Ekaterina A. Koroleva

The aim of this study was to investigate the effect of the innovative Russian antibacterial drug Fluorothiazinone, an inhibitor of the type III secretion system and flagellar function, on the virulence properties of urinary tract infection (UTI) pathogens. Materials and methods. Clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa species were obtained from 4 patients with recurrent UTI. Cytotoxicity analysis was performed on HeLa cells using the CytoTox 96 kit. Bacterial flagellar motility was assessed in 0.25% agar by measuring the zone of motility. Biofilms were cultivated in 96-well plates and stained with crystal violet and Congo red. Biofilm structure was assessed by microscopy, and the bacterial biofilm and exopolysaccharide matrix were quantified by measuring the optical density of the dye bound to the biofilm. Intracellular development was studied in PC-3 cells by determining the number of intracellular bacteria using a cell lysate plating method and an immunochemical method involving staining the bacteria with specific antibodies. Results. The Fluorothiazinone antibacterial specifically inhibited the cytotoxicity and motility of E. coli and P. aeruginosa. For K. pneumoniae, susceptibility to Fluorothiazinone was observed, associated with the suppression of cytotoxicity. For all the isolates studied, inhibition of biofilm formation on an abiotic surface was demonstrated. For E. coli and K. pneumoniae, a significant decrease in intracellular replication within human prostate adenocarcinoma cells and suppression of intracellular bacterial community formation were observed. Conclusion. Fluorothiazinone disrupts mechanisms that contribute to pathogen persistence in the formation of chronic UTI, including biofilm formation on abiotic surfaces such as catheters, stents, and drains, and the formation of intracellular bacterial communities and dormant intracellular reservoirs.

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.

In order to combat resistance, it is necessary to develop antimicrobial agents that act differently from conventional antibiotics. Fluorothiazinone, 300 mg tablet (The Gamaleya National Research Center), is an original antibacterial drug based on a new small molecule T3SS and flagellum inhibitor. A total of 357 patients with complicated urinary tract infections (UTIs) were divided into two groups and given Fluorothiazinone 1200 mg/day or a placebo for 7 days to evaluate the efficacy and safety of the drug. Additionally, all patients were given Cefepime 2000 mg/day. Fluorothiazinone with Cefepime showed superiority over placebo/Cefepime based on the assessment of the proportion of patients with an overall outcome in the form of a cure after 21 days post-therapy (primary outcome), overall outcome in cure rates, clinical cure rates, and microbiological efficacy at the end of therapy and after 21 days post-therapy (secondary outcomes). In patients who received Fluorothiazinone, the rate of infection recurrences 53 and 83 days after the end of the therapy was lower by 18.9%, compared with patients who received placebo. Fluorothiazinone demonstrated a favorable safety profile with no serious unexpected adverse events reported. The results showed superiority of the therapy with Fluorothiazinone in combination with Cefepime compared with placebo/Cefepime in patients with cUTIs.

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.

The development of new approaches and drugs for effective control of the chronic and complicated forms of urogenital chlamydia caused by Chlamydia trachomatis, which is suspected to be one of the main causes of infertility in both women and men, is an urgent task. We used the technology of single-domain antibody (nanobody) generation both for the production of targeting anti-chlamydia molecules and for the subsequent acquisition of anti-idiotypic nanobodies (ai-Nbs) mimicking the structure of a given epitope of the pathogen (the epitope of the Chlamydial Type III Secretion System Needle Protein). In a mouse model, we have shown that the obtained ai-Nbs are able to induce a narrowly specific humoral immune response in the host, leading to the generation of intrinsic anti-Chlamydia antibodies, potentially therapeutic, specifically recognizing a given antigenic epitope of Chlamydia. The immune sera derived from mice immunized with ai-Nbs are able to suppress chlamydial infection in vitro. We hypothesize that the proposed method of the creation and use of ai-Nbs, which mimic and present to the host immune system exactly the desired region of the antigen, create a fundamentally new universal approach to generating molecular structures as a part of specific vaccine for the targeted induction of immune response, especially useful in cases where it is difficult to prepare an antigen preserving the desired epitope in its native conformation.

Uropathogenic Escherichia coli (UPEC) is the most common pathogenic bacterium associated with urinary tract infection. Due to the development of antibiotic resistance and MDR, UPEC infection has become a serious problem in the last decade. In order to combat resistance, it is necessary to develop innovative antimicrobial agents that act by different mechanisms than conventional antibiotics. Among the new therapeutic strategies, suppression of pathogen virulence has become a promising alternative, since it fundamentally reduces selective pressure and the development of resistance. In our study, we showed that the compound Fluorothiazinon suppressed UPEC’s ability to form biofilms and to move using the flagellum, as well as to penetrate into cells. Prophylactic use with subsequent treatment of FT in rodent models led to an improvement in survival and significantly reduced the bacterial load in the organs of the urinary system, thereby inhibiting the development of ascending infection and preventing the development of pathological changes in prostate tissues. These results suggest that FT affects several UPEC virulence factors at once and if similar results can be found in clinical trials it can potentially be used as a new drug against UPEC.

Chlamydia trachomatisimposes serious health problems and causes infertility. Because of asymptomatic onset, it often escapes antibiotic treatment. Therefore, vaccines offer a better option for the prevention of unwanted inflammatory sequelae. The existence of serologically distinct serovars ofC. trachomatissuggests that a vaccine will need to provide protection against multiple serovars.Chlamydiaspp. use a highly conserved type III secretion system (T3SS) composed of structural and effector proteins which is an essential virulence factor. In this study, we expressed the T3SS needle protein ofChlamydia muridarum,TC_0037, an ortholog ofC. trachomatisCdsF, in a replication-defective adenoviral vector (AdTC_0037) and evaluated its protective efficacy in an intravaginalChlamydia muridarummodel. For better immune responses, we employed a heterologous prime-boost immunization protocol in which mice were intranasally primed with AdTC_0037 and subcutaneously boosted with recombinant TC_0037 and Toll-like receptor 4 agonist monophosphoryl lipid A mixed in a squalene nanoscale emulsion. We found that immunization with TC_0037 antigen induced specific humoral and T cell responses, decreasedChlamydialoads in the genital tract, and abrogated pathology of upper genital organs. Together, our results suggest that TC_0037, a highly conserved chlamydial T3SS protein, is a good candidate for inclusion in aChlamydiavaccine.