, so it is importatnt to study the biological properties of cholera phages along with their genetic structure. This information is necessary to predict the phage life cycle and assess the prospects of its practical use in experiments, phagodiagnostics and phagoprophylaxis.

Materials and methods. The presence or absence of genes characteristic of temperate bacteriophages was tested using a database created by the authors and developed software "PhageAnalyzer", which allows for rapid analysis of bacteriophage genome-wide sequencing data and prediction of their life cycle.

Results and discussion. The morphological structure of experimental diagnostic cholera phages is represented by head bacteriophages of various morphogroups. Negative colonies phage differed in diameter, shape and degree of transparency. No genetic determinants of resistance factors and toxins have been found in the genomes of bacteriophages Rostov-1, Rostov-6, Rostov 7, and Rostov M3. Results of phylogenetic analysis demonstrated that the studied experimental cholera bacteriophages resemble headphages from the genus Vibrio, but are unique, since they lie outside “cluster groups”. Vibrio phages Rostov-1 and Rostov M3 are appeared to be lytic. Genes characteristic of moderate bacteriophages were found in cholera phages Rostov-6 and Rostov 7.

Conclusion. The experimental cholera bacteriophage Rostov-1 can be used to differentiate cholera vibrion O1 the serogroup of the El Tor biovar, and Vibrio phage Rostov M3 can be used to differentiate the Classical biovar. Both bacteriophages are lytic and promising components for creating prophylactic drugs against cholera. Vibrio phages Rostov-6 and Rostov 7 can be successfully used only in experimental activities, as well as for monitoring cholera vibrions in the environment. Complete genomic sequences are deposited and available in the international database Genbank (NCBI).

Journal of microbiology, epidemiology and immunobiology. 2021;98(3):290–297
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Evaluation of azoximer bromide (polyoxidonium) influence on the adhesive properties of the Yersinia pestis EV NIIEG vaccine strain by atomic force microscopy

Shchukovskaya T.N., Goncharova A.Y., Bugorkova S.A., Erokhin P.S., Kudryavtseva O.M.


Aim. To characterize the influence of azoximer bromide (polyoxidonium, PO) in cultivation conditions on the morpho- and nanomechanical cell surface properties of Y. pestis EV NIIEG vaccine strain and its derivatives Y. pestis КМ218 (pYT–, pYV–, pYP–), Y. pestis КМ216 (pYT–, pYV–, pYP+), Y. pseudotuberculosis, Y. enterocolitica by atomic force microsopy (AFM), as well as on the adhesion of cells Y. pestis EV NIIEG to human collagen type IV.

Materials and methods. The measurements were carried out using the Solver P47-PRO probe microscope (NT-MDT, Russia), standard methods of semi-contact AFM and AFM imaging analysis program. The adhesion of Y. pestis EV NIIEG to type IV collagen was determined by the number of cells binding to glass slides covered with human collagen type IV.

Results. The introduction of PO in the cultivation environment caused changes in the morphometric parameters of the cells of Y. pestis EV NIIEG vaccine strain and its isogenic derivatives (increase in volume, flatten ingested (S/H), index I (W/H). These changes were accompanied by the transformation of nanomechanical properties of the cell surface (reducing the root mean square, adhesion force), which countenance was associated with the plasmid profile. The lesser decrease of adhesion force in the absence of changes of the index I was observed in cells Y. pseudotuberculosis and Y. enterocolitica with plasmid pYV. In the strain Y. enterocolitica KM383 (pYV–) PO did not induce significant changes in the indicators studied. The introduction of the PO into the cultivation environment decreased the ability of Y. pestis EV cells to bind to human collagen type IV. Modification by PO the adhesive properties of the vaccine strain Y. pestis EV NIIEG was accompanied by an increase in its immunogenicity.

Journal of microbiology, epidemiology and immunobiology. 2021;98(3):298-307
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Molecular genetic analysis of West Nile virus variants circulating in European Russia between 2010 and 2019

Baturin A.A., Tkachenko G.A., Ledeneva M.L., Lemasova L.V., Bondareva O.S., Kaysarov I.D., Shpak I.M., Boroday N.V., Korol' E.V., Teteryatnikova N.N.


Aim. A study of the prevalence of West Nile virus (WNV) genetic lineages and genovariants in the south of European Russia between 2010 and 2019.

Materials and methods. The study was carried out on 311 WNV containing biological samples from patients, vectors and reservoirs of infection. WNV typing was carried out using reverse transcription and real-time polymerase chain reaction with designed three pairs of primers and three probes and by the sequencing of the 277 bp WNV genome region corresponding to the 5'-untranslated region and locus of the polyprotein gene encoding the capsid protein C. Sequencing results were analyzed using the Nucleotide BLAST software (NCBI).

Results. As a result of typing, out of 311 WNV RNA isolates taken for the study, 15 (4.82%) were assigned to lineage 1 (from Astrakhan and Volgograd regions, Krasnodar and Stavropol Territories, Republic of Tatarstan), 285 (91.64%) to lineage 2 (from Astrakhan, Volgograd, Voronezh, Kursk, Lipetsk, Penza, Rostov and Saratov regions, Krasnodar and Stavropol Territories, Republics of Kalmykia and Crimea), and 11 (3.54%) to lineage 4 (from the Volgograd region, Republics of Kalmykia and Crimea). The predominance of viral lineage 2 was demonstrated. The identified isolates of the viral lineage 1 belonged to the «Astrakhan» variant, isolates of lineage 2 belonged to «Russian» and «European» variants. Previously uncommon WNV variants of lineages 1 and 4 were also found.

Conclusion. Lineage 2 of WNV prevailed in the south of European Russia in the last decade. The «Russian» variant is most common and its area is expanding. The circulation of various WNV genetic lineages in Russia indicates the need for further study of their spread and improving diagnostic methods and test systems for identifying and differentiating pathogen strains.

Journal of microbiology, epidemiology and immunobiology. 2021;98(3):308-318
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A systematic review of outbreaks of bloodborne infections (hepatitis B and C, HIV) transmitted from patient to patient in healthcare settings

Sacuk A.V., Solopova G.G., Ploskireva A.A.


Healthcare facilities have always played an important role in transmission of bloodborne infections. Procedures involving blood and blood fluids pose a risk of transmitting hepatitis B, hepatitis C and HIV not only to healthcare workers, but also to patients. To assess the role of healthcare facilities in transmission of bloodborne infections and to identify risk groups among patients as well as transmission factors, a total of 75 outbreaks of hepatitis B, hepatitis C and HIV have been analyzed with reference to the data published in different countries in 2008–2020. The comparative analysis was conducted for the outbreaks in the United States during 1992–2008 and 2008–2019. Most of the outbreaks of bloodborne infections at healthcare facilities were caused by non-adherence to standard precautions among healthcare workers: Reusing disposable items; improper handwashing; reusing gloves; non-disinfecting surfaces, reusable equipment and devices; non-sterilizing reusable instruments. In terms of bloodborne infections, high-risk facilities include hemodialysis centers, oncohematology clinics, outpatient clinics, nursing homes, residential care facilities, and diabetes treatment centers. High-risk groups include patients undergoing hemodialysis, oncohematological patients, and patients with diabetes. Diagnosis of bloodborne infections on a regular basis, hepatitis B vaccination among high-risk patients, investigation of outbreaks, adoption of rules and procedures combined with training and compliance control of healthcare workers contribute to solution of the problem associated with nosocomial transmission of bloodborne infections.

Journal of microbiology, epidemiology and immunobiology. 2021;98(3):319-330
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Disturbances of intestinal microbiota in autism spectrum disorders: new horizons in search for pathogenetic approaches to therapy. Рart 3. Рotential strategies of influence on gut-brain axis for correction of symptoms of autism spectrum disorders

Blagonravova A.S., Zhilyaeva T.V., Kvashnina D.V.


The third part of the literature review on the role of intestinal microbiota disturbances in the pathogenesis of autism spectrum disorders (ASD) is devoted to the analysis of published literature on possible interventional approaches for intestinal microbiota in ASD and the evaluation of the effectiveness of various types of interventions that have been studied in experiment and in clinical practice. Presented are available data on the possibility of correcting the intestinal microbiota in ASD with diet, taking pre- and probiotics, antibiotic therapy, as well as the effectiveness of transplantation of intestinal microbiota. An analysis of published data suggests that further development of approaches for correcting intestinal dysbiosis in ASD may provide safe and probably effective strategy for behavioral symptoms, but this requires further randomized controlled trials to confirm efficacy and safety from the perspective of evidence-based medicine, since available to date studies are small and scattered, and therefore they can only be considered preliminary.

Journal of microbiology, epidemiology and immunobiology. 2021;98(3):331-338
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Development and application of the technique for identification of Borrelia miyamotoi surface antigens

Mironov K.O., Titkov A.V., Kuleshov K.V., Kolyasnikova N.M., Bondarenko E.I., Platonov A.E.


Introduction. Borrelia miyamotoi is a pathogen causing erythema-free ixodid tick-borne borreliosis (ITBB), a disease widespread in Russia. The genome of B. miyamotoi contains genes of multiple variable major proteins (Vmps). Vmps fall into two families — Vsps and Vlps (with subfamilies δ, γ, α and β). At a particular time, a single B. miyamotoi expresses only one variant of Vmp gene.

The purpose of the work is to develop a technique for identification of the Vmp present at the expression site.

Materials and methods. The technique is designed in the format of a real-time multiplex PCR. It was tested by using B. miyamotoi DNA samples extracted from blood collected from 172 ITBB patients and 109 ticks. The samples were collected in 14 regions of Russia.

Results. The new technique made it possible to identify the expressed Vmp in 82% of the examined samples, thus having demonstrated its efficiency. Negative results were much less often observed with samples from patients than with samples from ticks. At the same time, the percentage of samples with one type of Vmp is identical for clinical samples and ticks, while the percentage of samples containing concurrently two types of Vmps is significantly higher among samples from patients with the most frequent occurrence of the Vlp-δ and Vsp combination.

Discussion. The frequent occurrence of the combination of two Vmp types in the blood samples can indicate the concurrent presence of several subpopulations of B. miyamotoi in ITBB patients. A new antigenic Vmp variant is synthesized after protective antibodies have been produced for the major protein of the strain transmitted by a tick. This phenomenon known as immune evasion allows the pathogen to persist within a host.

Conclusion. The developed technique of real-time multiplex PCR allows to simultaneous detect of several antigenic variants of the variable basic surface proteins of B. miyamotoi. The study of the antigenic spectrum of B. miyamotoi strains in comparison with the characteristics of conserved regions of the genome by the method of multilocus sequencing will clarify the stages of evolution and distribution of B. miyamotoi sensu lato.

Journal of microbiology, epidemiology and immunobiology. 2021;98(3):339-350
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Prevalence of tuberculosis among perinatally exposed to HIV and HIV-infected children and predictors of its development

Kukarkina V.A., Golubkova A.A., Podymova A.S.


Purpose of the study. To establish risk factors for tuberculosis (TB) in children perinatally exposed to HIV and HIV-infected for the development of corrective measures.

Materials and methods. Outpatient records (form No. 025/y) of 216 children perinatally exposed to HIV and 121 children with HIV infection and their parents (281 individuals) were analyzed. The control group consisted of 100 healthy children. Epidemiological (descriptive, evaluative and analytical) and statistical research methods were used in the study.

Results. The likelihood of contact with a patient with an active form of TB was significantly higher in perinatally exposed to HIV and HIV-infected children compared to children in the control group (p < 0.001). The risk of TB in HIV-exposed children was 5.3 times higher in foci where both parents were HIV-infected than in foci formed by discordant couples (RR = 5.3; 95% CI 1.7–21.7). Children in study groups who were not vaccinated with BCG had the highest risk of TB compared to children in the control group (RR = 1.9; 95% CI 1.6–2.2).

Conclusion. Risk factors for TB in children perinatally exposed to HIV are untimely vaccination against TB or its absence and living in the foci where both parents are HIV-infected. The predictors of the development of TB in HIV-infected children are household contacts with TB patient , late diagnosis of HIV infection, and late prescription of highly active antiretroviral therapy after the formation of severe immunosuppression.

Journal of microbiology, epidemiology and immunobiology. 2021;98(3):351-361
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In memory of Hans-Dieter Klenk

Editorial a.



Journal of microbiology, epidemiology and immunobiology. 2021;98(3):362-363
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In memory of Luydmila Pavlovna Zueva

Editorial a.



Journal of microbiology, epidemiology and immunobiology. 2021;98(3):364
pages 364 views


To the 90th anniversary of the academician of the Russian Academy of Sciences Dmitry Konstantinovich Lvov

Editorial a.



Journal of microbiology, epidemiology and immunobiology. 2021;98(3):365-366
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