Vol 97, No 6 (2020)

Currently, a lot of attention is given to SARS-CoV-2 subpopulations and their coexistence with different genomic variants within the same patient. In this study, we performed next-generation whole-genome sequencing and assembly of viruses from samples representing swabs or autopsy specimens obtained from patients diagnosed with СOVID-19, which were initially confirmed by the real-time polymerase chain reaction (Ct = 10.4–19.8). Samples were prepared for sequencing by using the SCV-2000bp protocol. The obtained data were checked for presence of more than one SARS-CoV-2 genetic variants in a sample. Variants of nucleotide substitutions, coverage for each variant, and location of the variable position in the reference genome were detected with tools incorporated in the CLC Genomics Workbench program. In our search for variable nucleotide positions, we assumed that the sample had two genetic variants (not more); the threshold value ≥ 90% was set for probability of the identified variant. Variants represented by less than 20% of the reads in the total coverage were not taken into consideration. The obtained results showed that 5 samples had variability, i.e. they had several genetic variants of SARS-CoV-2. In 4 samples, both of the detected genomic variants differed only in one nucleotide position. The fifth sample demonstrated more substantial differences: a total of 3 variable positions and one three-nucleotide deletion. Our study shows that different genetic variants of SARS-CoV-2 can coexist within the same patient.

Aim. To study the sensitivity of biofilms of vaccine and freshly isolated strains of Bordetella pertussis to antibiotics.

Materials and methods. Vaccine and freshly isolated strains of B. pertussis were used. Cultures of strains grown on dense nutrient medium were used as inoculate for biofilms production. The intensity of biofilm formation in round-bottomed polystyrene 96-well plates was estimated by staining with 0.1% gentian-violet solution. The following antibiotics were used in experiments: penicillins (ampicillin), cephalosporins (ceftriaxone), aminoglycosides (gentamicin), macrolides (erythromycin).

Results. The highest resistance to antibiotics was demonstrated by the vaccine strain No. 305 and freshly isolated strain No. 211, sensitive only to erythromycin. Vaccine strain No. 703 was sensitive to gentamicin and ampicillin and showed resistance to erythromycin and ceftriaxone. Vaccine strain No. 475 was sensitive to all tested antibiotics. The Tohama 1 strain was resistant to ampicillin and sensitive to other antibiotics. Freshly isolated strains No. 178 and No. 162 were resistant to ceftriaxone and sensitive to gentamicin, erythromycin and penicillin. Minimal inhibitory concentrations of tested antibiotics ranged from 0.2 μg/ml to 5.0 μg/ml.

Conclusion. These data indicate the heterogeneity of vaccine and freshly isolated strains of B. pertussis in sensitivity to antibiotics. The greatest activity was shown by erythromycin, which suppressed the growth of biofilms of 6 out of 7 strains. The least effective was ceftriaxone, which suppressed the growth of biofilms of only 2 strains.

Introduction. Listeriosis is a foodborne infection, especially dangerous for people in at-risk groups. Susceptibility to listeria infection is determined by a complex of reasons: environmental factors, host immune status, and pathogen virulence. The susceptibility to listeriosis can also be aggravated by previous infections, especially viral infections, which demonstrate a steadily increasing number of identified pathogens.

The aim of our study was to present molecular and genetic characterization of pathogens causing sporadic invasive listeriosis in a megalopolis, primarily during the peak of influenza and ARVI incidence.

Materials and methods. Listeria monocytogenes isolates were collected from 18 hospitalized patients at hospitals in Moscow, from November 2018 to October 2019. The first comparison group was represented by isolates from food products and fish preserves. The second comparison group included previously examined environmental isolates. The clinical isolates were examined by using multilocus sequence typing techniques, including the standard MLST scheme extended by loci of internalin genes. Isolates of the autochthonous genotype (ST7) were compared through whole-genome sequencing and subsequent analysis of the core genome (cgMLST).

Results. In cases of invasive listeriosis, 44% of isolates were isolated from patients with listeriosis; 27% of isolates were obtained from patients with meningitis. L. monocytogenes of phylogenetic lineage II prevailed in these groups of cases that occurred when the epidemic threshold for influenza was crossed during the 2018/2019 season. Listeria pneumonia identified in the senior age group occurred during the season of autumn ARVI and was primarily caused by L. monocytogenes of phylogenetic lineage I. The examination of genomes of ST7 isolates demonstrated identity between the core genomes of bacteria isolated from the mother-infant pair. Out of ST7 food isolates most closely related to the clinical ones was the isolate from meat (23 locus differences, the common deletion in the MFS transporter locus). Analyzing invasive listeriosis, the comparison between the list of the identified genotypes and the data from European countries showed that each country had its own specific range of genotypes, though ST7 was detected in all the examined samples.

Conclusions. Along with the monitoring of food manufacturing and storage, timely vaccination against seasonal respiratory infections and use of personal protective equipment in public spaces can reduce the risk of listeriosis incidence in at-risk groups.

Objective — comparative study of protein expression in typical and genetically altered Vibrio cholerae strains of O1 serogroup, biovar El Tor by means of proteomic analysis.

Materials and methods. Clinical V. cholerae strains — typical strain, M106 (Astrakhan, 1970) and genetically altered one, M1509 (Moscow, 2012) — were used as model ones. Strains were cultivated in LB broth (pH7.2). Then, cell and exoprotein lysate fractions were obtained and investigated in 2D electrophoresis. Different protein stains were examined using mass spectrometry. Survivability of V. cholerae strains under osmotic and oxidative stresses was studied during incubation of the strains in 3 M NaCl solution or 20 mM H₂O₂ solution.

Results and discussion. When analyzing cell lysates, significant differences in protein expression with known function between studied strains were not detected. The great majority of identified proteins in the lysates is functionally associated with carbohydrate metabolism, amino acid metabolism, and energy processes in a cell. At the same time, exoprotein fraction of M1509 genovariant contained increased amount of proteins (peroxidase, superoxide dismutase, thioredoxin, outer membrane proteins OmpW, OmpT) protecting the cells of cholera vibrio from effect of stress factors of the environment. Further study of the resistance to osmotic and oxidative stresses revealed better survivability in the genovariant when exposed to the stated factors.

Conclusion. The data of proteomic analysis of the typical and genetically altered V. cholera strains, biovar El Tor, testify to high levels of expression of the proteins that provide for vibrio resistance to the effect of environmental stress factors in genovariants, which is possibly one of the causes of their wide dissemination. In addition, the results obtained will allow for identification of new biomarkers which can be used for differentiation of typical strains and genovariants of V. cholerae, biovar El Tor.

Introduction. Currently, there is insufficient data on the prevalence of Klebsiella pneumoniae strains with virulence factors genes uge and fim among women and newborns. This indicates the need for a study of the prevalence of K. pneumoniae (uge⁺, fim⁺) and the degree of heterogeneity of the bacterial population isolated from children and adults.

The aim of the study was to perform a phylogenetic analysis of the uge and fim genes of the K. pneumoniae strains.

Materials and methods. Total 65 strains of K. pneumoniae isolated from samples of feces, blood, urine, placenta, cervical canal, pharynx, suture of 39 newborns and 24 women were studied. Two blood cultures were obtained from one patient with an interval of two weeks, and two isolates were obtained from the separated cervical canal and suture of one patient. The presence of genes was detected by PCR, nucleotide sequences of the genes were determined by Sanger sequencing.

Results. The frequency of detection of the uge gene was 53.8% (35 of 65), fim gene — 23.1% (15 of 65), which indicates a higher prevalence of uge gene strains compared to fim (p < 0.001). The phylogenetic analysis of 18 nucleotide sequences of the uge gene and 4 of the fim gene demonstrated that the strains were distributed in 7 and 4 clusters, respectively. It was established that for, there are No clear clustering by time and place of isolation, patient age, and type of biological material was observed for both uge and fim genes.

Discussion. The results of phylogenetic analysis demonstrate the genetic heterogeneity of the studied population of K. pneumoniae, which is confirmed by the wide geography and time variations in detection of the most genetically close bacterial isolates.

To maintain viability under stressful conditions of existence and the implementation of protective strategies, bacteria must receive signals and respond quickly to extreme changes in environmental parameters. The results of recent experimental studies complement the paradigm that has dominated since the 1970s on the predominant role of phospholipids (PL) as molecular building blocks in the formation of the cell wall of bacteria. Specific transformations of these lipid domains have shown to have a significant effect on the shape and function of cells, membrane remodeling, and the ability of bacteria to adapt to environmental stresses. The physiological role of bacterial PLs is pleiotropic and determines both cell integrity and cell function. In addition to the key structural role of membrane PL in the cell, their intermediate metabolites are able to act as secondary messengers and perform important signaling and regulatory functions. Modern studies of the mechanisms of detection and integration of signals from the environment that cause stationary-dynamic changes in phospholipid homeostasis and form pleiotropic resistant cellular bacterial phenotypes are of fundamental and practical interest. PL homeostasis was proved to be crucial for the pathogenesis of bacterial infections and is necessary not only to maintain the viability of bacteria, but also to ensure their growth during infection. The suppression of the biosynthesis of these macromolecules reduces the viability of bacteria. In recent decades, one of the main advances in the concept of "liquid mosaic" model of biological membranes has been the understanding of their domain structure. This discovery is of fundamental and practical interest, since phospholipid domains are a promising target for modern antimicrobial strategies. The aim of this review is to summarize modern ideas about the structural, metabolic and signaling role of membrane PL in the implementation of the protective mechanisms of bacteria and maintaining their viability in adverse environmental conditions.

Background. The influence of such factors as population density, practices for testing for the SARS-CoV-2 (combined with quarantine/self-isolation for infected individuals and their contacts) and ambient temperature on the spread of the novel coronavirus infection and related mortality in the 85 different regions of the Russian Federation isn’t well characterized.

Materials and methods. Population density in the different regions of the Russian Federation is measured as the number of persons per square kilometer of settled areas; ambient temperature is measured as the mean for January and July values; practices for testing for SARS-CoV-2 are characterized via case-fatality rates (the percent of deaths among cases with known outcome (recovered + fatal)) — under more active testing for SARSCoV-2, greater numbers of mild/moderate cases of infection are detected, resulting in the decline in case-fatality rates, i.e. the intensity of testing is inversely proportional to the case-fatality rate.

Results. The correlation between population density and rates of mortality for COVID-19 per 100,000 persons on November 22, 2020 in the 85 different regions of the Russian Federation is 0.53 (0.36; 0.67); the correlation between case-fatality rates and rates of mortality for COVID-19 per 100,000 persons on Nov. 22, 2020 in the different regions of the Russian Federation is 0.62 (0.47; 0.74). Results of the linear regression suggest a positive association between population density, as well as case-fatality rates and rates of mortality for COVID-19 in the different regions of Russia, and a negative association between ambient temperature and rates of mortality for the novel coronavirus infection.

Conclusions. Lower population density, more active testing for SARS-CoV-2 and higher ambient temperature are associated with lower rates of mortality for COVID-19. In particular, additional measures should be implemented towards testing of different categories of individuals for SARS-CoV-2, including those seeking testing on their own initiative, those seeking medical help with respiratory symptoms, and contacts of confirmed COVID-19 cases.

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