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Introduction. Infections of the lower respiratory tract by bacteria of the Pseudomonadota phylum: Pseudomonas aeruginosa, Burkholderia spp., Achromobacter spp. are critical to the quality and life expectancy of patients with cystic fibrosis (CF). When the infection is chronic, eradication of bacteria with existing antibacterial drugs is practically impossible. To explore alternative drugs, trials are needed on bacteria isolated from CF patients and characterized using genomic approaches. The objective of our study was a comparative analysis of virulence factors of 6 isolates of bacteria of the Pseudomonadota phylum and testing the efficacy of the innovative drug Fluorothiazinone (FT) in suppressing the pathogenicity of bacteria in vitro. Materials and methods. Isolates of A. ruhlandii ST36, A. xylosoxidans ST555, B. cepacia ST2140, B. gladioli ST2141, P. aeruginosa ST859 and ST198 were examined using whole-genome sequencing and bioinformatics analysis to search for resistance and virulence determinants. The FT drug was tested for its effect on bacteria in in vitro experiments on cytotoxicity on HeLa cells, motility and biofilm formation. Results. Genomic studies have confirmed the arsenal of resistance determinants, especially the efflux systems of bacteria isolated from patients with CF, and the diversity of virulence factors, among which we identified factors in the categories of motility, signals of quorum-sensing systems, secretion systems, exotoxins, as the most essential for the adaptation of bacteria to conditions of the lower respiratory tract. In vitro tests of the FT drug showed its effectiveness in suppressing cytotoxicity (2.6–4.0 times), motility (2.0–3.6 times) and the process of biofilm formation (2.0–7.7 times). Conclusion. For the first time, the effectiveness of the innovative antibacterial drug Fluorothiazinone has been shown against bacteria of the Pseudomonadota phylum, isolated from chronically infected patients with CF, with the described potential of virulence factors.

Introduction. Carbapenemase-producing Klebsiella pneumoniae (CP-Kp), which are international high-risk clones, have become a problem of utmost importance. CP-Kps, adapting to the hospital environment, evolve into convergent pathotypes. Such variants combine traits of two genetic lineages: multidrug resistant (MDR) and hypervirulent. The pathotypes, along with MDR K. pneumoniae, pose an exceptional threat to young patients during systemic infection. The objective of this study is the detailed molecular genetic analysis of MDR isolates of K. pneumoniae detected during the monitoring of resistant Gram-negative bacteria at the National Medical Research Center for Children’s Health in 2014–2021. Materials and methods. Whole-genome sequencing with a subsequent bioinformatics analysis of eight MDR isolates from the bloodstream and cerebrospinal fluid. Results. MDR isolates belonged to 4 sublineages (SL): SL307, SL395, SL29 and SL1198. In the genomes of 6 pangrug-resistant (PDR) isolates, genes associated with resistance to all categories of antibiotics recommended for Enterobacteriaceae therapy were identified. Plasmids were present in all genomes. In 6 isolates, plasmids contained heavy metal ion resistance operons in addition to antibiotic resistance genes. Prophages within the plasmids were also involved in the transfer of resistance genes. The ST395 isolate from the cerebrospinal fluid belonged to the convergent pathotype in terms of resistance and virulence. Comparison of genomes within SLs revealed recombination events in the K- and O-locus regions and the Yersiniabactin operon. Conclusion. Thus, in a sample of resistant K. pneumoniae isolated from bloodstream and cerebrospinal fluid, 6 PDR isolates were detected, one of which belongs to the convergent pathotype ST395.

Introduction. Invasive listeriosis is a rare disease posing a threat to high-risk groups and often leading to a fatal outcome. Its causative agent is Listeria monocytogenes, a ubiquitous saprophyte that has turned into an important foodborne pathogen with the growing industry of semi-cooked and ready-to-eat products. The aim of the study is the characterization of L. monocytogenes isolates in the Moscow region and identification of possible causes of susceptibility to infection Materials and methods. The multicenter monitoring of L. monocytogenes was conducted in the Moscow metropolitan area, using bacteriological and genomic methods for description of the pathogen, medical history collection and detailed analysis of patient case summaries. Results. In the cohorts of patients with perinatal listeriosis (PL) and meningitis-septicemia (MS), invasive listeriosis had a year-round occurrence with slight upswings in MarchApril and JulyNovember. During the COVID-19 pandemic, in the MS group, the minimum age of patients decreased to 31 years and the proportion of deaths increased 1.57-fold compared to 20182019. During the pandemic, an increase in the diversity of L. monocytogenes genotypes was observed, along with changes in the spectrum of pathogen genotypes throughout the pandemic stages. During the monitoring, a total of 73 L. monocytogenes clinical isolates belonging to 24 genotypes were described. Seven genotypes belonged to the first phylogenetic lineage (PLI); 14 genotypes belonged to PLII. The PL cohort had the highest proportion of PLI genotypes (52%). In the MS cohort, the group of men had the widest diversity of genotypes, 6 of which were identical to genotypes of food isolates. In the analysed set of isolates, 12 new profiles of internalin genes were identified and described. The whole genome sequencing detected the presence of plasmids in 9 of 58 genomes of clinical isolates. The comparison of core genomes revealed an epidemic relationship between isolates of the same genotype for ST4, ST21, and ST425. Conclusion. The performed study presents a detailed description of the diversity and virulence of L. monocytogenes circulating in the Moscow metropolitan area, thus providing information for timely diagnosis and treatment of invasive listeriosis.

Listerias of the phylogenetic lineage II (PLII) are common in the European environment and are hypovirulent. Despite this, they caused more than a third of the sporadic cases of listeriosis and multi-country foodborne outbreaks. L. monocytogenes ST37 is one of them. During the COVID-19 pandemic, ST37 appeared in clinical cases and ranked second in occurrence among food isolates in the Moscow region. The aim of this study was to describe the genomic features of ST37 isolates from different sources. All clinical cases of ST37 were in the cohort of male patients (age, 48–81 years) with meningitis–septicemia manifestation and COVID-19 or Influenza in the anamnesis. The core genomes of the fish isolates were closely related. The clinical and meat isolates revealed a large diversity. Prophages (2–4/genome) were the source of the unique genes. Two clinical isolates displayed pseudolysogeny, and excided prophages were A006-like. In the absence of plasmids, the assortment of virulence factors and resistance determinants in the chromosome corresponded to the hypovirulent characteristics. However, all clinical isolates caused severe disease, with deaths in four cases. Thus, these studies allow us to speculate that a previous viral infection increases human susceptibility to listeriosis.

Virus-neutralizing antibodies are one of the few treatment options for COVID-19. The evolution of SARS-CoV-2 virus has led to the emergence of virus variants with reduced sensitivity to some antibody-based therapies. The development of potent antibodies with a broad spectrum of neutralizing activity is urgently needed. Here we isolated a panel of single-domain antibodies that specifically bind to the receptor-binding domain of SARS-CoV-2 S glycoprotein. Three of the selected antibodies exhibiting most robust neutralization potency were used to generate dimeric molecules. We observed that these modifications resulted in up to a 200-fold increase in neutralizing activity. The most potent heterodimeric molecule efficiently neutralized each of SARS-CoV-2 variant of concern, including Alpha, Beta, Gamma, Delta and Omicron variants. This heterodimeric molecule could be a promising drug candidate for a treatment for COVID-19 caused by virus variants of concern.

Abstract Background Microbes infecting cystic fibrosis patients’ respiratory tract are important in determining patients’ functional status. Representatives of Burkholderiales order are the most dangerous. The goal of our investigation was to reveal the diversity of Burkholderiales, define of their proportion in the microbiome of various parts of respiratory tract and determine the pathogenicity of the main representatives. Results In more than 500 cystic fibrosis patients, representing all Federal Regions of Russia, 34.0% were infected by Burkholderia cepacia complex (Bcc), 21.0% by Achromobacter spp. and 12.0% by Lautropia mirabilis. B. cenocepacia was the most numerous species among the Bcc (93.0%), and A. ruhlandii was the most numerous among Achromobacter spp. (58.0%). The most abundant genotype in Bcc was sequence type (ST) 709, and in Achromobacter spp. it was ST36. These STs constitute Russian epidemic strains. Whole genome sequencing of strains A. ruhlandii SCCH3:Ach33–1365 ST36 and B. cenocepacia GIMC4560:Bcn122 ST709 revealed huge resistomes and many virulence factors, which may explain the difficulties in eradicating these strains. An experience of less dangerous B. cenocepcia ST710 elimination was described. Massively parallel sequencing of 16S rDNA amplicons, including V1-V4 hypervariable regions, was used to definite “healthy” microbiome characteristics. Analysis of maxillary sinus lavage of 7 patients revealed infection with Proteobacteria of the same ST as pathogens from sputum, suggesting that the maxillary sinus is a source of infection in cystic fibrosis patients. Conclusions Characterization of the Russian epidemic bacterial strains in the sputum and sinuses of cystic fibrosis patients have better defined the importance of Burkholderiales bacteria. This information may aid in the development of effective approaches for treatment of this disease.

Since the beginning of the 2021 year, all the main six vaccines against COVID-19 have been used in mass vaccination companies around the world. Virus neutralization and epidemiological efficacy drop obtained for several vaccines against the B.1.1.7, B.1.351 P.1, and B.1.617 genotypes are of concern. There is a growing number of reports on mutations in receptor-binding domain (RBD) increasing the transmissibility of the virus and escaping the neutralizing effect of antibodies. The Sputnik V vaccine is currently approved for use in more than 66 countries but its activity against variants of concern (VOC) is not extensively studied yet. Virus-neutralizing activity (VNA) of sera obtained from people vaccinated with Sputnik V in relation to internationally relevant genetic lineages B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.617.3 and Moscow endemic variants B.1.1.141 (T385I) and B.1.1.317 (S477N, A522S) with mutations in the RBD domain has been assessed. The data obtained indicate no significant differences in VNA against B.1.1.7, B.1.617.3 and local genetic lineages B.1.1.141 (T385I), B.1.1.317 (S477N, A522S) with RBD mutations. For the B.1.351, P.1, and B.1.617.2 statistically significant 3.1-, 2.8-, and 2.5-fold, respectively, VNA reduction was observed. Notably, this decrease is lower than that reported in publications for other vaccines. However, a direct comparative study is necessary for a conclusion. Thus, sera from “Sputnik V”-vaccinated retain neutralizing activity against VOC B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.617.3 as well as local genetic lineages B.1.1.141 and B.1.1.317 circulating in Moscow.

Chromobacterium species are common in tropical and subtropical zones in environmental samples according to numerous studies. Here, we describe an environmental case of resident Chromobacterium vaccinii in biofilms associated with Carex spp. roots in Moscow region, Russia (warm-summer humid continental climate zone). We performed broad characterization of individual properties as well as surrounding context for better understanding of the premise of C. vaccinii survival during the winter season. Genome properties of isolated strains propose some insights into adaptation to habit and biofilm mode of life, including social cheaters carrying ΔluxR mutation. Isolated C. vaccinii differs from previously described strains in some biochemical properties and some basic characteristics like fatty acid composition as well as unique genome features. Despite potential to modulate membrane fluidity and presence of several genes responsible for cold shock response, isolated C. vaccinii did not survive during exposure to 4 °C, while in the complex biofilm sample, it was safely preserved for at least half a year in vitro at 4 °C. The surrounding bacterial community within the same biofilm with C. vaccinii represented a series of psychrophilic bacterial species, which may share resistance to low temperatures with other species within biofilm and provide C. vaccinii an opportunity to survive during the cold winter season.

Marinomonas primoryensis KMM 3633T, extreme living marine bacterium was isolated from a sample of coastal sea ice in the Amursky Bay near Vladivostok, Russia. The goal of our investigation is to study outer membrane channels determining cell permeability. Porin from M. primoryensis KMM 3633T (MpOmp) has been isolated and characterized. Amino acid analysis and whole genome sequencing were the sources of amino acid data of porin, identified as Porin_4 according to the conservative domain searching. The amino acid composition of MpOmp distinguished by high content of acidic amino acids and low content of sulfur-containing amino acids, but there are no tryptophan residues in its molecule. The native MpOmp existed as a trimer. The reconstitution of MpOmp into black lipid membranes demonstrated its ability to form ion channels whose conductivity depends on the electrolyte concentration. The spatial structure of MpOmp had features typical for the classical gram-negative porins. However, the oligomeric structure of isolated MpOmp was distinguished by very low stability: heat-modified monomer was already observed at 30 °C. The data obtained suggest the stabilizing role of lipids in the natural membrane of marine bacteria in the formation of the oligomeric structure of porin.