Among the non-traditional antibacterial agents in development, only a few targets critical Gram-negative bacteria such as carbapenem-resistant Pseudomonas aeruginosa, Acinetobacter baumannii or cephalosporin-resistant Enterobacteriaceae. Endolysins and their genetically modified versions meet the World Health Organization criteria for innovation, have a novel mode of antibacterial action, no known bacterial cross-resistance, and are being intensively studied for application against Gram-negative pathogens. The study presents a multidisciplinary approach, including genetic engineering of LysECD7-SMAP and production of recombinant endolysin, its analysis by crystal structure solution following molecular dynamics simulations and evaluation of antibacterial properties. Two types of antimicrobial dosage forms were formulated, resulting in lyophilized powder for injection and hydroxyethylcellulose gel for topical administration. Their efficacy was estimated in the treatment of sepsis, and pneumonia models in BALB/c mice, diabetes-associated wound infection in the leptin receptor-deficient db/db mice and infected burn wounds in rats. In this work, we investigate the application strategies of the engineered endolysin LysECD7-SMAP and its dosage forms evaluated in preclinical studies. The catalytic domain of the enzyme shares the conserved structure of endopeptidases containing a putative antimicrobial peptide at the C-terminus of polypeptide chain. The activity of endolysins has been demonstrated against a range of pathogens, such as Klebsiella pneumoniae, A. baumannii, P. aeruginosa, Staphylococcus haemolyticus, Achromobacter spp, Burkholderia cepacia complex and Haemophylus influenzae, including those with multidrug resistance. The efficacy of candidate dosage forms has been confirmed in in vivo studies. Some aspects of the interaction of LysECD7-SMAP with cell wall molecular targets are also discussed. Our studies demonstrate the potential of LysECD7-SMAP therapeutics for the systemic or topical treatment of infectious diseases caused by susceptible Gram-negative bacterial species and are critical to proceed LysECD7-SMAP-based antimicrobials trials to advanced stages.

BACKGROUND: During the coronavirus disease 2019 pandemic, the sale of antimicrobials (AMPs) in the pharmacy network and their purchases by healthcare facilities of the Russian Federation have significantly increased. Microbiological monitoring conducted at the City Clinical Hospital No. 67 named after L.A. Vorokhobov of the Moscow Health Department in 2020–2021 revealed an urgent problem: gram-negative bacteria, which are dominated by multidrug-resistant (MDR) strains, are now mainly isolated from patients with healthcare-associated infections (HAIs). Pharmacoeconomic studies have shown that the cost of one course of targeted antibiotic therapy for HAIs can increase by 6–12 times compared with a similar course of therapy in the absence of nosocomial MDR and extensively drug-resistant (XDR) pathogens. AIM: To increase the effectiveness of preventive measures aimed at preventing the spread of hospital-acquired pathogen strains in the intensive care unit (ICU) using effective bacteriophages against MDR strains of Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii, which were selected in accordance with an individualized algorithm. MATERIALS AND METHODS: The study included two groups, totaling 20 patients, who were on prolonged mechanical ventilation in the ICU of City Clinical Hospital No. 67 named after L.A. Vorokhobov of the Moscow Health Department in 2022. The second group received 20 mL of bacteriophages via an intragastric tube for five consecutive days. The concept of a personalized approach to phage therapy for various forms of HAIs caused by nosocomial MDR pathogens in patients of a multidisciplinary hospital has been proposed and tested. RESULTS: With this medical technology, the effectiveness of phage therapy for various nosological forms of HAIs caused by nosocomial MDR and XDR strains increased by 30%, and the effectiveness of the initial antibiotic therapy regimen was ≥70%. The economic effect of the combination of antibiotic therapy and phage therapy (booster therapy) amounted to more than 3 million rubles compared with the use of AMP alone for treating patients with HAIs caused by nosocomial MDR/XDR strains. CONCLUSIONS: Booster therapy as a variant of IIB therapy reduces the cost of treatment of AMPs of various forms of HAIs by ≥2.5 times compared with AMPs alone.

Pharmacokinetics and safety studies of innovative drugs is an essential part of drug development process. Previously we have developed a novel drug for intravenous administration (lyophilizate) containing modified endolysin LysECD7-SMAP that showed notable antibacterial effect in different animal models of systemic infections. Here we present data on pharmacokinetics of endolysin in mice after single and multiple injections. Time-concentration curves were obtained, and pharmacokinetic parameters for preparation (C0, kel t1/2, AUC0-∞, MRT, ClT, Vss) were calculated. It was shown that although endolysin is rather short-lived in blood serum (t1/2 = 12.5 min), the therapeutic concentrations of LysECD7-SMAP (in degraded and non-degraded form) were detected for 60 minutes after injection that is sufficient for antibacterial effect. Based on the obtained data, it was proposed that endolysin distributes presumably in murine blood, degrades in blood and liver, and is eliminated via glomerular filtration. Safety profile of the preparation relating to general toxicity, immunotoxicity and allergenicity was assessed in rodents. It was demonstrated that LysECD7-SMAP in potential therapeutic (12.5 mg/kg), 10-fold (125 mg/kg) and 40-fold (500 mg/kg) doses showed no signs of intoxication and significant abnormalities after single and repeated i.v. administrations, preparation was non-immunogenic and induced minor and reversible allergic reaction in animals.

Antibacterial therapy with phage-encoded endolysins or their modified derivatives with improved antibacterial, biochemical and pharmacokinetic properties is one of the most promising strategies that can supply existing antibacterial drugs array. Gram-negative bacteria-induced infections treatment is especially challenging because of rapidly spreading bacterial resistance. We have developed modified endolysin LysECD7-SMAP with a significant antibacterial activity and broad spectra of action against gram-negative bacteria. Endolysin was formulated in a bactericidal gel for topical application with pronounced effectivity in local animal infectious models. Here we present preclinical safety studies and pharmacokinetics of LysECD7-SMAP-based gel. We have detected LysECD7-SMAP in the skin and underlying muscle at therapeutic concentrations when the gel is applied topically to intact or injured skin. Moreover, the protein does not enter the bloodstream, and has no systemic bioavailability, assuming no systemic adverse effects. In studies of general toxicology, local tolerance, and immunotoxicology it was approved that LysECD7-SMAP gel local application results in the absence of toxic effects after single and multiple administration. Thus, LysECD7-SMAP-containing gel has appropriate pharmacokinetics and can be considered as safe that supports the initiation of the phase I clinical trials of novel antibacterial drug intending to treat acute wound infections caused by resistant gram-negative bacteria.

Aim evaluation of specific T-cell immunity against SARS-CoV-2 in primary and secondary response to virus antigens by screening method.MATERIALS AND METHODS:Patients were tested 11.5 months after COVID-19 and 610 months before and after vaccination. Healthy volunteers were screened before, 26 times during the vaccination course, and 68 months after revaccination with the Sputnik V vaccine. IgG and IgM antibodies to SARS-CoV-2 were detected by ELISA using commercially available kits (Vector-Best, Russia). Antigenic (AG) activation of T cells in the fraction of bloods mononuclear cells was assessed by IFN- production after AG stimulation in the wells of plates from ELISA kits intended for detection of antibodies against SARS-CoV-2. Data were processed by MS Excel and Statistica 10.0 software.RESULTS:AG-specific T cells were detected in 88.5% of vaccinated healthy volunteers, half of whom were found to have T cells appearing earlier than antibodies to AG. After 6-8 months, the level of AG activation decreases. Following the revaccination, the level of AG activation of memory T cells in vitro increases within six months in 76.9100.0% of vaccinated subjects. On the contrary, after COVID-19, 86.7% of individuals had in their blood the AG-specific T cells with high activity at the time of vaccination. The activity of T cells recognizing the RBD domain of the SARS-CoV-2 S protein and the proportion of individuals who had these cells in their blood increased after the vaccination of reconvalescents.CONCLUSION:T-cell immunity against SARS-CoV-2 antigens has been shown to persist for 6 months after illness. In vaccinated individuals without history of COVID-19, such duration of the preservation of AG-specific T cells in blood was only achieved after the revaccination.

Endolysin-based therapeutics are promising antibacterial agents and can successfully supplement the existing antibacterial drugs array. It is specifically important in the case of Gram-negative pathogens, e.g., ESKAPE group bacteria, which includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, and are highly inclined to gain multiple antibiotic resistance. Despite numerous works devoted to the screening of new lytic enzymes and investigations of their biochemical properties, there are significant breaches in some aspects of their operating characteristics, including safety issues of endolysin use. Here, we provide a comprehensive study of the antimicrobial efficacy aspects of four Gram-negative bacteria-targeting endolysins LysAm24, LysAp22, LysECD7, and LysSi3, their in vitro and in vivo activity, and their biological safety. These endolysins possess a wide spectrum of action, are active against planktonic bacteria and bacterial biofilms, and are effective in wound and burn skin infection animal models. In terms of safety, these enzymes do not contribute to the development of short-term resistance, are not cytotoxic, and do not significantly affect the normal intestinal microflora in vivo. Our results provide a confident base for the development of effective and safe candidate dosage forms for the treatment of local and systemic infections caused by Gram-negative bacterial species.

Abscess formation is a common complication of severe life-threatening infections caused by obligate anaerobes. Fusobacterium necrophorum is among the frequently detected anaerobic pathogens from clinical specimens associated with liver abscesses, skin and soft tissue infections, or oral abscesses. The antimicrobial therapy for this kind of infection needs to be optimized. Here, we examined the possibility of treating F. necrophorum-induced abscess wound infections with candidate therapeutics based on three endolysins with activity against a broad spectrum of aerobe Gram-negative pathogens. Antibacterial gel containing three Gram-negative bacteria-targeting endolysins, LysAm24, LysAp22, and LysECD7, was formulated for topical use. Abscess formation was induced in rabbits with F. necrophorum and caused systemic infection. The survival and lifespan of the animals, general parameters, and biochemical and hematological blood tests were analyzed to assess the effectiveness of the gel treatment for the wound infection. The administration of the investigated gel twice per day for 5 days resulted in less acute inflammation, with decreased leukocytes and segmented neutrophils in the blood, retardation of infection progression, and an almost two-fold increase in the lifespan of the animals compared to the placebo group. The results indicate that endolysin-based therapy is an effective approach to treat anaerobic bacterial infections. The use of endolysins as independent pharmaceuticals, or their combination with antibiotics, could significantly reduce the development of complications in infectious diseases caused by sensitive bacterial species.

The bactericidal activity of copper and copper alloys is well appreciated and was already exploited in medical practice in 19th century. However, despite of being an essential nutrient required by organisms to perform life functions, excess copper is extremely toxic and detrimental to health. Recent studies have shown that superhydrophobic surfaces have a significant antibacterial potential for reduction of nosocomial infections. At the same time, the prolonged contact with biological liquids may cause a degradation of the superhydrophobic copper surface and corrosion with increasing egress of toxic copper ions. These aspects are poorly studied so far. In this paper, we analyze the evolution of the properties of both the superhydrophobic copper surface and the suspension of Escherichia coli bacteria during their prolonged contact and study the impact of such contact on the bactericidal activity of the surface. It is shown that by controlling the corrosion resistance and the wettability of the superhydrophobic copper substrate, it becomes possible to sustain the bactericidal action of copper substrates for a long time, simultaneously avoiding the excessive corrosive degradation and release of copper ions in the environment.

The use of recombinant endolysins is a promising approach for antimicrobial therapy capable of counteracting the spread of antibiotic-resistant strains. To obtain the necessary biotechnological product, diverse peptide tags are often fused to the endolysin sequence to simplify enzyme purification, improve its ability to permeabilize the bacterial outer membrane, etc. We compared the effects of two different types of protein modifications on endolysin LysECD7 bactericidal activity in vitro and demonstrated that it is significantly modulated by specific permeabilizing antimicrobial peptides, as well as by widely used histidine tags. Thus, the tags selected for the study of endolysins and during the development of biotechnological preparations should be used with the appropriate precautions to minimize false conclusions about endolysin properties. Further, modifications of LysECD7 allowed us to obtain a lytic enzyme that was largely devoid of the disadvantages of the native protein and was active over the spectra of conditions, with high in vitro bactericidal activity not only against Gram-negative, but also against Gram-positive, bacteria. This opens up the possibility of developing effective antimicrobials based on N-terminus sheep myeloid peptide of 29 amino acids (SMAP)-modified LysECD7 that can be highly active not only during topical treatment but also for systemic applications in the bloodstream and tissues.

The aim of the study was to develop some approaches to evaluate the basic parameters of the humoral and cellular immune response to a bacteriophage, taking into account the multifactorial aspects of its interaction with both the pathogen and the macroorganism. The necessary reagents were obtained and a line of diagnostic ELISA test systems was designed to allow semi-quantitative assessment of the anti-bacteriophage IgG-antibody level in serum or other biological human fluids, as well as in preparations obtained from human blood. The need for neutralization reaction to determine the effect of detected antibodies on phage activity against a target bacterium has been proven. Testing the approaches used in the investigation of patients' blood sera showed that antibodies to bacteriophages synthesized during phage therapy are not always neutralizing. Also approaches have been developed to evaluate cell immunity reactions to bacteriophage namely to identify T-lymphocytes (T-helpers and cytotoxic lymphocytes) that can be activated in the presence of the phage under study (by expressing the early activation marker (CD69) and by the ability to produce IFNγ). Approbation of the technique in the study of lymphcytes in patients during phage therapy showed the presence of activated cells by both the CD69 expression and IFNγ production, the dynamics of which depended on the timing and frequency of therapy. The appearance of neutralizing anti-phage antibodies and corresponding activated T-lymphocytes should be taken into account in phage therapy, the effectiveness of which can directly depend not only on the activity of the phage against the target bacterium, but also on the response of the patient's immune system to the bacteriophage.

In the present study, we investigate the biological properties and genomic organization of virulent bacteriophage AM24, which specifically infects multidrug-resistant clinical Acinetobacter baumannii strains with a K9 capsular polysaccharide structure. The phage was identified as a member of the family Myoviridae by transmission electron microscopy. The AM24 linear double-stranded DNA genome of 97,177 bp contains 167 open reading frames. Putative functions were assigned for products of 40 predicted genes, including proteins involved in nucleotide metabolism and DNA replication, packaging of DNA into the capsid, phage assembly and structural proteins, and bacterial cell lysis. The gene encoding the tailspike, which possesses depolymerase activity towards the corresponding capsular polysaccharides, is situated in the phage genome outside of the structural module, upstream of the genes responsible for packaging of DNA into the capsid. The data on characterization of depolymerase-carrying phage AM24 contributes to our knowledge of the diversity of viruses infecting different capsular types of A. baumannii.

A method based on nanosecond laser processing was used to design superhydrophilic and superhydrophobic copper substrates. Three different protocols were used to analyze the evolution of the bactericidal activity of the copper substrates with different wettability. Scanning electron microscopy was used to study the variation of cell morphology after the attachment to superhydrophilic and superhydrophobic surfaces. The dispersions of Escherichia coli K12 C600 and Klebsiella pneumoniae 811 in Luria Bertani broth in contact with the superhydrophilic copper surface showed enhanced bacterial inactivation, associated with toxic action of both hierarchically textured copper surface and high content of Cu2+ ions in the dispersion medium. In contrast, the bacterial dispersions in contact with the superhydrophobic copper substrates demonstrated an increase in cell concentration with time until the development of corrosion processes. The resistance of bacterial cells to contact the copper substrates is discussed on the basis of surface forces, determining the primary adhesion and of the protective action of a superhydrophobic state of the surface against electrochemical and biological corrosion.

Fibrin glue has been used clinically for decades in a wide variety of surgical specialties and is now being investigated as a medium for local, prolonged drug delivery. Effective local delivery of antibacterial substances is important perioperatively in patients with implanted medical devices or postoperatively for deep wounds. However, prolonged local application of antibiotics is often not possible or simply inadequate. Biofilm formation and antibiotic resistance are also major obstacles to antibacterial therapy. In this paper we test the biocompatibility of bacteriophages incorporated within fibrin glue, track the release of bacteriophages from fibrin scaffolds, and measure the antibacterial activity of released bacteriophages. Fibrin glue polymerized in the presence of the PA5 bacteriophage released high titers of bacteriophages during 11 days of incubation in liquid medium. Released PA5 bacteriophages were effective in killing Pseudomonas aeruginosa PA01. Overall, our results show that fibrin glue can be used for sustained delivery of bacteriophages and this strategy holds promise for many antibacterial applications.

Bacteriophages are highly efficient in treatment of intestinal and respiratory infections caused by community-acquired antibiotic-resistant pathogens. However, mass production of bacteriophages does not consider a rapid turnover of circulating strains causing healthcare-associated infections, formation of anti-phage immunity, the focus of infection in human body, phage pharmacokinetics, and a number of other issues. Therefore, we developed an original algorithm for personalized phage therapy which includes three consecutive stages: 1) determination of bacterium sensitivity to a number of widely applied bacteriophage strains using spot test and modified Gratia’s assay; 2) measurement of neutralizing anti-phage IgG in the patients’ serum utilizing enzyme-linked immunosorbent assay and neutralization test; 3) personalized selection of the phage strain for therapy including assessment of the optimal route of delivery and phage pharmacokinetic properties. Implementation of aforementioned algorithm for patients with healthcare-associated infections in intensive care units resulted in 40% increase in efficiency of phage therapy (up to 72 per cent).

A method based on nanosecond laser treatment was used to design superhydrophobic and superhydrophilic aluminum alloy substrates showing enhanced cytotoxic activity with respect to Escherichia coli K12 C600 strain. It was shown that the survival of cells adhered to the superhydrophobic substrates was significantly affected by the presence of organic contaminants, which are ubiquitous in hospital practice and the food industry. The peculiarities of the texture also played a notable role in antibactericidal activity. It was found that the superhydrophilic surfaces had much higher toxicity than the superhydrophobic ones, which was explained by the mechanisms of adhesion of cells to the surface. Scanning electron microscopy and tomographic reconstruction of the adhered cells were used to study the variation of cell morphology after attachment to surfaces with different wettability. It was shown that the cytotoxicity of superhydrophobic surfaces could be significantly enhanced by using the combined antimicrobial action of bacteriophages and the superhydrophobicity of the objects.

The relevance of bioassay standardization results from the lack of consistent national regulatory requirements for evaluation of recombinant human erythropoietin quality and the need to harmonize these requirements with international ones. Precision studies were carried out in 6 experiments on Balb/C mice. The factors that can influence the accuracy of the method were altered during the experiments. Each experiment included three levels: 20, 40 and 80 IU/ml, and 8 replicates for the reference and test samples. The trueness was estimated by bias relative to the reference value at 5 levels: 10, 20, 40, 80 and 160 IU/ml, and 4 replicates for the reference and test samples at each level. The test samples were prepared by a series of independent dilutions of the reference standard. Reticulocyte count was performed using a flow cytometer. 5 μmol acridine orange solution was used as a dye. Experimental study of accuracy and optimization of erythropoietin bioassay procedure helped to obtain two validation characteristics (trueness and precision). It was shown that logarithms of erythropoiesis registered values could reasonably be used in statistical calculations of erythropoietin specific activity and evaluation of the method's validation parameters. The theoretically and experimentally justified test procedure includes three levels of doses: 20, 40 and 80 IU/ml, and 8 animals for each level, which is consistent with the international requirements for accuracy. According to the results of experimental studies, the trueness is characterized by a bias of no more than 9 % and does not exceed the range of the calculated activity (80-125 %). Statistical processing of the test results by the parallel-line method makes it possible to check the assumption of equivalence of the test and reference samples and to calculate the test sample activity. The confidence limit of the calculated activity for intra-laboratory precision of 5.6 % is equal to 76-131 % which complies with the proposed range (64-156 %, P=0.95).