RU2813626C1 - Modified endolysin and antibacterial compositions based thereon for treating infections caused by acinetobacter baumannii, pseudomonas aeruginosa, klebsiella pneumoniae, escherichia coli bacteria - Google Patents

Abstract

FIELD: biotechnology; biochemistry.

SUBSTANCE: group of inventions relates to biotechnology, biochemistry, molecular biology, microbiology. Disclosed is a modified endolysin polypeptide having the amino acid sequence SEQ ID NO: 1, having antimicrobial activity with respect to Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli. There are also created an antibacterial composition, a drug and a method for treating diseases caused by bacteria Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, using modified endolysin polypeptide.

EFFECT: group of inventions provides the creation of an effective agent having bactericidal action on gram-negative bacteria of the species Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli, including those having multiple antibiotic resistance.

11 cl, 6 dwg, 1 tbl, 8 ex

Description

Field of technology

The group of inventions relates to biotechnology, biochemistry, molecular biology, microbiology and can be used in the creation of antibacterial drugs for the prevention and treatment of infections caused by bacteria Acinetobacter baumannii , Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli.

State of the art

The demand for new approaches to the treatment and prevention of bacterial infections is increasing with the spread of the problem of acquired antibiotic resistance among microorganisms around the world. This is especially true in the fight against infections associated with the provision of health care, since in their case, there is a significant selection pressure of a closed system of hospital organizations and a predisposition to the transmission of resistance factors among pathogens. In light of the growing problem of antibiotic resistance, the search and development of new classes of antibiotics with new targets and mechanisms of action that do not exhibit cross-resistance to existing classes of antibiotics is of paramount importance [1,2]. Among them, an actively studied class are enzyme-based antibacterial drugs (enzybiotics), which are lytic enzymes from various sources that can destroy components of the cell walls of bacterial cells and lead to their lysis. Such enzymes include endolysins of bacteriophages, which destroy bacteria due to hydrolysis of the peptdoglycan layer and subsequent osmotic lysis of pathogen cells, which are actively being studied in the light of the prospects for combating infections associated with the provision of medical care, prone to spread and acquisition of multidrug resistance [3,4]. . For example, endolysins have been shown to have high activity against gram-negative and gram-positive bacteria, regardless of their resistance status to antibiotic drugs [5]. In addition, they have significant antibiofilm activity [6-8] and, apparently, do not provoke bacteria to rapidly develop resistance, since they act on highly conserved areas of the cell wall [9,10].

The literature describes examples of the use of endolysins as antimicrobial agents. In addition, clinical studies of endolysins CF-301 and N-Rephasin® SAL200 are ongoing in patients with Staphylococcus aureus bacteremia (ClinicalTrials.gov Identifiers: NCT03089697; NCT03163446).

Several examples of solutions based on endolysins are known from the prior art.

There is a known application for an invention (RU 202128276 A, published: 03/20/2022), in which modified variants of endolysin PlySs2 have been developed that inhibit growth, reduce the population or lead to the destruction of at least one type of gram-positive bacteria, as well as a corresponding expression vector, antibacterial composition and method preventing or treating a bacterial infection caused by at least one type of gram-positive bacteria, including in combination with antibiotics.

There is a known patent for an invention (RU 2715694 C1, published: 03/02/2020), which presents a pharmaceutical composition for the treatment of eye infections caused by methicillin-resistant strains of Staphylococcus aureus , including as the active principle the N-terminal CHAP domain of endolysin of bacteriophage K Staphylococcus aureus . The claimed pharmaceutical composition is non-toxic and more effective than the antibiotic vancomycin.

There is a known patent for a group of inventions (RU 2735103 C2, published: 10.28.2020), relating to methods for preventing the formation, as well as destruction or eradication of biofilms of gram-positive bacteria. A method is proposed for preventing, destroying or eradicating a biofilm comprising one or more of the bacteria Staphylococcus and Streptococcus , and a method for preventing or treating an infection associated with a biofilm.

It is believed that these inventions can eliminate mature streptococcal or staphylococcal biofilms, and prevent the formation of de novo biofilms on the surface of devices, implants, and separation membranes.

All of the above solutions are intended to treat infections caused by gram-positive pathogens, and the results of clinical studies are mixed. Thus, for the endolysin derivative LSVT-1701 (tonabacase), phase 2 CTs were completed ahead of schedule, without announcing the results, due to a sponsor’s decision to withdraw, not related to any safety problems of the drug. At the same time, it is known from the literature that lysines active against gram-positive bacteria often do not have a wide spectrum of action, and can only effectively act on bacteria within the genus, and sometimes even species or certain strains.

At the same time, a wide spectrum of activity has been shown for lysines isolated from bacteriophages acting against gram-negative bacterial species [11]. However, in the case of Gram-negative pathogens, the interaction of lysine with its substrate peptidoglycan can be complicated by the presence of an outer membrane and often requires additional permeabilizing compounds in the dosage form.

This problem can be solved by modifications of native endolysin polypeptides by introducing permeabilizing peptides into the sequence of the recombinant protein.

For gram-negative bacteria, a patent for an invention is known (RU 2725809 C2, published 07/06/2020), in which the F307 polypeptide and its derivatives, including those conjugated with antimicrobial peptides, are used as an antimicrobial composition for the treatment of bacterial infections caused by A. baumannii and others species of gram-negative bacteria ( A. baumannii , E. coli , P. aeruginosa , S. aureus , B. anthracis and K. pneumoniae ). Methods for treating a subject, disinfecting an object, and inhibiting the formation or destruction of bacterial biofilm are disclosed. However, in in vitro tests for bacteria of the species A. baumannii and B. anthracis, the reduction in inoculations after the use of these polypeptides was no more than 4 orders of magnitude, and in the case of E. coli, P. aeruginosa and S. aureus, the polypeptides were practically ineffective (decrease in CFU less than 1.5 orders of magnitude). In vivo testing has only been conducted in infection models using A. baumannii, and therefore, true efficacy against a broad range of Gram-negative pathogens cannot be determined.

There is a known patent for an invention (RU 2703043 C1, published 10/15/2019) relating to the modification of native endolysin of the antipseudomonas bacteriophage KPP10 with a modified fragment of the sheep myeloid antimicrobial peptide SMAP-29 [K2,7,13]-SMAP-29(1-17), with for the purpose of use as an antibacterial composition exhibiting activity against gram-negative bacteria Pseudomonas aeruginosa . Its activity against a wide range of bacteria has also not been established.

There is a known application for an invention (RU 2020118691 A, publication date 01/13/2022), which describes the use of a pharmaceutical composition according to claim 1 or 2 for the elimination of P. aeruginosa , which is a solution, suspension, emulsion, inhalable powder, aerosol or spray. Its use is limited to action against P. aeruginosa .

The application for invention (RU 2015122812 A, publication date 01/10/2017) describes recombinant endolysin rEPA as an antibacterial agent against infectious diseases caused by P. aeruginosa and a pharmaceutical composition based on it for the treatment of eye infections caused by this bacterium. Its use is limited to infections caused by P. aeruginosa .

There is a known application for an invention (RU 2020129467 A, publication date 05/04/2022), which shows the sequences of 34 different endolysins, including those modified with various peptides. Minimum inhibitory concentrations were determined for these proteins on one strain of P. aeruginosa . Six of them were evaluated in combination with commonly used therapeutic antibiotics in an in vitro experiment. However, the production of these endolysins is not shown, their effectiveness has not been assessed in models of experimental infections, and the compositions of pharmaceutical compositions with these proteins are not given.

There is a known patent for an invention (RU 2730613 C1, published on August 24, 2020), in which an antibacterial agent based on endolysin LysAm24 was developed. It is proposed to use the recombinant bacteriophage endolysin protein, including in combination with pharmaceutically acceptable carriers and/or substances that increase membrane permeability, as an antimicrobial agent directed against the bacteria Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Salmonella typhi and Staphylococcus haemolyticus . Antibacterial compositions were obtained based on this protein, including one fused with an 8-histidine tag. The invention ensures effective penetration of endolysin without the addition of various permeabilizers or covalently bound penetrating peptides through the outer membrane of these bacteria and cleavage of peptidoglycan, which ultimately leads to the death of the bacteria. However, this protein significantly loses activity against bacteria in the stationary growth phase, as well as in complex mixtures, in the presence of high concentrations of salts, and, therefore, may be less effective when administered systemically with drugs based on it.

Currently, there are no drugs based on bacteriophage endolysins approved for use in the treatment of infectious diseases. The claimed technical solution is more effective than the original unmodified protein, since it retains its antibacterial properties in the presence of physiological salt concentrations, which makes it possible to increase the effectiveness of antibacterial therapy against bacteria A. baumannii , P. aeruginosa , K. pneumoniae , E. coli , including including for use as an injection form of the drug.

There is a known patent for an invention (RU 2781050 C1, published 10/04/2022), in which a bactericidal composition of a certain composition in the form of a gel for topical use was developed based on modified endolysin LysECD7-SMAP, hydroxyethylcellulose and polyethylene glycol, which has activity against gram-negative bacteria when applied topically. This composition is intended for use as an antimicrobial agent for wound and burn infections. However, the active substance of this composition does not have an effect on bacterial films, which is an important component of the treatment of wound infections, and the antibacterial effect in combination with antibiotics has not been established.

Disclosure of the invention

The technical objective of the claimed invention is to expand the arsenal of antimicrobial agents with lytic action against gram-negative bacteria, including those with multiple drug resistance to antibacterial agents used in therapy.

The technical result consists in creating an effective antimicrobial agent that has a bactericidal effect against gram-negative bacteria of the species K. pneumoniae, P. aeruginosa, A. baumannii and E. coli, including those with multiple resistance to antibiotics. This agent can be used as an antibacterial composition, used alone or in combination with other drugs, where combinations with other drugs have a synergistic or additive effect. In addition, this product can be used to prevent and treat infectious diseases caused by gram-negative bacteria, as well as to eliminate and prevent the formation of bacterial biofilms.

This technical result is achieved by obtaining a recombinant modified endolysin polypeptide (GRC-ML07) containing a wild-type LysAm24 endolysin polypeptide and an antimicrobial peptide RKLRRLKRKIAHKVKKY, wherein the modified endolysin polypeptide has the amino acid sequence SEQ ID NO:1.

In addition, antibacterial compositions were obtained containing the specified polypeptide in various concentrations (from 0.001 mg/ml to 10 mg/ml). Also, antibacterial compositions, in addition to the polypeptide, may contain various pharmaceutically acceptable excipients, permeabilizers (tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid (EDTA) and/or its salts), antimicrobials (antibiotics and synthetic antibacterial agents - polymyxins, penicillins, chloramphenicol and others; antimicrobial peptides - SMAP-29 (and its variants), LL37, gramicidin, magainin, protegrin, colicin, cecropin and others) and other lytic enzymes (native or modified lysines of bacteriophages AM24, AP22, ECD7, SI3 or other bacteriophages, lysostaphin, lysozyme ) to improve therapeutic properties.

A medicinal product has also been developed in the form of a solution, lyophilisate or gel containing the specified antibacterial composition.

In addition, the use of a modified lysine polypeptide as an antimicrobial agent directed against the bacteria Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli is disclosed.

A method has also been developed for the treatment of diseases caused by bacteria Acinetobacter baumannii , Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, which consists of administering to a subject in need of it an effective amount of an individual antibacterial composition containing a modified endolysin polypeptide, or an effective amount of an antibacterial composition as part of a combination therapy with antibiotics of the classes of polymyxins, penicillins, cephalosporins or chloramphenicol.

Brief description of drawings

Figure 1 is a graph showing the bactericidal activity of various concentrations of 1 μg/ml, 10 μg/ml and 100 μg/ml of the GRC-ML07 polypeptide against strains A. baumannii Ts 50-16, P. aeruginosa Ts 38-16 and E. coli ATCC 25922. Antibacterial activity (%) of the polypeptide under study was calculated relative to the number of bacteria in the control incubated with buffer instead of protein. For all experiments, the average standard deviation values from three independent experiments are shown.

Figure 2 is a graph showing the bactericidal activity of the GRC-ML07 polypeptide at a concentration of 100 μg/ml against various strains of Gram-negative bacteria. Antibacterial activity (%) compared to control is given. The results are presented in range diagrams: lines - medians; boxes - interquartile range; mustache - min-max.

In fig. Figure 3 shows micrographs of a suspension of bacteria A. baumannii Ts 50-16 after incubation with the GRC-ML07 polypeptide at a concentration of 100 μg/ml or with a PBS control buffer for 30 minutes. Total increase 16,000.

Figure 4 shows a graph of the activity of GRC-ML07 at various concentrations (0.1 and 1 mg/ml) against the formed 24-hour biofilms of K. pneumoniae Ts 104-14 after incubation with the polypeptide or control buffer for 2 hours. The activity is expressed in a decrease in the optical density (OD600) of biofilms stained with crystal violet dye. For all experiments, mean values with SD from three independent experiments are shown. * - significant bactericidal effect relative to control (p<0.05), Kruskal-Wallis test.

Figure 5 presents the results of an experimental study of the antibacterial effect of GRC-ML07 in an infection model (infected wound). A. Dynamics of wound healing, presented as a change in wound area, expressed as a percentage of the value on 1 day before the start of treatment. For all experiments, mean values with SEM are shown. A significant effect relative to the control was observed on day 3 in the GRC-ML07 gel group (p=0.033), two-way analysis of variance. B. Bacterial load of washings from the wound surface. Mean values with SD are shown for all experiments. **** - significant bactericidal effect relative to control (p<0.0001), two-way analysis of variance.

Figure 6 presents the results of an experimental study of the antibacterial effect of GRC-ML07 on an infection model (sepsis) in monotherapy and in combination with an antibiotic. A. Survival of mice during the experiment (in each group n=4). B. Cultures from the blood and spleens of animals during the experiment. For all experiments, mean values with SEM are shown. **** - significant effect relative to control (p<0.0001), two-factor analysis of variance.

Carrying out the invention

The product is a modified endolysin polypeptide GRC-ML07, a recombinant enzyme obtained biotechnologically. Obtaining a polypeptide consists of several stages. The original coding sequence of GRC-ML07 was artificially synthesized in the vector pALTA-GRC-ML07 and integrated into the expression vector pET-42b(+), resulting in plasmid pET42b-GRC-ML07. The correctness of the assembly of vector constructs was verified by Sanger sequencing.

The protein substance was obtained using the following technology: transformation of cells of the producer strain with a plasmid using the heat-shock method, cultivation of the resulting producer cells E. coli Bl21(DE3)pLysS in Erlenmeyer flasks with induction of protein expression using isopropyl-β-D-1-thiogalactopyranoside, clarification culture fluid by centrifugation, obtaining soluble fractions of producer cell biomass lysates, their two-stage chromatographic purification (cation exchange chromatography on an SP-sepharose carrier, followed by gel exclusion chromatography on a Superdex 75 carrier).

Thus, the pharmacologically active substance of the GRC-ML07 polypeptide was obtained in the form of a solution in sterile phosphate-buffered saline, pH 7.4, in a concentration range from 1 to 30 mg/ml. The purity and identity of the resulting recombinant polypeptide was confirmed by vertical polyacrylamide gel electrophoresis under denaturing conditions, and the concentration was measured spectrophotometrically taking into account the theoretical extinction coefficient.

The authors of the invention obtained antibacterial compositions based on the above substance, containing the specified polypeptide in various concentrations (from 0.001 mg/ml to 10 mg/ml).

Sample formulations supplemented with various pharmaceutically acceptable carriers (eg, water, saline, phosphate-buffered saline, tris(hydroxymethyl)aminomethane hydrochloride) have been developed. These compositions were included in drug samples in the form of a solution, in the form of a lyophilisate, and also in the form of a gel (hydroxyethylcellulose, sodium carboxymethylcellulose, sodium alginates). They have been shown to have a strong antibacterial effect against various types of bacteria.

These compositions can be supplemented with permeabilizers (tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid and/or its salts), antimicrobials (antibiotics and synthetic antibacterial agents - polymyxins, penicillins, chloramphenicol and others; antimicrobial peptides - SMAP-29 (and its variants) , LL37, gramicidin, magainin, protegrin, colicin, cecropin and others) and other lytic enzymes (native or modified lysines of bacteriophages AM24, AP22, ECD7, SI3 or other bacteriophages, lysostaphin, lysozyme) to improve therapeutic properties.

The implementation of the invention is confirmed by the following examples.

Example 1. Study of antibacterial activity of GRC-ML07

The spectrum of action and the degree of antibacterial activity of GRC-ML07 was assessed in experimentsin vitro against various representatives of gram-negative bacteria with varying degrees of antibiotic resistance. To do this, a suspension of bacteria in the exponential growth phase in PBS, pH 7.4, was mixed with endolysin and incubated for 30 minutes at 37°C, followed by seeding on Petri dishes with an agar nutrient medium and counting colonies after incubation in a thermostat. The polypeptide exhibited a dose-dependent antibacterial effect on strainsP. aeruginosa, A. baumanniiAndE. coli(Fig. 1), significantly reducing the number of bacteria at concentrations from 1 μg/ml to 10 mg/ml. At a concentration of 100 μg/ml, endolysin exhibited significant bactericidal activity (more than 30%) against representatives of gram-negative bacteria speciesK. pneumoniae(n=4), P. aeruginosa(n=4), A. baumannii(n=4) AndE. coli(n=2) (Fig. 2). Thus, the spectrum of antibacterial activity of the modified endolysin includes almost all representatives of the socially significant group of pathogenic bacteria ESKAPE, as well as other important pathogens of infectious diseases of various etiologies.

Example 2. Bactericidal effect of the enzyme

The antibacterial activity of the polypeptide consists of a bactericidal type of action on bacterial cells, which was shown using scanning electron microscopy.

After incubating the polypeptide at a concentration of 100 μg/ml with a suspension of bacteria of the model strain A. baumannii for 30 minutes and preparing microslides, lysis of bacteria under the influence of GRC-ML07 was shown, in contrast to bacteria incubated with PBS buffer (Fig. 3).

Example 3. Advantages of the GRC-ML07 polypeptide compared to unmodified endolysin

It was shown that the GRC-ML07 polypeptide at a concentration of 10 μg/ml has 100% antibacterial activity on bacterial cells of the model strain A. baumannii in a buffer with a sodium chloride concentration of 150 mM (phosphate-buffered saline, pH 7.4), in contrast to the unmodified endolysin, whose activity is completely inhibited by the addition of NaCl at a concentration of 50 mM. This advantage of the GRC-ML07 polypeptide allows it to be used to create pharmaceutical compositions for parenteral use.

Example 4. Effect of GRC-ML07 on formed bacterial biofilms.

The antibiofilm activity of endolysin was assessed on 24-hour biofilms of various bacterial species ( K. pneumoniae, P. aeruginosa, A. baumannii ). For this purpose, biofilms were grown in the wells of a 96-well plate, washed from planktonic cells and exposed to an endolysin solution at concentrations of 100 and 1000 μg/ml for 2 hours. Further staining with crystal violet solution showed that endolysin GRC-ML07 significantly reduced biofilm density relative to control in a dose-dependent manner, destroying up to 100% of biofilm. An example of the effect of the polypeptide on formed 24-hour biofilms of K. pneumoniae is presented in Fig. 4.

Example 5. Synergism of the action of the GRC-ML07 polypeptide and antibacterial drugs.

During the work, the potential of using a combination therapy approach based on classical antibiotics and modified endolysin of the bacteriophage GRC-ML07 against gram-negative pathogens was assessed in vitro .

The synergistic effect of various concentrations of GRC-ML07 and the antibiotics polymyxin B, chloramphenicol, ampicillin and cefotaxime was shown using the checkerboard method on various representatives of gram-negative bacteria (two strains each of the species K. pneumoniae, P. aeruginosa, A. baumannii ), including those with multidrug resistance.

To do this, two-fold dilutions of endolysin solution were incubated in the wells of a 96-well plate with two-fold dilutions of the antibiotic for 16-20 hours and bacterial growth inhibitory combinations were evaluated. For each combination, ∑FIC (fractional inhibitory concentration index) was calculated.

∑FIC = MIC _AB / MIC _A + MIC _BA / MIC _B ,

where MIC _A is the MIC of antibiotic A;

MIC _B - MIC of antibiotic B;

MIC _AB - MIC of antibiotic A in the presence of antibiotic B;

MIC _BA - MIC of antibiotic B in the presence of antibiotic A.

The results obtained were interpreted as follows:

x ≤ 0.5 Synergy 0.5 < x ≤ 4 Additive effect x > 4 Antagonism

In the dose range of GRC-ML07 from 512 μg/ml to 64 μg/ml and antibiotics from 16 μg/ml to 0.015 μg/ml, a strong synergistic or additive effect was observed on all three bacterial species (Table 1). Moreover, no cases of antagonism were shown for either combination.

Table 1 - Sensitivity of the studied strains to four antibiotics (AMP - ampicillin, CHL - chloramphenicol, PMB - polymyxin B, CTX - cefotaxime), as well as the results of studying the combined effect of antibiotics with GRC-ML07 (Lys), expressed in minimal ∑FIC. R - resistant, S - sensitive strain; SYN - synergism, ADD - additive effect, NE - no effect, "-" - no data. Bacterial strain A. baumannii 50-16 A. baumannii 53-16 K. pneumoniae M-9 K. pneumoniae F104-14 P. aeruginosa ATCC 9027 P. aeruginosa 38-16 Ampicillin Strain sensitivity to AMP R R R R R R ∑FIC Lys-AMP 0.75 R 0.56 R 0.63 R Interpretation ADD NE ADD NE ADD NE Chloramphenicol Strain sensitivity to CHL R R S R R R ∑FIC Lys-CHL 0.31 0.63 0.56 0.75 0.5 R Interpretation SYN ADD ADD ADD SYN NE Polymyxin B Strain sensitivity to PMB S S S S S S ∑FIC Lys-PMB 0.31 0.18 0.12 0.09 0.12 0.12 Interpretation SYN SYN SYN SYN SYN SYN Cefotaxime Strain sensitivity to CTX S R S R R R ∑FIC Lys-CTX 0.56 - - - - - Interpretation ADD - - - - -

We have shown that combinations can not only have a synergistic effect (in the case of a combination of lysine and polymyxin), but also restore the sensitivity of the microorganism to the antibiotic. For example, for three strains it was possible to show an additive effect in the combination of lysine with ampicillin, to which all strains were initially resistant. Similar results were shown for chloramphenicol, and it should be noted that A. baumannii and P. aeruginosa are naturally resistant to it, while we showed a strong synergism or additive effect. At the same time, the mechanism of the antimicrobial action of endolysins assumes that bacteria do not develop resistance to it, which means this therapeutic regimen is safe when used in a course. These studies may provide the basis for the development of new treatment regimens for severe and chronic infections caused by antibiotic-resistant pathogens.

Example 6. Preparation of samples of antibacterial compositions based on GRC-ML07

Samples of compositions with the following composition were obtained:

1. Solutions were obtained by diluting the polypeptide concentrate (substance) in an appropriate buffer to the required concentration.

Polypeptide solutions with concentrations of 0.001 mg/ml, 0.01 mg/ml, 0.1 mg/ml, 1 mg/ml, 10 mg/ml in phosphate-buffered saline with pH 5.0, 6.0, 7.0 , 7.5, 8.0, 9.0.

Polypeptide solutions with concentrations of 0.001 mg/ml, 0.01 mg/ml, 0.1 mg/ml, 1 mg/ml, 10 mg/ml 20 mM Tris hydrochloride solution with pH 5.0, 6.0, 7.5 , 8.0, 9.0.

Polypeptide solutions with concentrations of 0.001 mg/ml, 0.01 mg/ml, 0.1 mg/ml, 1 mg/ml, 10 mg/ml in physiological solution.

The solutions were supplemented with substances that improve the permeability of bacterial membranes (permeabilizers) - tris(hydroxymethyl)aminomethane at a concentration of 40-100 mM, ethylenediaminetetraacetic acid (EDTA) or its sodium salt at a concentration of 1-100 mM.

2. Lyophilisates with the polypeptide were obtained from the above solutions by freeze drying.

3. Polypeptide gels were prepared by introducing the above polypeptide solutions into a previously prepared 0.5% - 1% sodium alginate solution, 1 - 3% hydroxyethylcellulose solution and 1 - 3% sodium carboxymethylcellulose solution.

The antibacterial activity of the samples was assessed in in vitro experiments using a model strain of A. baumannii. For example, solutions of the polypeptide in PBS, Tris-HCl in the pH range 5.0 - 9.0, as well as saline at a concentration of 1 μg/ml have an activity of 80 - 100%. The addition of permeabilizers leads to an increase in activity up to 100% in all cases. The claimed compositions can be obtained by specialists in practice and their use ensures the implementation of the stated purpose.

Example 7. Experimental study of the antibacterial effect of GRC-ML07 on an infection model (infected wound).

For the experiment, a gel was prepared based on algal alginate (1%) containing 1 mg/ml endolysin GRC-ML07. BALB/c mice (females, 18-20 g) had their hair removed with a clipper one day before the experiment. The wound defect was made on the backs of animals under anesthesia (zoletyl-xylazine) with a special biopsy instrument (d=8 mm, Medax, Italy). After 5 minutes, a suspension of the multiresistant strain P. aeruginosa Ts 38-16 was applied to the surface of the wound at a concentration of 5×10 ⁸ CFU/wound. After 24 hours, treatment was started with cutaneous application of the drug (n = 6), placebo (gel without polypeptide, n = 6) or standard drug (gentamicin ointment, n = 6) in accordance with the experimental plan. To do this, drugs in a volume of 100 μl were applied to the wound surface twice a day every day for 5 days. On days 1, 3 and 7, pathomorphological changes in the wound surface were studied and its area was measured (planimetry), and the microbial load was also assessed by washings from the surface of the wound. The resulting swabs were sown in PBS and diluted on a solid nutrient medium with further cultivation for 18 hours at 37°C to assess their contamination.

When assessing the results of the study, taking into account the dynamics of changes in the area of wounds and the bacterial load of washings, a direct effect of the studied antibacterial composition on wound healing in the formation of infected wounds was revealed. A change in the contamination of the wound and a significant decrease in the bacterial load under the influence of the gel with GRC-ML07 were shown. Treatment with GRC-ML07 gel showed a significant reduction in wound area compared to the control. Moreover, after the end of treatment, the therapeutic effect in the GRC-ML07 group was maintained much better than in the placebo group. Treatment with gentamicin ointment significantly worsened the course of infection, wound healing and contamination of wound surfaces. The results of the experiment are presented in Fig. 5.

Example 8. Experimental study of the antibacterial effect of GRC-ML07 on an infection model (sepsis) in monotherapy and in combination with an antibiotic.

Female BALB/c mice (18-20 g) were intraperitoneally injected with a suspension of cells of the K. pneumoniae M9 strain at a dose of 1×10 ⁵ CFU/animal. 2 hours after infection, the study drug GRC-ML07 in PBS pH=7.5 (12.5 mg/kg, n = 4), a combination of GRC-ML07 and polymyxin B (0.05 mg/kg, n = 4) was administered intravenously ) or placebo (PBS, n = 4) in a volume of 100 μl. Treatment was carried out daily, 5 days, 2 times a day. The animals were observed for 3 days, their death was taken into account, and survival curves were constructed.

Blood samples were taken from dead animals and the spleen was removed; surviving animals were euthanized by cervical dislocation and the same samples were collected on the last day of the experiment. The contamination of blood and spleen homogenates was assessed by bacteriological inoculation of various dilutions on a solid nutrient medium with further cultivation for 18 hours at a temperature of 37°C, counting the grown bacterial colonies.

When assessing the results of the study, a significant antibacterial effect of the study drug GRC-ML07 was revealed both in monotherapy and in combination with an antibiotic at a dosage of 0.5 MIC. Survival in the control group was 0% (all died on the second day of the experiment), while in the GRC-ML07 group survival on day 3 was 75%, and in the group with the addition of a subtherapeutic dose of polymyxin B it reached 100%. A significant reduction in the bacterial load in the blood under the influence of GRC-ML07 was also shown. No significant differences were shown in the spleen, however, the trend towards a decrease in seedings was pronounced. The results of the experiment are presented in Fig. 6.

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List of sequences

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Epidemiology and Microbiology named after honorary academician N.F. Gamaleya"

Ministry of Health of the Russian Federation</ApplicantName>

<ApplicantNameLatin>federalnoe gosudarstvennoe biudzhetnoe

uchrezhdenie Natsionalnyi issledovatelskii tsentr epidemiologii i

mikrobiologii imeni pochetnogo akademika N F Gamalei Ministerstva

zdravookhraneniia Rossiiskoi Federatsii</ApplicantNameLatin>

<InventionTitle languageCode="en">Modified endolysin and

antibacterial compositions based on it for the treatment of infections,

caused by bacteria Acinetobacter baumannii, Pseudomonas aeruginosa,

Klebsiella pneumoniae, Escherichia coli</InventionTitle>

<SequenceTotalQuantity>1</SequenceTotalQuantity>

<SequenceData sequenceIDNumber="1">

<INSDSeq>

<INSDSeq_length>243</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..243</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q2">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MDILKFNSRGDLVVHLQKKLASLGYKLNPDGIFGKATQEAVIHFQRQNG

LTSDGIVGKLTWSMLDSKTSSIGKTRTISQNGINFIKSFEGLRLRAYDDGVGVITIGYGTTRYPNGHKVQ

LGDTCTEKQAEQYLSNDLVKFEKAVNELVKVPVNQNQYDALVSFTYNVGVGALSTSKALKLLNAGDYTGC

AKAMLSWNKGRVGGKLVEIGGLTRRRNAEKDLFLKLERKLRRLKRKIAHKVKKY</INSDSeq_sequen

ce>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (11)

1. A modified endolysin polypeptide having the amino acid sequence SEQ ID NO:1 and having antimicrobial activity against Acinetobacter baumannii , Pseudomonas aeruginosa , Klebsiella pneumoniae , Escherichia coli. 2. The modified endolysin polypeptide according to claim 1, where said sequence contains a linker between the endolysin polypeptide and the antimicrobial peptide, consisting of at least one amino acid residue. 3. Antibacterial composition against bacteria Acinetobacter baumannii , Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli , including a modified endolysin polypeptide according to any one of claims. 1, 2, in amounts from 0.001 to 10 mg/ml. 4. The antibacterial composition according to claim 3, further comprising a pharmaceutically acceptable carrier. 5. Antibacterial composition according to any one of paragraphs. 3, 4, additionally including permeabilizers selected from tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid (EDTA) and/or its salts. 6. Antibacterial composition according to any one of paragraphs. 3-5, additionally including antibiotics, in particular polymyxins, penicillins, chloramphenicol and/or cephalosporins. 7. Antibacterial composition according to any one of paragraphs. 3-6, further including SMAP-29 or variants thereof, LL37, gramicidin, magainin, protegrin, colicin or cecropin. 8. Antibacterial composition according to any one of paragraphs. 3-7, additionally including native or modified lysines of bacteriophages AM24, AP22, ECD7, SI3, lysostaphin, lysozyme. 9. A medicinal product containing an antibacterial composition according to any one of paragraphs. 3-8, in the form of a solution, lyophilisate or gel. 10. Use of a modified endolysin polypeptide according to any one of claims. 1, 2 as an antimicrobial agent directed against bacteria Acinetobacter baumannii , Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli . 11. A method for treating diseases caused by bacteria Acinetobacter baumannii , Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, which consists in administering to a subject in need of it an effective amount of an individual antibacterial composition according to any one of claims. 3-8, containing a modified endolysin polypeptide according to any one of paragraphs. 1, 2, or an effective amount of an antibacterial composition as part of combination therapy with antibiotics of the classes of polymyxins, penicillins, cephalosporins and/or chloramphenicol.