Background/Objectives Bacterial ghosts (BGs), non-living empty envelopes of bacteria, are produced either through genetic engineering or chemical treatment of bacteria, retaining the shape of their parent cells. BGs are considered vaccine candidates, promising delivery systems, and vaccine adjuvants. The practical use of BGs in vaccine development for humans is limited because of concerns about the preservation of viable bacteria in BGs. Methods: To increase the efficiency of Klebsiella pneumoniae BG formation and, accordingly, to ensure maximum killing of bacteria, we exploited previously designed plasmids with the lysis gene E from bacteriophage φX174 or with holin–endolysin systems of λ or L-413C phages. Previously, this kit made it possible to generate bacterial cells of Yersinia pestis with varying degrees of hydrolysis and variable protective activity. Results: In the current study, we showed that co-expression of the holin and endolysin genes from the L-413C phage elicited more rapid and efficient K. pneumoniae lysis than lysis mediated by only single gene E or the low functioning holin–endolysin system of λ phage. The introduction of alternative lysing factors into K. pneumoniae cells instead of the E protein leads to the loss of the murein skeleton. The resulting frameless cell envelops are more reminiscent of bacterial sacs or bacterial skins than BGs. Although such structures are less naive than classical bacterial ghosts, they provide effective protection against infection by a hypervirulent strain of K. pneumoniae and can be recommended as candidate vaccines. For our vaccine candidate generated using the O1:K2 hypervirulent K. pneumoniae strain, both safety and immunogenicity aspects were evaluated. Humoral and cellular immune responses were significantly increased in mice that were intraperitoneally immunized compared with subcutaneously vaccinated animals (p < 0.05). Conclusions: Therefore, this study presents novel perspectives for future research on K. pneumoniae ghost vaccines.

Background: The emergence of multidrug-resistant hypervirulent Klebsiella pneumoniae (hvKp) has made it difficult to treat and control infections caused by this bacterium. Previously, the therapeutic effectiveness of phage-encoded depolymerase Dep_kpv74 in a mouse model of K. pneumoniae-induced thigh soft tissue infection was reported. In this study, the effect of Dep_kpv74 on blood parameters in mice, the proliferation and subpopulation composition of spleen lymphocytes, and the activity and stability of the enzyme at different pH and temperatures were further explored. Results: The stability tests showed that Dep_kpv74 remained active in the temperature range from 8 °C to 55 °C. The optimal pH value for maintaining the activity of Dep_kpv74 ranged from 5.0 to 9.0. The depolymerase was detected in the blood, spleen, and lungs of mice 10 min after intraperitoneal administration, reaching maximum activity values after 1–3 h and maintaining activity a day after administration. The introduction of Dep_kpv74 at the therapeutic dose (10 μg/mouse) or at a 10-fold higher dose did not lead to reliable changes in bloodstream cell content compared with the reference values of intact mice. The biochemical results of the studies indicated that Dep_kpv74 did not exert any toxic effects on liver and kidney functions. The results of the analysis of lymphocyte proliferative activity demonstrated that Dep_kpv74 depolymerase has a mild immunomodulatory effect. Conclusions: Thus, the results of this study provide one more confirmation that depolymerase Dep_kpv74 is a potential candidate for the treatment of infections caused by hvKp expressing K2 capsular polysaccharides.

Live anthrax vaccine containing spores from attenuated strains STI-1 of Bacillus anthracis is used in Russia and former CIS (Commonwealth of Independent States) to prevent anthrax. In this paper we studied the duration of circulation of antibodies specific to spore antigens, the protective antigen (PA), the lethal factor (LF) and their domains (D) in donors’ blood at different times after their immunization with live anthrax vaccine. The relationship between the toxin neutralization activity level and the level of antibodies to PA, LF and their domains was tested. The effect of age, gender and number of vaccinations on the level of adaptive post-vaccination immune response has been studied. It was shown that antibodies against PA-D1 circulate in the blood of donors for 1 year or more after immunization with live anthrax vaccine. Antibodies against all domains of LF and PA-D4 were detected in 11 months after vaccination. Antibodies against the spores were detected in 8 months after vaccination. A moderate positive correlation was found between the titers of antibodies to PA, LF, or their domains, and the TNA of the samples of blood serum from the donors.

While the pandemic is behind us, the world community faces a global threat of bacterial resistance outbreak. One of the key ways to combat the spread of multi-resistant bacteria is infection prevention and control tactics using modern antiseptic and disinfectant compositions. Herein, we continue the path to unravel the structure–activity relationship (SAR) of potent pyridine-derived biocide class bis-quaternary ammonium compounds (QACs). In this study, twenty dihydroxynaphthalene-derivative bis-QACs were subjected to extensive microbiological analysis on planktonic cells and biofilms of the ESKAPE microorganisms. Among them, hit compounds were superior in their bacteriostatic and bactericidal action to commercial mono-QACs and were comparable to the best bis-QAC antiseptic on the market. SAR analysis indicated that the linker conformation does not significantly affect the activity, though structure symmetry and especially lipophilicity had an influence on antibacterial performance. Furthermore, we delve deeper in investigation of the antimicrobial potential of bis-QACs and conducted a variety of assays, including time–kill kinetics, bacterial resistance formation, cell morphology, and cytotoxicity. Studies showed promising results for compounds 5d and 6d, indicating 2 to 3-fold less cytotoxicity and hemotoxicity compared to commercial QACs. Moreover, SEM imaging revealed that bis-QACs can cause severe membrane damage to S. aureus and P. aeruginosa strains, confirming great potential of novel compounds as antiseptic and disinfectant.

Francisella tularensis is an intracellular bacterium that causes tularemia. Progress in creating a safe and effective vaccine for the prevention of tularemia is challenging due to a lack of knowledge about immunological parameters indicative of protective adaptive immunity. Objective of the research was to assess the effect of modifications of the F. tularensis 15 NIIEG genome on the immunogenic and protective properties of F. tularensis 15/23-1ΔrecA and F. tularensis 15/23-1/sodBΔrecA strains. Materials and methods. Multi-parameter flow cytometry and the measurement of secreted cytokines were used to characterize the responses of mouse spleen lymphocytes in response to re-stimulation of F. tularensis with acid-insoluble complex (AIC) in vitro. Also, the titers of specific antibodies to F. tularensis lipopolysaccharide in blood serum were analyzed by enzyme-linked immunosorbent assay. Results and discussion. It has been shown that immunization with the studied strains led to a significant increase in CD4+ and/or CD8+ T cells capable of expressing functional markers: CD69, CD25 and/or CD28; an increase in the subpopulation of T-helpers synthesizing IFN-γ. In the body of immune mice, a pool of B-lymphocytes was formed, capable of secreting IFN-γ in response to their stimulation with AIC. Immunization with the strain 15/23-1/sodBΔrecA provided 70% protection in mice from intranasal infection with a virulent strain of F. tularensis SchuS4. More pronounced protective properties were associated with the activation of not only B-lymphocytes and T-helpers, but also with the simultaneous activation of cytotoxic T-lymphocytes.

Neutralization of the lethal toxin of Bacillus anthracis is an important topic of both fundamental medicine and practical health care, regarding the fight against highly dangerous infections. We have generated a neutralizing monoclonal antibody 1E10 against the lethal toxin of Bacillus anthracis and described the stages of receptor interaction between the protective antigen (PA) and the surface of eukaryotic cells, the formation of PA oligomers, assembly of the lethal toxin (LT), and its translocation by endocytosis into the eukaryotic cell, followed by the formation of a true pore and the release of LT into the cell cytosol. The antibody was shown to act selectively at the stage of interaction between Bacillus anthracis and the eukaryotic cell, and the mechanism of toxin-neutralizing activity of the 1E10 antibody was revealed. The interaction between the 1E10 monoclonal antibody and PA was found to lead to inhibition of the enzymatic activity of the lethal factor (LF), most likely due to a disruption of true pore formation by PA, which blocks the release of LF into the cytosol.