Ryzhova Natalia Nikolaevna

The global threat of carbapenemase-producing K. pneumoniae (CP-Kp) has recently been amplified by the evolution of the convergent pathotype, combining the extensive antimicrobial resistance with the hypervirulence (CP-hvKp). The high-risk CP-Kp ST39 clone has demonstrated rapid nosocomial transmission in the European Economic Area in 2018–2022. CP-hvKp ST39 was first identified in Moscow hospitals in 2018, and the next clone was identified in 2022. The aim of our study was to analyse the genetic determinants of resistance and virulence in later of K. pneumoniae ST39 isolates from 2023–2025, characterise their episomal architecture, and evaluate their biofilm-formation capacity, and susceptibility of biofilm to the last-resort antimicrobials and to the new antivirulence agent fluorothiazinone. Overall, four nosocomial K. pneumoniae ST39 isolates were identified convergent based on whole genome analysis. Plasmidome diversity contributed significantly to the resistome and virulome of the isolates, and to the ability of CP-hvKp ST39 to develop mature, highly structured biofilms, leading to tolerance, even to last-resort agents. Fluorothiazinone reliably reduced biofilm accumulation to weak or moderate baseline levels across all ST39 isolates. The combination of traditional antibiotics with a lipophilic, tissue-distributing, cell-penetrating, and safe well-tolerated antivirulence drug could facilitate the effective eradication of biofilm forming CP-hvKp ST39.

Introduction. Mycoses (fungal infections) represent a serious global health threat: more than 6.5 million severe invasive cases of such diseases are registered annually worldwide, accompanied by extremely high mortality (on average 68%). The high degree of threat determines the relevance of intensifying research into the mechanisms of fungal drug resistance, the pathogenesis of the fungal diseases, as well as the development of more effective therapeutic approaches. The implementation of methods that combine high analysis speed with accurate quantitative assessment of viable microorganisms is a key condition for obtaining reproducible research results both at the stage of culture preparation for in vivo infection modeling and when evaluating the efficacy of antifungal drugs. Aim of this study was to develop and evaluate the application of flow cytometry for rapid quantification of microorganisms using Candida albicans fungi as an example, both in pure culture and in multicomponent samples, in comparison with the CFU counting method. Materials and Methods. To model infection, female BALB/c mice were intravenously inoculated with C. albicans isolate GIMC100:CaMK2014SRP at doses of 0.5 × 10⁶, 1.0 × 10⁶, and 2.0 × 10⁶ CFU/animal. The number and viability of C. albicans cells were assessed by culture-based and flow cytometric methods using species-specific FITC-labeled antibodies and propidium iodide. Results. Flow cytometry allows rapid counting of C. albicans in samples, showing measurement accuracy comparable to the CFU counting method across a wide range of pathogen concentrations (from 100 to 1 million cells). The use of pathogen-specific fluorescently labeled antibodies and a viability dye makes it possible to reliably detect live C. albicans cells in samples regardless of the presence of impurities (debris, eukaryotic cells, dead C. albicans cells), without reducing counting accuracy. Conclusion. Having no alternatives with similar characteristics, the proposed method appears to be highly demanded for the standardization of C. albicans samples for modeling candidiasis in laboratory animals. Furthermore, this method is seen as a promising alternative to the conventional method of CFU counting on solid nutrient media for determining the number of live C. albicans cells when analyzing biological samples or evaluating the efficacy of antifungal drugs in culture.

Chronic lung infection caused by Pseudomonadota (PCH) in patients with cystic fibrosis (pwCF) is difficult to eradicate. CFTR modulators have a potential r

Biofilms pose a significant challenge to antimicrobial therapy. Bacteria in biofilms differ from planktonic counterpart in their altered metabolism, collective behavior, protective role of extracellular matrix and diversified microbial subpopulations. These attributions significantly influence bioavailability and activity of antibiotics. The presence of bacterial aggregates during acute infections expands the problem to many other conditions previously not discussed in the biofilm context. Klebsiella pneumoniae is a leading cause of life-threatening hospital-acquired infections and is included in the WHO Bacterial Priority Pathogens List due to increasing antimicrobial resistance. The combination of antimicrobial resistance and the ability to form biofilms severely limits the efficacy of antibiotic treatments. In this study, we investigated the in vitro susceptibility of mature biofilms to 13 antimicrobials of K. pneumoniae clinical isolates from a single hospital. The resistance profiles of the local clinical isolates were consistent with the global epidemiology of K. pneumoniae . Minimal biofilm eradication concentrations (MBEC) for mature biofilms were defined with two assays (biomass and metabolic activity measurements) and brought into relation with susceptibility breakpoints and plasma ( C _max ). Colistin sulfate, tigecycline, cephalosporins and combination of imipenem with cilastatin were the most potent biomass eradicators, while suppression of metabolic activity was barely reachable. Moreover, we observed a notable increase in metabolic activity upon exposure to sub-MBEC concentrations of antibiotics. Finally, our data broach a subject of antibiotic prioritization with respect to biofilm tolerance. IMPORTANCE: This study addresses the critical gap between standard antibiotic susceptibility testing and the tolerance of biofilm and microbial aggregates during infections caused by K. pneumoniae. By systematically evaluating mature biofilms from a significant number of clinical isolates, we demonstrate that colistin and tigecycline show potent activity against both biofilm biomass and metabolic activity, whereas cephalosporins primarily reduce biomass without effectively suppressing bacterial metabolism, and other drugs have only weak effects on biofilms at clinically achievable concentrations. Furthermore, the alarming observation that sub-inhibitory biofilm eradication concentration (sub-MBEC) of antibiotic can paradoxically increase the metabolic activity of biofilms highlights a potential risk factor for therapy failure and resistance development. Our findings contribute to the necessary evidence base for prioritizing existing antibiotics in the limited armamentarium against biofilm-forming K. pneumoniae .

Background/Objectives: Flaviviruses, including West Nile virus (WNV), pose global health challenges due to their worldwide distribution, pathogenicity, and lack of effective treatments or vaccines. Today, WNV is considered the most important causative agent of viral encephalitis worldwide. This study investigated the different forms of the main WNV antigen—the preM/E protein—in the context of its immunogenic and protective properties. Methods: The recombinant adenovirus type 2 (rAd2) vectors expressing different forms of the WNV preM/E genes were obtained using standard molecular biology techniques. Immunogenicity in mice was assessed by enzyme-linked immunosorbent assay (ELISA) and virus neutralization assay. Immunological efficacy was evaluated in a mouse viral challenge model. Results: The rAd2 vector expressing the West Nile virus preM/E gene with mutations in the fusion loop exhibited robust immunogenicity when administered intramuscularly either once or in a homologous prime-boost regimen. This antigen form, as part of an adenoviral vector, protected mice from death in viral challenge experiments, providing 100% survival following WNV challenge. Conclusions: We believe that a vaccination strategy involving a recombinant adenoviral vector based on human adenovirus type 2 and the WNV antigen represented by the preM/E gene with mutations in the fusion loop may be a promising approach for combating West Nile virus infection.

Chronic lung infection with Pseudomonadota (PCH) in patients with cystic fibrosis (pwCF) is difficult to eradicate. CFTR modulators have a potential role in the prevention of airway infections, but their ability to eradicate chronic infection remains to be investigated. The aim of our study was to evaluate the impact of combination (antibacterial (AT) and modulator (MT)) therapy on the lung microbiome composition (LMC) in the pwCF cohort. The microbiome of sputum samples longitudinally collected from Russian adult pwCF chronically infected with Pseudomonadota CF pathogens (PCH) was analyzed. MT resulted in a trend of bacterial load reduction. LMC did not undergo significant changes in PCH pwCF receiving MT for less than three years. Two-component MT resulted in a temporary decrease in the proportion of the CF pathogen only when combined with a course of AT. Three-component MT has been successful in inducing favorable microbiome changes (with abundance and diversity of anaerobic taxa) over a period of more than 3 years, but not for all cases of Burkholderiales infection. Respiratory system damaged by bronchiectasis is susceptible to new infections, so patient management requires constant monitoring of the LMC and replenishment of the therapeutic landscape with both new modulators and new antibacterial drugs.

Introduction. Adenovirus infection occurs globally in the form of sporadic cases and isolated outbreaks. Human adenovirus type 55 (HAdV-55), endemic in China and South Korea, causes acute respiratory viral infections (ARVI) of varying severity, both among the civilian population and in military units in different countries of the world. Genomic research facilitates reliable identification of HAdV-55. The aim of this study was to identify HAdV isolated in Moscow in 2022, as well as to conduct whole-genome sequencing and comparative genomic research. Materials and methods. HAdV-55 was isolated from a sample of a patient hospitalized with pneumonia and analyzed using restriction fragment length polymorphism analysis and whole-genome sequencing. Bioinformatics comparative analysis was performed on a sample of sequences of 83 isolates. Results. The whole-genome sequencing of first isolated in Russia HAdV-55 was conducted. The sequence of isolate SCV3008:Ad55 was deposited in GenBank (Accession Number PQ641625). Unique mutations in the SCV3008:Ad55 genome were identified, one of which resulted in a conservative T29A substitution in the penton that did not affect its functions. Phylogenetic analysis showed clustering of SCV3008:Ad55 with isolates of clade II, which included representatives of 7 countries on different continents, indicating a wide distribution of HAdV-55. Isolates from endemic regions of China and South Korea formed separate clades. The study of microsatellite length polymorphism in untranslated regions of the genome became an additional tool for distinguishing closely related genomes. Conclusion. The obtained genomic information laid the foundation for further monitoring for HAdV-55 in Russia and demonstrated the informativeness and significance of whole-genome studies for monitoring adenoviruses. The development and implementation of genotyping methods aimed at detecting HAdV-55 and other clinically relevant genotypes will significantly improve the effectiveness of the diagnosis of adenovirus infections with the threat of developing bronchopneumonia.

Extraintestinal pathogenic Escherichia coli causes community-acquired and nosocomial pneumonia and poses a risk of infection, especially for patients with impaired lung function, such individuals with cystic fibrosis (CF). When chronic infection develops, eradication of the pathogen is difficult even with aggressive antibacterial therapy and targeted CF treatment. A new agent, Fluorthiazinone (CL-55), an inhibitor of bacterial virulence, was registered in Russia in 2024. The aim of our study was to characterize the genomes of E. coli ST648 isolated from long-term-infected CF patients, describe virulence factors, and investigate the effect of CL-55 on two CF isolates in vitro. Comparison of the genomes of hypermucoviscous isolates showed that, in the presence of a large number of core genes, the isolates have adaptive differences both in their chromosomes and the composition and genes of their plasmidomes. Both isolates formed mature biofilms on an abiotic surface and were able to survive and proliferate inside macrophages. CL-55 in in vitro experiments was effective in suppressing E. coli ST648 in both the aggregate and intracellular states, allowing us to propose the use of Fluorthiazinone as a combinative therapy to facilitate eradication of pathogenic microorganisms in the respiratory tract in patients with CF.

Extraintestinal pathogenic Escherichia coli causes community-acquired and nosocomial pneumonia, and poses a risk of infection, especially for patients with impaired lung function, such as patients with cystic fibrosis (CF). When chronic infection develops, eradication of the pathogen is difficult even with aggressive antibacterial therapy and targeted CF treatment. A new compound, Fluorthiazinone (CL-55), an inhibitor of bacterial virulence, was registered in Russia in 2024. The aim of our study was to characterize the genomes of E. coli ST648 isolated from long-term infected CF patients, describe AMR and virulence factors, and investigate the effect of CL-55 on CF isolates in vitro. Comparison of the genomes of hypermucoviscous isolates showed that, in the presence of a large number of core genes, the isolates have adaptive differences both in chromosome and in the composition and genes of the plasmidome. Both isolates formed mature biofilms on an abiotic surface and were able to survive and proliferate inside macrophages. CL-55 in in vitro experiments was effective in suppressing E. coli ST648 in both the aggregate and intracellular states, which allows us to propose the use of Fluorothiazinone as a combinative therapy to facilitate eradication of pathogenic microorganisms in the respiratory tract of patients with CF.

INTRODUCTION. Marburg and Ebola viruses cause severe haemorrhagic fever in humans and primates. Currently, there are no licensed prophylactic vaccines that can simultaneously prevent the spread or reduce the severity of both diseases caused by these filoviruses. The development of effective prophylactic vaccines requires studies aimed at selecting the most immunogenic forms of protective antigens.AIM. This study aimed to evaluate humoral immune induction in animals after administration of recombinant adenoviral vectors expressing various forms of Ebola and Marburg virus glycoproteins (GPs).MATERIALS AND METHODS. Samples of recombinant human adenovirus type 5 (rAd5) were obtained using homologous recombination in Escherichia coli, growth in HEK293 cells, and purification by CsCl gradient ultracentrifugation. The resulting rAd5 samples were characterised in terms of their identity (PCR and whole-genome sequencing), the concentration of viral particles (fluorescence spectroscopy), and the concentration of infectious viral particles (TCID₅₀ assay). Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the GP-specific IgG titres in the sera of immunised mice.RESULTS. The authors constructed rAd5 samples, and each construct contained an expression cassette with a GP gene form encoding a full-length GP, a GP without the mucin-like domain, or a GP without both the glycan cap and the mucin-like domain. Each of these forms was studied using the GPs of four filoviruses, including Zaire Ebola virus, Sudan Ebola virus, Bundibugyo Ebola virus, and Marburg virus. Neither of the forms had a critical effect on the rAd5 replicative capacity. Three weeks after immunisation, the highest GP-specific IgG production was induced by the rAd5 samples encoding either the full-length GP or the GP without the mucin-like domain. The GP without both the glycan cap and the mucin-like domain was the least immunogenic antigen regardless of the filovirus species.CONCLUSIONS. The most promising constructs for the development of filovirus vaccines based on recombinant adenoviral vectors are the constructs that include the genes encoding the fulllength GP or the GP without the mucin-like domain.