Method for disrupting and preventing the formation of bacterial biofilms using insect antimicrobial peptide complexes
The present invention relates to the fields of medicine, hygiene, cosmetology and veterinary medicine and can be used to destroy pathogenic biofilms formed by a wide variety of bacteria on the surface of the skin and other organisms, medical products, implants and catheters.
Many antibiotics and antibacterial agents which are toxic to bacteria and belong to different classes of organic compounds (β -lactams, macrolides, tetracyclines, fluoroquinolones, sulfonamides, amidoglycosides, imidazoles, peptide compounds, quaternary ammonium salts, etc.) are used in medicine and veterinary medicine, most of the antibiotics and antibacterial agents known today are effective in the case of planktonic (free-standing) bacterial forms when a biofilm (a multicellular bacterial community surrounded by a matrix and adhered to the external or internal surface of a host organism or inanimate object) is formed, the bacteria acquire resistance to antibiotics and antibacterial agents [1,2] and become unable to be destroyed by immune system cells [3] thus, it is very difficult to treat and prevent diseases promoted by biofilms [4,5] it is well known that biofilms cause about 80% of human bacterial infections [6] as well as a variety of inflammatory diseases and autoimmune and neoplastic diseases related thereto, cardiovascular system damage.
The use of natural and synthetic antimicrobial peptides is considered to be one of the advanced trends to solve this problem [6-9 ]. However, the practical realization of this idea has met with considerable difficulties related to low efficiency, high cost in the use of these peptides, the risk of cross-resistance in bacteria to endogenous antibacterial peptides in humans and animals, and many other problems [10-12 ]. In order to improve the efficiency of therapy, it has been proposed to create a combination of antibacterial peptides and conventional antibiotics, wherein the combination of peptides and antibiotics show a synergistic effect on biofilms. In particular, synthetic antimicrobial peptides have been proposed for use in combination with various antibiotics to treat bacterial biofilm-induced infections [13 ]. For the same purpose, the use of phage lysine proteins in combination with antibiotics is suggested [14 ]. This Method of disrupting biofilms (RU 2014149348A, 05.09.2013, "Method for harvesting, dispersing and processing a biofilm with bacterial biofilm lysine)" is the closest technical solution to the claimed invention and was chosen as the prototype, where the peptide (protein) product of protein synthesis is used as an antibiotic synergist. It is emphasized that both of the above inventions use a single compound (synthetic peptide 13 or natural protein 14) with peptide properties and the method of disrupting the biofilm is to use the compound in combination with an antibiotic. Such multi-component combinations have not been used to date due to the technical complexity and high manufacturing costs of developing such combinations. Meanwhile, it is well known that the natural mechanism of genetic immunization of multicellular animals includes complex aggregates of antibacterial peptides. This provides a number of key advantages not seen with single antibacterial peptides and antibiotics, in particular preventing the development of bacterial resistance [16 ].
The technical problem underlying the present invention is to develop a method for disrupting biofilms using the synergistic or sequential action of natural complexes of antimicrobial peptides and known antibiotics. The technical solution of the present invention is to improve the efficiency of treating human and animal diseases caused by pathogenic biofilms. This result is achieved because the sensitivity of the biofilm to the antibiotic is increased by the antimicrobial peptide complex, the therapeutically effective concentration of the antibiotic is reduced, and the level of toxicity to the patient is reduced. To solve this problem, it has been proposed to use a purified natural complex of an antimicrobial peptide synthesized from a culture of dipteran insects. The obtained sample is used in conjunction or sequentially with an antibacterial means (or combination of antibacterial means) selected from the group consisting of antibiotics or antibacterial agents toxic to the given bacterial species. Thus, antimicrobial means that exhibit a synergistic effect with the antimicrobial peptide complex when tested in vitro on a biofilm of a given species are preferred.
Essence of the claimed invention
Two basic forms are known to be specific for most bacteria: planktonic forms (providing independent living cells infected by the host organism) and biofilms (multicellular communities of one or more microorganisms immersed in a matrix released by these microorganisms, which communities adhere to a variety of surfaces). Bacterial colonies may persist in the host organism indefinitely as part of the biofilm, forming planktonic cells and transmitting the infection from the primary site if necessary [1-5 ].
The primary means of treating bacterial infections, including biofilms, is antibiotics. However, as mentioned above, most antibiotics have low or zero activity against biofilms compared to planktonic forms of the same strain. Strains with genetic resistance to antibiotics are widespread and even largely limit the possibility of treating biofilm infections. To solve this problem, antibacterial peptides have been proposed in the literature as potentiators to enhance the anti-biofilm activity of antibiotics [6-9, 13,14 ]. The current evaluation of synergy ("checkerboard method") is mainly used to develop binary compositions (1 peptide +1 antibiotic). This essentially limits the possibilities of methods currently available for disrupting bacterial biofilms. In particular, such combinations have a narrow spectrum of therapeutic activity. Thus, the use of the bacteriophage lysine [14] is only applicable to biofilms formed by gram-positive bacteria of the Staphylococcus aureus type, but not gram-negative bacteria.
As with known methods, the method of disrupting biofilms claimed herein is based on the use of a synergistic effect of an antimicrobial peptide and an antibiotic. The main difference is that the antimicrobial peptide purification complex of diptera insects is suggested to be used as an antibiotic booster, rather than the antimicrobial peptide alone (e.g. synthetic cationic peptide [13] or phage lysine [14 ]). The idea is based on the study of the authors of the present invention in the field of immunology of insects, in particular of the larvae of blowfly red-head blowfly (Calliphora vicina). The authors have indicated that this type of larvae respond to bacterial contamination while synthesizing and accumulating complexes of antimicrobial peptides including defensins, cecropins, dipteramicidin and proline rich peptides in haemolymph [15, 16 ]. Some of these peptides selectively disrupt the cell wall of gram-positive (defensins) and gram-negative (dipterans, cecropins) bacteria, others interfere with protein and RNA synthesis in the bacterial cell (proline-rich peptides). These four classes are typical of the immune system of dipteran insects [15 ]. Thus, as shown by the real-time test results given in examples 1 and 2, complexes of antimicrobial peptides obtained from a variety of species can be used to embody the invention claimed herein. In addition to red-head blowfly, species that can be used for this purpose include black-cheek blowfly (c. vomitoria), Lucilia sericata (Lucilia sericata), Lucilia bifida (l.caesar) (diptera family of blowdiels), housefly (Musca domestica) (diptera family of muscidae), Hermetia illucens (hermetiillucens) (diptera family of dielliferae). Like blowfly, these species have the unique feature that they all belong to the family of domestic saprophagous double-wing insects, living in an environment that is maximally populated by pathogenic microflora of humans, farm animals and pets. Thus, the antimicrobial complex of domestic saprophages exhibits high activity with respect to this species of microbial flora [15-16 ]. The ecological characteristics of red-headed blowfly and other above listed diptera feeding insects make it possible to culture them on an industrial scale with inexpensive feeding substrates, which makes the biosynthesis of the antibacterial complex claimed in this application technically and economically feasible.
A unique feature of the insect antimicrobial peptide complex, exemplified by blowfly, is the complexity of its composition. The results of the study conducted, summarized in example 3, indicate that not less than 11 antimicrobial peptides involved in the immune response of the insect are present in the composition of the complex. This fact leads to red-headed blowfly and the approximate species of diptera being particularly advantageous for achieving the method of disrupting and preventing biofilm formation claimed herein. However, the evolutionary conservation of the natural immune mechanisms allows for the hypothesis that antimicrobial peptide complexes of other organisms may also be used to implement the methods claimed herein.
It is well known that the antimicrobial peptide complex of blowfly red head is capable of disrupting biofilms formed by a variety of bacteria, but this requires the production of high concentrations of complex (1.5-7.6g/1, depending on the bacterial species) [18 ]. This fact essentially limits the possibilities of using the compound in medical and other fields. Theoretically, this limitation can be eliminated by combining the natural complex of antimicrobial peptides with antibiotics or antimicrobials to form a synergistic pair (synergist pair). However, this hypothesis is not considered in the literature, nor has it been experimentally investigated. Separately, methods for disrupting bacterial biofilms based on the use of antimicrobial peptide complexes of diptera insects in combination with other antimicrobial means are not known. This problem was first addressed and solved by the authors of the present invention. The results of the corresponding experimental study are given in a specific embodiment.
In view of this object, the authors have studied the complex of the Drosophila erythropolis antibacterial peptide (CAMP) with the amino glycosides, β -lactamsAntibiotic activity in combination with antibiotics of the glycopeptides, macrolides, lincosamides, fluoroquinolones, amidoalcohols (amphenicols) and tetracyclines type, and with antibacterials from the cetrimides type (quaternaries). Detailed descriptions of the experiments are given in examples 4-5. The main technique of the study is to analyze the interaction of various antibacterial means in vitro systems, which is widely used to perform similar studies ("checkerboard" technique). Biofilms resistant to antibiotics, which are formed by the bacteria Escherichia coli (Escherichia coli), Staphylococcus aureus (Staphylococcus aureus), Acinetobacter baumannii (Acinetobacter baumannii), Pseudomonas aeruginosa (Pseudomonas aeruginosa), were used as biological models. The biofilm formed by these bacteria causes most clinically significant bacterial infections. Table 1 summarizes the data showing the effect of antimicrobial peptide complexes on the anti-biofilm activity of antibiotics and antimicrobials. MBIC90The value (antibiotic concentration inhibiting 90% of the metabolic activity in the standard TTC test) is a quantitative standard for the anti-biofilm activity.
Of the 20 variants of CAMP combinations studied with different classes of antibacterial means, 8 combinations showed additive effects (FICI >0.5) and 11 combinations showed synergistic effects (FICI < 0.5). In only one case (polymyxin B on E.coli biofilm), no antibiotic efficacy enhancement was shown. Both types of interactions allow for disruption of the biofilm and reduction of the therapeutically effective concentration of antibiotics. The results obtained from the experimental studies clearly demonstrate, demonstrate and confirm the 14 sub-claims of the independent claim 1 of the claimed method for disrupting biofilms. Several of the most advanced embodiments of the present invention should be extracted according to the type of biofilm.
Thus, the biofilm of Staphylococcus aureus is resistant to the aminoglycoside drug β -lactam meropenem, amikacin and kanamycin (MBIC)90>500. mu.g/ml), lincosamide clindamycin (MBIC)90>250 μ g/ml) was absolutely insensitive. Those antibiotics show high anti-biofilm activity in the presence of CAMP (MBIC respectively)90< 0.1, 1.5, 3 and 12. mu.g/ml). Therefore, this application is toThe claimed invention allows the use of those antibiotics to treat the widest group of bacterial infections, i.e. the biofilm of staphylococcus aureus. In the case of contamination with particularly dangerous methicillin-resistant (MRSA) and vancomycin-resistant (VRSA) staphylococcus aureus, it is particularly important to expand the range of antibiotics to treat such infections. From the data obtained, the combination of CAMP + aminoglycoside is expected to disrupt MRSA biofilms in the case of VRSA infection, as well as CAMP + lincosamides.
Some of the antibiotics used in the studies were toxic to planktonic cells of staphylococcus aureus and when used at higher concentrations, remained active against biofilms of that species. Within this group, mention should be made in particular of vancomycin and ampicillin, the CAMPs of which act as powerful synergists and reduce the concentration of inhibitory Staphylococcus aureus biofilms from 38-24 to below 1. mu.g/l. The convenience of using CAMP in combination with those antibiotics to inhibit s.aureus biofilms appears to be significant. It should be noted that the lower amplification factor value (C)a<10) Can greatly improve the treatment of the staphylococcus aureus biofilm infection.
Thus, like other antiseptics, benzalkonium chloride antiseptics used to disinfect wound surfaces exhibit high toxicity. The possibility of reducing its concentration by a factor of 8 while maintaining anti-biofilm activity allows to reduce its toxic effects on the tissues surrounding the wound, thus improving the wound infection treatment protocol.
The biofilm formed by another broad pathogen enterobacter coli showed relative sensitivity to all studied antibiotics (although reduced compared to the planktonic cells of the bacterium.) in these cases its combination with CAMP provided an enhancement to all studied antibiotics however, it should be noted that CAMP was particularly high in level of synergy with the β -lactams meropenem and cefotaxime (187 and 62 times lower effective concentrations respectively). according to this index, the combination of CAMP with meropenem and other β -lactams seems particularly promising.
Combining CAMP with antibiotics can also provide another technically important result, namely lowering the effective concentration of CAMP. The combined use of CAMP with amikacin (effective concentration reduced by more than 48-fold) and vancomycin, kanamycin and tetracycline (effective concentration reduced by 16-fold to over 24-fold) showed the best results from the point of view of disrupting the biofilm of staphylococcus aureus. With respect to biofilms formed by other research bacteria (e.coli, pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii), the effective concentration of CAMP can also be reduced by 2-fold to 43-fold or more, depending on the type of bacteria and the type of antibiotic, in combination with various classes of antibiotics.
In addition to disrupting mature biofilms, the method claimed herein provides another technologically important result, namely the removal of independently living (planktonic) cells of bacteria and thus the prevention of biofilm formation by these cells (example 6). The synergistic or additive effects of CAMP and antibiotics on planktonic cultures, together with preventive measures, can significantly reduce the therapeutically effective concentration of antibiotics necessary to prevent biofilm formation, thereby avoiding the adverse consequences associated with administration of large doses of antibiotics.
It should be noted that the complex of the antimicrobial peptide and the antibiotic may be included in one pharmaceutical composition and applied to the surface of the biofilm at the same time. Instead, the antibiotic may be introduced into the organism parenterally, orally, or by any other method that is medically acceptable, separately from the sample containing the antimicrobial peptide complex, and ensures that the antibiotic is in contact with the biofilm to be disrupted.
The claimed method can also be achieved by introducing insect antimicrobial peptide complexes into the composition of various medical devices (wound and burn treatment covers, catheters, implants, etc.) to disrupt and prevent the formation of pathogenic biofilms.
Furthermore, the method claimed herein can be achieved by introducing a complex of insect antimicrobial peptides into a composition of a skin care cosmetic to prevent skin damage caused by biofilm formation.
It is also apparent that the methods claimed herein can be used in veterinary medicine to treat bacterial infections in animals similar to human diseases.
In general, the method claimed in the present application allows to significantly expand the technical scope and to improve the efficiency of the treatment of bacterial infections in general for the medical and related fields. The main advantages of the method claimed in the present application are:
1. due to the use of natural complexes of antimicrobial peptides, it is possible to create multicomponent compositions. As such a complex, a dipteran antimicrobial peptide combination comprising four different classes of peptides (defensins, cecropins, dipterans and proline rich peptides), each represented by several different forms, can be used. The methods of disrupting biofilms known today are limited by the "a peptide plus an antibiotic" regimen, which is characterized by a narrow spectrum of antibacterial activity and an increased risk of developing resistance in bacteria.
2. Due to the potentiating (synergistic or additive) effect of the insect antimicrobial peptide complex, it is possible to reduce the concentration of antibiotics required to disrupt the biofilm. This may reduce the therapeutic dose of the antibiotic and thus reduce the risk of adverse reactions resulting from administration of the antibiotic.
3. In particular, the results of scientific research and specific test examples show that β -lactam antibiotics and aminoglycosides exhibit anti-biofilm activity in the presence of CAMP, and that these two key antibiotics are now believed to be of little use in the treatment of biofilm infections.
4. The mode of administration of the antibiotic was varied. Currently, most antibiotics are used systemically by parenteral or oral administration. Topical administration of antibiotics is limited by insufficient clinical efficiency. In systemic administration, large doses of antibiotics are used in order to produce the desired concentration in the infected lesion, which leads to death of the patient's normal flora and risks triggering other adverse events (nephrotoxicity, neurotoxicity, cardiotoxicity, etc.). The method claimed herein allows to increase the efficiency of antibiotics while being administered directly to the foci of infection (for example, by application on the surface of a biofilm), thus eliminating the necessity of their systemic administration in certain cases experimentally verified.
5. The antibiotics studied in turn enhance the anti-biofilm action of the antimicrobial peptide complexes of insects, allowing to reduce their therapeutically effective concentration and to enlarge the range of possible applications.
6. At present, it has been experimentally proven that the combination of 19 CAMP and antibiotics is reasonable and confirms the results obtained. It is evident from practice that this list will be significantly expanded from now on in later scientific and experimental studies. The method claimed herein also allows for varying the balance between the therapeutic dosage and the mode of administration of CAMP and the antibiotic.
7. Summarizing all the above, the method claimed herein enables to substantially and drastically expand the possibilities of personalized treatment of bacterial infections, taking into account the characteristics of the patient and the characteristics of the disease.
The results of the extensive testing are confirmed by the specific exemplary embodiments given below.
Example 1
Samples of the refined complexes containing dipteran antimicrobial peptides were prepared and analyzed for antimicrobial activity.
The technique of generating the sample corresponds to the procedure described previously [16 ]. For this purpose, four insects from the order diptera were used, including three blowflies from the families callimastidae red-head blowfly (Calliphora vicina), black-cheek blowfly (C.vomitooria) and Lucilia sericata (Lucilia sericata) and housefly (Musca domestica) from the family Musca. The procedure for preparing the samples is as follows. Larvae were immunized by introducing a bacterial cell suspension into the body cavity and incubated for 24 hours. After that period of time, hemolymph was collected from the larvae through the incision of the epidermis and used to release the complex of antimicrobial peptides. For this purpose, collected hemolymph was acidified with 0.1% trifluoroacetic acid (TFA) and insoluble precipitates were removed by centrifugation. The supernatant obtained was applied to a column with C-18 adsorbent pre-stabilized with 0.05% TFA (Waters, 35CC SepPack column), washed with 0.05% TFA and eluted with 50% acetonitrile/0.05% TFA. The eluate was lyophilized and used as a purified complex of antimicrobial peptides in this example and the following examples. The antibacterial activity of the complexes was determined using serial dilution techniques [19 ]. The mean Minimum Inhibitory Concentration (MIC) of the planktonic culture, e.coli 774.1, among three independent measurements is shown in table 2. All four complexes showed the expressed antibacterial activity. Accordingly, the greatest activity was found in the complexes from blowfly rubra. This sample has been selected for the following study as the best embodiment of the invention.
Example 2
Antibacterial activity of Hermetia illucens (Hermetia illucens) samples
Samples containing the antimicrobial peptide complex from hermetia illucens were prepared according to the technique described in example 1. Determination of the antibacterial Activity of the complexes Using agar plates [19]. For this purpose, 7.5ml of Luria-Bertany nutrient solution (1% bacto tryptone, 0.5% yeast extract, 1% NaCl) with agarose (Invitrogen) were inserted into sterile petri dishes (diameter 9 cm). Before curing, 2X 105Bacterial plankton cultures of the corresponding strains of the individual cells (Table 3) were introduced into warm medium. The test material was applied to the surface of the solidification medium in a volume of 2. mu.l. The dishes were incubated at +37 ℃ for 24h and the diameter of the zone of inhibition of bacterial growth was measured. Samples from red-headed blowfly were used as reference samples. The data in table 3 show that, under the conditions of the given experiment, the samples from hermetia illucens showed activity against pseudomonas aeruginosa, differently from the samples from blowfly rubra. Thus, samples from hermetia illucens may have advantages in treating bacterial infections induced by a given bacterium.
Example 3
Composition analysis of antimicrobial peptide complexes
Samples containing the antimicrobial peptide complexes from blowfly rubra were produced according to the technique described in example 1. The composition of the antimicrobial peptides was studied using the earlier described techniques such as liquid chromatography, mass spectrometry and transcriptome analysis [18 ]. The structures of the 11 peptides responsible for the antibacterial activity of the complex have been determined (table 4). Of these, 5 peptides (seq. ID No.1, 2, 4, 9, 11) [18,20] were characterized earlier, while the other 6 were novel findings in science. In addition, the composition includes an antimicrobial peptide having a molecular weight of 6773-6973 daltons, whose structure has not been broken, and possibly other minor components having antimicrobial activity. According to the classification accepted in the literature [21,22], the combination of activities from blowfly red head belongs to four classes of insect antimicrobial peptides: defensins (seq. ID No.1), cecropins (seq. ID No.2, 3), dipteran (seq. ID No.4-8) and proline rich peptides (seq. ID No. 9-11).
Example 4
Biofilms formed by pathogenic bacteria are disrupted when exposed to CAMP from blowfly rubra and various antibiotics. TTC test
In this study, the strains Escherichia coli ATCC25922, Staphylococcus aureus 203, Pseudomonas aeruginosa ATCC 27583, Klebsiella pneumoniae 145, Acinetobacter baumannii 28 with enhanced biofilm-forming ability [18]]. The technique for producing the biofilm corresponds to that described in the publication. Biofilms were generated in 96-well microplates. With a cell concentration of 5X 105The wells were filled with a bacterial suspension of CFU/ml and the suspension was incubated at 37 ℃ for 24 h. LB broth (Invitrogen) was used as a negative control. Red head blowfly CAMP samples were prepared according to the protocol described in example 1. The interaction of red-headed blowfly CAMP in combination with antibiotics on biofilms was studied using a modified cross-titration technique. For this purpose, the 24-hour biofilm in the microplate was washed 3 times with 200. mu.l of PBS solution and dried. The red-headed blowfly CAMP and antibiotic combination was prepared in another 96-well microplate by placing two-fold dilutions of the sample in the horizontal row of wells and two-fold dilutions of the antibiotic in the vertical row of wells. In addition, 100. mu.l of the contents from each well of the microplate were transferred to a microplate with a biofilm and incubated at 37 ℃ for 24 h. Biofilm formation was assessed by staining it with tetrazolium chloride (TTC). For this purpose, 11. mu.l of a 0.2% TTC solution were added to all of the platesIn the hole. After incubation at 37 ℃ for 1h, OD was measured using a reader of a microplate reader epoch (BioTek)540. OD of 48h biofilm without having undergone antibacterial compound540The values served as controls. All experiments were performed in duplicate. The minimum inhibitory concentration (MBIC) of the biofilm was evaluated as MBIC90I.e., the concentration of the sample that inhibits 90% of cell viability. The Fractional Inhibitory Concentration Index (FICI) for each sequence in the combination is determined according to the following expression: the FICI is equal to the Minimum Inhibitory Concentration (MIC) of antibiotic a in combination with another antibiotic divided by the MIC of antibiotic a alone, and the FICB is equal to the Minimum Inhibitory Concentration (MIC) of antibiotic B in combination with another antibiotic divided by the MIC of antibiotic B alone. The FICI is explained as follows: FICI<Synergistic Effect at 0.5, FICI>Additive effect, FICI, at 0.5 ≦ 1>1. ltoreq.4, FICI>4 has antagonistic effect. The anti-biofilm activity of a total of 1470 CAMP and antibiotic combinations has been studied using the TTC test.
The results are summarized in Table 5. experiments with Staphylococcus aureus showed that the combination with CAMP had a significant synergistic effect on the anti-biofilm activity of aminoglycosides (amikacin and kanamycin), β -lactams (ampicillin and meropenem), the glycopeptide vancomycin, macrolide erythromycin, lincosamide clindamycin, chloramphenicol. CAMP had an additive effect on the efficacy of oxacillin and antibacterial agents such as benzalkonium chloride. thus, CAMP enhanced the effect of all studied antibiotics and antibacterial agents on the biofilm of Staphylococcus aureus. experiments with Escherichia coli have determined that CAMP exerted a synergistic effect on the destruction of such biofilms by meropenem and cefotaxime, and an additive effect on the efficacy of gentamicin, ciprofloxacin, chloramphenicol and tetracycline. it has also been determined that, when the biofilm formed by Pseudomonas aeruginosa and Acinetobacter baumannii was destroyed, CAMP showed a synergistic effect with meropenem, and an additive effect on Klebsiella pneumoniae (K. Pnenoni. the additive effect of this antibiotic exerted no more than the antibiotic type B of the same type of antibiotic B, and no antibiotic type of the same type of antibiotic B.
Thus, CAMP is in fact a versatile means of enhancing the anti-biofilm activity of antibiotics. The method of disrupting biofilm with the combination of CAMP and antibiotic claimed herein may be implemented in various combinations, taking into account the type of biofilm, reducing the threshold of antibiotic therapeutic dose and/or altering the method of administering it to the infected site.
Example 5
Biofilms formed by pathogenic bacteria are disrupted when exposed to CAMP from blowfly rubra and various antibiotics. CV testing
In this example, the interaction between red-headed blowfly CAMP and antibiotics was studied using another technique for analyzing anti-biofilm activity, crystal violet staining of biofilms (CV test). The analysis technique corresponds to one of the previously described techniques [18]]. Unlike the TTC test, which evaluates effects according to the level of reduction in the metabolic activity of cells, the CV test allows the degree of biofilm disruption (thickness) to be evaluated according to the amount of the staining agent bound to the biofilm. Minimum inhibitory concentrations of CAMP, antibiotics, or various combinations thereof were used as efficiency standards, versus control (BIC)90) In contrast, this concentration reduced the amount of bound CV by 90%. Biofilms incubated for 24h in 96 well plates were washed 3 times with 200 μ l sterile PBS solution and air dried. Sterile series were prepared by two dilutions of CAMP and antibiotic in PBS, 100 μ l of each concentration was added to each well, respectively, and the plates were incubated at 37 ℃ for 24 h. Then, the medium residues were removed and the wells were washed 3 times with 200 μ l PBS, dried in air and stained with 0.1% CV in water (Lenreaktiv, russia) within 2 min. The stained biofilm was washed 3 times with 200. mu.l PBS, dried in air, and then the stain was diluted with 200. mu.l 95% ethanol over 1 h. The optical density of the stain solution bound by the biofilm was then measured at a wavelength of 570nm on an instrument Epoch reader (BioTek). Two independent replicates were made for each measurement.
The data obtained are summarized in table 6. As in the TTC test (table 5), the combination with CAMP enhanced the antibiotic activity against biofilms of all the strains studied. Thus, synergy was enhanced in 8 cases and additive effects were increased in 2 cases. The evaluation of the interaction types revealed in the TTC and CV tests was consistent in all test examples. Thus, the data from the CV test confirm the conclusion based on the TTC test results that CAMP is a versatile means of enhancing the anti-biofilm activity of antibiotics. Comparison of the results of the TTC and CV tests also shows that the method of disrupting biofilm as claimed herein provides for both removal of bacterial cells and disruption of biofilm components including the matrix. The last fact demonstrates another important advantage of the proposed method-the possibility of accelerating the removal of bacterial metabolites and thus reducing the inflammation and allergic reactions that accompany biofilm infections.
Example 6
Preventing biofilm growth when planktonic forms of bacteria come into contact with the red-headed blowfly CAMP and various antibiotics
Biofilms are formed by independently living (planktonic) bacterial cells present on the surface of biological or non-biological objects. The removal of planktonic cells in infected lesions is the most reliable method to prevent biofilm formation. Currently, antibiotics and antimicrobials are used for this purpose. The experimental purpose discussed in this example was to determine the bactericidal effect of AMPV from red-headed blowfly administered in combination with antibiotics and antimicrobials on planktonic cells of pathogenic bacteria. In these experiments, strains of Escherichia coli ATCC25922, Staphylococcus aureus 203, Pseudomonas aeruginosa ATCC 27583, Klebsiella pneumoniae 145, Acinetobacter baumannii 28, all of which have the ability to form biofilms, were used [18]]. A planktonic culture was produced by incubating cells overnight in liquid LB broth (Invitrogen) at 37 ℃. The interaction between CAMP of blowfly rubropus and antibiotic combinations in planktonic cultures was studied using cross-titration techniques. For this purpose, the combination of red-headed blowfly CAMP and antibiotics was prepared in 50 μ l of liquid nutrient solution in a 96-well microplate, two-fold dilutions of the sample were placed in the horizontal row of wells, and two-fold dilutions of the antibiotics were then placed in the vertical row of wells. In addition, 50. mu.l of a bacterial suspension with a cell concentration of 106CFU/ml was introduced into each well together with the sample, and the plates were incubated at 37 ℃ for 24 h. By subjecting it To Tetrazolium Chloride (TTC)Staining to assess cell growth. For this purpose, 11 μ l of a 0.2% TTC solution was added to all wells of the plate. After incubation at 37 ℃ for 1h, OD was measured using a reader of a microplate reader epoch (BioTek)540. OD of 48h suspension culture without having undergone antibacterial compound540The values served as controls. All experiments were performed in duplicate. Minimal Inhibitory Concentration (MIC) evaluation of the Plankton cultures as MIC90I.e., the concentration of the sample that inhibits 90% of cell viability.
The results are summarized in table 7. Experiments with staphylococcus aureus have shown that the combination with CAMP has a significant synergistic effect on the antibiotic activity of chloramphenicol and tetracycline. CAMP has additive effects on amikacin, kanamycin, ampicillin, meropenem, vancomycin, erythromycin, clindamycin and benzalkonium chloride bactericide. Thus, CAMP potentiates the effects of almost all studied antibiotics and antimicrobials in planktonic cultures of staphylococcus aureus. In experiments with e.coli it has been determined that CAMP has a synergistic effect on the activity of polymyxin and cefotaxime and an additive effect on the efficacy of ciprofloxacin, chloramphenicol and tetracycline. It has also been determined that CAMP exhibits a synergistic effect with meropenem in inhibiting the growth of planktonic cells of acinetobacter baumannii, as well as an additive effect on staphylococcus aureus, pseudomonas aeruginosa and klebsiella pneumoniae.
Thus, CAMP significantly enhanced the bactericidal effect of all studied antibiotics on bacterial planktonic cells.
As shown by the results of the tests carried out in real time mode and under real conditions, the method claimed in the present application allows to prevent the growth of biofilms early in the infection process before the formation of mature biofilms, which, as mentioned above, is actually of great scientific and practical significance for the prevention, treatment of various diseases.
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TABLE 1 Effect of Red-headed blowfly (C.vicina) antibacterial peptide Complexes (CAMP) on the anti-biofilm Activity of antibiotics
AG-aminoglycosides, AM-aminoalcohols, BL- β lactams, FQ-fluoroquinolones, GP-glycopeptides, LA-lincosamides, ML-macrolides, PM-polymyxins, TC-tetracycline, BC-benzalkonium chloride
TABLE 2 antibacterial Activity of preparations obtained from different insect species of the Diptera order
Producer species
|
MIC, mg/L (mean. + -. mean error)
|
Red head blowfly (C.vicina)
|
250±0.0
|
Black cheek blowfly (C.vomitooria)
|
420±80
|
Lucilia sericata (L.sericata)
|
420±80
|
Housefly (M.domestica)
|
2000±0 |
TABLE 3 comparative Activity of antibacterial complexes of Red head blowfly and Hermetia illucens
TABLE 4 Structure of antibacterial peptide contained in Red head blowfly
Table 5. effect of red-headed blowfly CAMP preparation on the anti-biofilm activity of antibiotics. TTC test
MBIC of the studied CAMP Red head blowfly and corresponding antibiotic combinations90And minimum value of FICI
TABLE 6 Effect of formulations containing the Drosophila erythropolis antibacterial peptide complex on the anti-biofilm activity of various types of antibiotics. Crystal violet test
Minimum biofilm clearance concentration
MBIC of combinations of CAMP Red head blowfly and corresponding antibiotics90And minimum value of FICI
Amplification factor (MBEC of antibiotics)90Antibiotic MBIC90+ CAMP)
TABLE 7 Effect of Red head blowfly CAMP on the activity of different classes of antibiotics for planktonic cells. TTC test
MBIC of the studied CAMP Red head blowfly and corresponding antibiotic combinations90And the minimum value of the FICI.
Sequence listing
<110> S.I.Cherlichi
<120> method for disrupting bacterial biofilm and preventing bacterial biofilm formation using insect antimicrobial peptide complex
<130>TPD00855A
<141>2020-02-06
<150>2017120258
<151>2017-06-08
<160>4
<210>1
<211>40
<212>PRT
<213> Calliphora vicina (Red head blowfly)
<220>
<223> Defensin (Defensin)
<400>1
Ala Thr Cys Asp Leu Leu Ser Gly Thr Gly Ala Asn His Ser Ala Cys
1 5 10 15
Ala Ala His Cys Leu Leu Arg Gly Asn Arg Gly Gly Tyr Cys Asn Gly
20 25 30
Lys Ala Val Cys Val Cys Arg Asn
35 40
<210>3
<211>39
<212>PRT
<213> Calliphora vicina (Red head blowfly)
<220>
<223> Cecropin (Cecropin)
<400>2
Gly Trp Leu Lys Lys Ile Gly Lys Lys Ile Glu Arg Val Gly Gln His
1 5 10 15
Thr Arg Asp Ala Thr Ile Gln Gly Leu Ala Val Ala Gln Gln Ala Ala
20 25 30
Asn Val Ala Ala Thr Ala Arg
35
<210>3
<211>63
<212>PRT
<213> Calliphora vicina (Red head blowfly)
<400>3
Met Asn Phe His Lys Val Phe Ile Phe Val Ala Leu Ile Leu Ala Val
1 5 10 15
Phe Ala Gly Gln Ser Gln Ala Gly Trp Leu Lys Lys Ile Gly Lys Lys
20 25 30
Ile Glu Arg Val Gly Gln His Thr Arg Asp Ala Thr Ile Gln Gly Leu
35 40 45
Ala Val Ala Gln Gln Ala Ala Asn Val Ala Ala Thr Ala Arg Gly
50 55 60
<210>4
<211>40
<212>PRT
<213> Calliphora vicina (Red head blowfly)
<220>
<223> Diptericin, N-terminal sequence (Dipteracidin, N-terminal sequence)
<400>4
Asp Ser Lys Pro Leu Asn Leu Val Leu Pro Lys Glu Glu Pro Pro Asn
1 5 10 15
Asn Pro Gln Thr Tyr Gly Gly Gly Gly Gly Ser Arg Lys Asp Asp Phe
20 25 30
Asp Val Val Leu Gln Gly Ala Gln...
35 40
<210>5
<211>111
<212>PRT
<213> Calliphora vicina (Red head blowfly)
<220>
<223> Diptericin (Dipterazosin)
<400>5
Met Lys Phe Val Tyr Leu Leu Ala Ile Ser Ala Leu Cys Met Ala Ala
1 5 10 15
Met Val Lys Ala Gln Asn Lys Pro Phe Lys Leu Thr Leu Pro Lys Glu
20 25 30
Glu Pro Lys Asn Leu Pro Gln Leu Tyr Gly Gly Gly Gly Gly Ser Arg
35 40 45
Lys Gln Gly Phe Asp Val Ser Leu Gly Ala Gln Gln Lys Val Trp Glu
50 55 60
Ser Gln Asn Lys Arg His Ser Val Asp Val Asn Ala Gly Tyr Ser Gln
65 70 75 80
His Leu Gly Gly Pro Tyr Gly Asn Ser Arg Pro Ala Tyr Asn Gly Gly
85 90 95
Val Gly Tyr Thr Tyr Lys Leu Val Asn Asp Cys Thr Ile Ser Gly
100 105 110
<210>6
<211>42
<212>PRT
<213> Calliphora vicina (Red head blowfly)
<220>
<223> Diptericin (Dipterazosin)
<400>6
Asp Ser Lys Pro Leu Asn Leu Val Leu Pro Lys Glu Glu Pro Lys Asn
1 5 10 15
Leu Pro Gln Leu Tyr Gly Gly Gly Gly Gly Ser Arg Lys Asp Gly Phe
20 25 30
Asp Val Ser Leu Gly Ala Gln Gln Arg Val
35 40
<210>7
<211>69
<212>PRT
<213> Calliphora vicina (Red head blowfly)
<220>
<223> Diptericin (Dipterazosin)
<400>7
Asn Leu Pro Gln Leu Tyr Gly Gly Gly Gly Gly Ser Arg Lys Asp Gly
1 5 10 15
Phe Asp Val Ser Leu Gly Ala Gln Gln Lys Val Trp Glu Ser Gln Asn
20 25 30
Lys Arg His Ser Val Asp Val Asn Ala Gly Tyr Ala Gln His Leu Ser
35 40 45
Gly Pro Tyr Gly Asn Ser Arg Pro Ala Tyr Ser Gly Gly Ala Ser Tyr
50 55 60
Thr Tyr Arg Phe Gly
65
<210>8
<211>70
<212>PRT
<213> Calliphora vicina (Red head blowfly)
<220>
<223> Diptericin (Dipterazosin)
<400>8
Met Asn Ser Phe Ile Phe Gly Asn Leu Cys Phe Ser Val Ala Ala Leu
1 5 10 15
Ala Lys Ala Asp Ser LysPro Leu Asn Leu Val Leu Pro Lys Glu Glu
20 25 30
Pro Lys Asn Leu Pro Gln Leu Tyr Gly Gly Gly Gly Gly Ser Arg Lys
35 40 45
Asp Gly Phe Asp Val Asn Leu Gly Ala Gln Gln Arg Val Trp Glu Ser
50 55 60
Glu Thr Asn Val Ile Gln
65 70
<210>9
<211>22
<212>PRT
<213> Calliphora vicina (Red head blowfly)
<220>
<223> Proline-rich peptide, N-terminal sequence
<400>9
Phe Val Asp Arg Asn Arg Ile Pro Arg Ser Asn Asn Gly Pro Lys Ile
1 5 10 15
Pro Ile Ile Ser Asn Pro...
20
<210>10
<211>56
<212>PRT
<213> Calliphora vicina (Red head blowfly)
<220>
<223> Proline-rich peptide
<400>10
Met Cys Gly Lys Lys Phe Phe Phe Phe Val Leu Met Ala Leu Met Ala
1 5 10 15
Leu Thr Thr Gln Leu Ala Ser Ala Ser Pro Phe Val Asp Arg Ser Arg
20 25 30
Arg Pro Asn Ser Asn Asn Gly Pro Lys Ile Pro Ile Ile Ser Asn Pro
35 40 45
Pro Phe Asn Pro Asn Ala Arg Pro
50 55
<210>11
<211>38
<212>PRT
<213> Calliphora vicina (Red head blowfly)
<220>
<223> Proline-rich peptide
<400>11
Ser Arg Asp Ala Arg Pro Val Gln Pro Arg Phe Asn Pro Pro Pro Pro
1 5 10 15
Lys Arg Glu Arg Pro Ile Ile Tyr Asp Ala Pro Ile Arg Arg Pro Gly
20 25 30
Pro Lys Thr Met Tyr Ala
35