Annotation of the results obtained in 2023
Within the framework of an interdisciplinary project, together with a scientific group led by Dr. Kayumov A.R. (KFU, Kazan), the synthesis of water-soluble conjugate forms based on meso-butane and meso-pentane BODIPY acids and myrtenol with a miramistin charged quaternary ammonium group was performed for the first time. The obtaining conjugates of a new type, the studing their spectral and biological properties, a scientific group led by Dr.Antina E.V. (ISC RAS, Ivanovo) synthesized the necessary working amount of BODIPY dyes containing butane or pentane acid residues and their esters in the meso-spacer, guided by the methods developed and tested in previous reporting periods of Project. The scientific groups carried out a cycle of joint synthetic work on the production of the myrtenol and thioterpenoid conjugates with alpha-propanoic acid of the BODIPY. For the synthesis of the corresponding conjugates, a scientific group from Ivanovo developed a working amount of the alpha-propane acid of the BODIPY. In order to develop new effective photosensitizers based on halogen-substituted BODIPY esters for antibacterial and antimycotic photodynamic therapy, a scientific group led by Dr. Antina E.V. (ISC RAS, Ivanovo) tested synthesis methods during the reporting period and obtained working quantities of 4,4'-dibromo- and 4,4'–diiodisubstituted BODIPY phosphors with ester (СН2)3СООСН3 group in the meso-spacer. Identification of all synthesized compounds for the fourth reporting period of the Project was carried out using H1, C13 NMR, mass and electron spectroscopy, as well as elemental analysis. The results of molecular docking and spectral studies being obtained during the reporting period by a scientific group led by Doctor of Chemical Sciences Antina E.V. (ISC RAS, Ivanovo) are summarized and analyzed. The process of molecular complexes formation of alpha- and meso-functionalized BODIPY and their terpene conjugates with serum albumins was established to be spontaneous and proceeds due to hydrogen and Van der Waals interactions. At the same time, alpha substitution of hydrogen atoms in the pyrrole nuclei of BODIPY by ester groups with terpenes and beta halogenation of the dipyrromethene backbone increases the affinity of phosphors to serum blood proteins. The results of spectral studies showed the introduction of carboxylic acid residues and their ester groups with myrtenol and thioterpene, including the miramistin charged quaternary ammonium group, into the alpha and meso positions of the dipyrromethene backbone of BODIPY doesn't to reduce the fluorescent characteristics of luminophores (up to φ ~96%) by comparison with the source BODIPY precursors. The introduction of propanoic acid residues into the alpha position of pyrrole nuclei and, especially, beta halogenation of luminophores significantly (up to ~50 nm) shifts the maxima of absorption bands and fluorescence to the red region compared with BODIPY meso-carboxylic acids and their esters. The most noticeable decrease in fluorescence is observed for dibromo- and diiodisubstituted BODIPY esters. The quantum yields of fluorescence of halogenated dyes are no more than ~2-32%, depending on the molecular structure and properties of the medium, which is due to the manifestation of the "heavy" atom effect. An important advantage of dibromo- and diiodisubstituted BODIPY esters is the high quantum yield of singlet oxygen generation: ΦΔ = 50-61% and 65-78%, respectively, which is of interest in the development of new theranostics based on them with the functions of PDT, APDT agents and fluorescent dyes. It is important to note that, unlike most hydrophobic BODIPY esters with alcohols, conjugates containing meso- or alpha-ester substituents, including along with the terpene radical a positively charged quaternary ammonium group, demonstrate satisfactory solubility for biological studies (up to c ~10-5 mol/l) in water and buffer solutions with pH from 1.65 to 9.18. However, dyes are chemically photostable in both acidic and alkaline aqueous media and retain high fluorescence (up to φ ~83%). The meso-substitution of the proton of the BODIPY methine bridge with residues of carboxylic acids and their esters was noted to increases the photostability of the chromophore system of dyes by almost ~2 times compared with meso-unsubstituted analogues. A marked decrease (up to ~ 2-4 times) in the photostability of dyes is observed in 2,6-dibromo- and diiodipyrromethene esters of BODIPY compared with non-halogenated analogues. An increase in the rate of photodestruction of halogenated chelates under UV irradiation may be facilitated by the effective generation of singlet oxygen by dyes due to the effect of a "heavy" atom. The introduction of a positively charged ammonium group into the meso-substituent of BODIPY conjugates was found to increased the affinity of the dye to hydrophilic media by almost ~9-14 times compared with uncharged analogues, as a result of increased in the hydration contribution to luminophore solvation. However, the combination of halogenation and meso-substitution increases the affinity of BODIPY esters to lipid biostructures by ~1.3 times and promotes the efficient transport of phosphors through the cell membrane layer compared with carboxylic acids of boron(III)dipyrromethenates. A scientific group led by Dr. Kayumov A.R. (KFU, Kazan) conducted in vitro biological studies to study the effectiveness of transport and localization of BODIPY conjugates in cellular organelles of pathogenic microorganisms. The conjugation of BODIPY fluorophore and methanol containing a miramistin fragment was found to favored the penetration of dyes into mycelial fungi Fusarium solani. However, it worsened their binding to S. aureus, K. pneumoniae and P. aeruginosa. The charged group of quaternary ammonium introduced between the myrtenol and fluorophore fragments was discovered to restored the staining of bacterial cells, but does not to affected the staining of fungi. It has been shown that alpha-functionalized BODIPY conjugates with methanol and triterpenoid, unlike structurally related carboxylic acid, are able to effectively penetrate the cytoplasm of C. albicans fungal cells. In this case, the conjugate with a triterpene residue most intensively stains the membrane structures of organelles (membranes of nuclei and mitochondria), in comparison with the myrtenol conjugate. As a result of determining the sensitivity of planktonic cells of the reference and clinical strains of yeast fungi to antifungal drugs, the alpha-substituted monoterpene conjugates of BODIPY was found to exhibiting effective antifungal activity, unlike carboxylic acid. The set of the results of theoretical (molecular docking) and experimental (in vitro) studies showed that conjugation of BODIPY with monoterpenoids is a promising way to increase the affinity of dyes to biostructures. In addition, BODIPY conjugates with monoterpenes are of interest using as fluorescent markers in the assessment of membrane structures of pathogen cells, as well as in determining effectors and modulating in vitro metabolic pathways of a fungal cell. It is relevant in studying of drug transport in vivo. The results of scientific research being obtained within the framework of the interdisciplinary Project for the fourth reporting period with the participation of two scientific groups (leader Dr. Antina E.V. (Institute of Chemical Chemistry RAS, Ivanovo) and leader Dr. Kayumov A.R. (KFU, Kazan)), are reflected in nine articles published in co-authorship and separately.

 

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Annotation of the results obtained in 2020
A retrospective microbiological analysis of biomaterial from patients with lesions of the oral mucosa was carried out. The following two main groups have been identified according to the age classification adopted by the World Health Organization (WHO): young patients 18-44 years old and middle-aged patients 45-59 years old. Additionally, each main group is divided into two subgroups: with damage to the mucous surface of the oral cavity 1) in acute and 2) chronic forms. The specific fraction of bacterial microflora in young patients was more than half of the total number of isolated microorganisms. In patients with chronic forms, the overwhelming majority was represented by gram-positive cocci (85%), 28.5% were representatives of the genus Staphylococcus and 71.4% were Streptococcus spp. Staphylococci were represented by 2 species, among which Staphylococcus aureus prevailed (26.9% of the number of gram-positive cocci and 22.9% of the total number of bacterial isolates). Among Streptococcus spp, hemolytic species were isolated most often in patients with chronic forms, 49.2%, in amounts from 10^2 to 10^3 CFU / swab. In addition, gram-negative bacteria were often found in the microbial landscape (16.2% of the total number of bacterial isolates). Among them, Klebsiella spp. (58.3% of the number of gram-negative). Appearance of E. coli was rare, only 27.3% of the total number of gram-negative bacteria and 1.3% of the total, in an amount of less than 10-100 CFU / swab. In chronic patients, fungal microflora was present in all samples from mucous membranes, where the main representative was the yeast Candida albicans (86.8% of the amount of fungal isolates). C. parapsilosis, C. tropicalis were also found. In 62% of cases, the amount of fungi was <100 CFU / swab. In patients of the first group with the acute period, the fraction of the bacterial microflora was also more than half of the number of isolated microorganisms. However, the microbial pattern was completely free of gram-negative bacteria, and the vast majority of gram-positive cocci were represented by Streptococcus spp. In addition, the fungal microflora was represented by 3 species (C. albicans, C. tropicalis, C. krusei) and consisted 27.6% of the total number of all isolates. In patients with chronic and acute forms, in general, no differences in the composition of the occurrence of isolates were found. Streptococcus spp. and Klebsiella spp were the leading gram-positive species. A frequent representative of the fungal flora was C. albicans. In vitro stable biofilms of S. aureus - E. coli, S. aureus - K. pneumonia, S. aureus and P. aeruginosa, as well as mixed fungal-bacterial biofilms of C. albicans with S. aureus, E. coli, K. pneumonia, and P. aeruginosa were obtained. Differences in the composition of mono- and two-species biofilms are shown. Thus, proteins predominate in the matrix of S. aureus and E. coli biofilms, while in the case of a mixed biofilm, α-polysaccharides increase. In the biofilm of S. aureus - K. pneumonia, the content of β-polysaccharides is almost 2 times higher than in the monoculture biofilm of K. pneumonia. The matrix of P. aeruginosa biofilm is dominated by α-polysaccharides, while in the mixed S. aureus - P. aeruginosa community, the amount of β-polysaccharides increases. In the matrix of mixed fungal-bacterial biofilms of C. albicans-S. aureus and C. albicans-E. coli, both α and β-polysaccharides and proteins prevail in approximately equal amounts. However, in the biofilms of C. albicans - K. pneumonia, the content of proteins prevails over the content of α-polysaccharides, and in the biofilms of C. albicans - P. aeruginosa, on the contrary. The effect of various antibiotics on mono- and polymicrobial biofilms of S. aureus and P. aeruginosa was investigated. Analysis of the distribution of S. aureus and P. aeruginosa in the biofilm after 24-hour treatment with vancomycin showed that, in contrast to the control, where S. aureus was mainly located in the upper layers of the biofilm, under conditions of vancomycin therapy, most cells moved to the lower and middle biofilm layers. Confocal microscopy also revealed a significant redistribution of S. aureus from the upper to the lower layers of the biofilm in the presence of antimicrobial drugs. The data obtained indicate that under the conditions of antimicrobial therapy, Staphylococcus aureus changes its localization in the structure of the polymicrobial biofilm, sinking into the lower layers, and at the same time becomes insensitive to most antimicrobial drugs of a narrow spectrum of action, using the phenotypic resistance of P. aeruginosa to these antimicrobial drugs. Similar results were obtained for S. aureus and K. pneumoniae, S. aureus and E. coli dimicrobial films. Thus, in the case of mixed biofilms, the use of broad-spectrum antibiotics is required. In this case, their efficiency is 2-4 times higher in compare to monocultures of bacteria, probably due to the antagonistic interactions of S. aureus with gram-negative bacteria in the polymicrobial biofilm. The use of antibiotics, effective only against S. aureus, leads to the transfer of bacteria to the lower layers of the mixed biofilm and to an increase in the resistance of staphylococcus to therapy due to the protection of the biofilm matrix of gram-negative bacteria. The considered effect may be due to the fact that S. aureus is able to synthesize metabolites that negatively affect P. aeruginosa cells and thereby increase their sensitivity to antibiotics. Indeed, the addition of antibiotics with the culture liquid of S. aureus resulted in the death of detached cells and cells in the biofilm of P. aeruginosa and K. pneumonia even at an antibiotic concentration of 0.03 × MBC, while the addition of aminoglycosides to the nutrient medium did not affect the viability of P. aeruginosa in biofilm and in a detached state even at a concentration of antibiotics equal to their 8 × MBC. In order to determine the most promising compound for further modification, a wide range of terpenes and terpenoids was tested. The structures of all new compounds were confirmed using NMR (1D and 2D) spectroscopy and high resolution mass spectrometry. The most promising compounds in terms of their antimycotic properties were the structures of myrtenol (compound 15) and 2 - (((1S, 2S, 4S) -1,7,7-trimethylbicyclo [2.2.1] heptan-2-yl) thio) ethane -1-ol (compound 17). It should be noted that compound 15 was tested in the form of individual enantiomers, and in the biological part it has the codes "S-15 +" and "S-15-". Connection 17 was assigned the code KS-1. Salts 20 (myrtenic acid salt) and 21 (myrtanic acid salt) were synthesized. The structures of compounds 17A and 17B were studied in detail using various methods of NMR spectroscopy; for compound 17B, X-ray structural analysis was performed. In order to study the biological properties of the oxidation products of terpene sulfide 17, its corresponding sulfoxides 23а, b and sulfone 24 were obtained. The structure of the obtained compounds was proved using NMR, mass spectrometry, and X-ray diffraction methods. Terpene 15 showed one of the best activities against microorganisms, therefore its fusion-combination with a BODIPY-fluorophore was obtained. The next stage of the work was to obtain a hybrid structure 27, which is a beta-hydroxysulfide with a terpene fragment from compound 15 and a hydroxysulfide fragment from compound 17. For a better understanding of the dependence of the activity of compounds on their structure, substances 26 and 28 were also obtained, which differ from sulfide 27 with only one atom in the beta position to the hydroxyl function. Thus, it was possible to modify the same terpene core with ethylene glycol, mercaptoethanol, and monoethanolamine. By analogy with the previous compounds 15 and 17, structure 27 was modified with a fluorescent label based on fluorophore B. The antimicrobial activity of S15 and S15 + terpenes, as well as of myrtenic acid and its quaternary salt, was evaluated. Among the tested compounds, the most active was compound S15, the minimum inhibitory concentration of which in relation to planktonic cells of S. aureus was 512 μg / ml, while for other compounds the MIC was 2048 μg / ml, while the studied compounds were inactive against gram-negative bacteria and their minimum inhibitory concentrations were above the detection limit. Synergy of S15 and S15 + with antibiotics has been shown. When combining S15 and S15 +, the FICI values were 0.31 and 0.50, respectively, confirming the synergistic effect of antimicrobial drugs. In combination with another antibiotic of the aminoglycoside series, gentamicin, the FICI values were 0.75 and 0.625, respectively, while the combined use of terpene derivatives with vancomycin exhibited an additive effect and the index values varied in the range 1.125-1.5. In contrast to bacteria, the growth of C. albicans fungi was inhibited by all compounds studied at relatively low concentrations. Fusarium cells were found to be more resistant to compounds. The fractional inhibitory concentration index (FICI) of compound S15 in combination with fluconazole was 0.3744-0.4992, which indicated the presence of synergism in relation to candida cells. An additive effect has been shown for Fusarium for the combined use of S15 with fluconazole. At a fixed concentration of terpenes (128 μg / ml), the MIC of benzalkonium chloride biocide decreases 2-8 times in relation to a mixed culture of staphylococcus and candida. Using confocal laser scanning microscopy and S15-lum2, it was shown that compound S15 is evenly distributed within S. aureus cells, while in the case of gram-negative E. coli and P. aeruginosa, fluorescence is identified only on the cell surface. Probably, the compound is unable to penetrate into the cells, due to the complex structure of the membrane, which explains the low antimicrobial activity of the compounds under study against gram-negative bacteria. When S15-lum was added to S. aureus cells, the time for the half-maximum penetration of the substance into the cell, t1 / 2, was 12 ± 1.3 min, while the half penetration of S15-lum into P. aeruginosa cells required 60 ± 1.6 min. Thus, the low efficiency of the studied terpene derivatives against gram-negative bacteria is explained by the low rate of penetration of compounds into the cells. Also, using confocal microscopy, the penetration of S15 carrying the BODIPY fluorophore (S15-lum) into the cells of yeast and micellial fungi was investigated. Microscopy also showed a high rate of penetration of the compound into fungal cells, followed by accumulation in cell organelles. By assessing the mebrane potential of cells, it was shown that the mechanism of action of S15 and S15 +, as for quaternary ammonium salts, is membrane damage.

 

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Annotation of the results obtained in 2021
Analysis of the microbiome of the oral mucosa of "conditionally healthy persons" and in patients diagnosed with bronchial asthma has revealed that normally the vast majority of bacteria are represented by gram-positive cocci (96%). Amog them 51.0% were Staphylococci (S. aureus and S. epidermidis) and 49.0% Streptococci, with trace cases of the occurrence of gram-negative bacetria. In 45% of the samples, fungal microflora was present, where the main representative was the yeast Candida albicans (81.8% of the number of fungal isolates), but only in 18.2% of cases with > 10^3 CFU / swab. In patients with bronchial asthma, the leading gram-positive species were Streptococcus spp., Among gram-negative ones - Klebsiella spp. and a frequent representative of the fungal flora was C. albicans. In 81% of cases, opportunistic microorganisms were in in etiologically significant amounts > 10^4-10^5 CFU / pad. The content of C. albicans and C. tropicalis significantly correlated with an increase in the content of leukocytes, lymphocytes, monocytes, eosinophils and neutrophils. The effect of extracellular metabolites of various Staphylococcus aureus strains on E. coli mature biofilms was analyzed. The addition of liquid at a concentration of 12.5% led to a 55% decrease in the respiratory activity of E. coli cells in the biofilm. The combination of amikacin and ciprofloxacin with the culture fluid of staphylococcus increased the effect of antibiotics by 4-8 times. The screening of optimal conditions for the maximum yield of antimicrobial metabolites from the culture liquid of staphylococcus was carried out. The optimized scheme for obtaining the fraction of antimicrobial peptides from the culture liquid of Staphylococcus aureus includes a pH decrease to 3.0, removal of cells by centrifugation, ultrafiltration with MWCO 10 kDa, solid-phase extraction with stepwise elution with acetonitrile solution with preservation of the elution fraction of 20-40% acetonitrile followed by purification on a HPLC column C-18. The ability of microscopic fungi Candida albicans and Fusarium solani to form mono- and polymicrobial biofilms was studied. It has been shown that the maximum biofilm biomass is formed by clinical isolates of fungi. At the same time, in the reference strains of fungi C. albicans and F. solani in monofilms, β-polysaccharides initially predominate in the composition. In the clinical strain of C. albicans, proteins and β-polysaccharides predominated in the monofilm, while the amount of proteins was 5 times higher than that of the reference strain. The amount of protein in polymicrobial films (C. albicans + F. solani) on the first day of incubation is statistically significantly higher than in monofilms. It was shown that monospecies biofilms formed by clinical isolates of F. solani and C. albicans are characterized by high density, tightly attached to the surface of the microplate compared to the reference strain. In a mixed culture, on the 5th day, the yeast cells of C. albicans densely wrapped around the hyphal structures of F. solani. The hyphae of F. solani F - 417 in biofilm provided architectural stability and acted as a frame structure of yeast cells and pseudohyphae. At the same time, the reference cultures of C. albicans and F. solani do not form a mature polymicrobial biofilm. C. albicans in biofilms exhibited resistance to all studied groups of drugs, and the MIC for biofilms was 25-100 times higher than the MIC for planktonic microorganisms. For F. solani biofilms formed on microplates, the MIC values were 25 times higher. For polymicrobial C. albicans + F. solani biofilms, the MIC values increased more than twofold compared to the monobiofilms. A number of structural analogs of miramistin carrying fragments of various terpenes both as a hydrophilic “head” and as a lipophilic “tail” have been obtained. Among all tested compounds, only one substance had an activity comparable to the reference drug and is of interest for further research. The concentrations of compounds leading to the half-maximal suppression of respiratory activity on HEK cells were significantly lower than the MIC, probably due to the quaternary ammonium group. The synergy of myrtenol with amikacin and benzalkonium chloride against S. aureus cells was assessed using the checkerboard assay. When amikacin was combined with myrtenol (-) c, a pronounced synergism (FICI 0.3-0.5) was observed on 5 strains out of 12, with myrtenol (+) - on 9 strains, regardless of their status (MRSA or MSSA). By suppressing the formation of S. aureus biofilm, in combination of amikacin with myrtenol (-) synergism was shown on 5 strains out of 12, with myrtenol (+) - on 4 strains. When benzalkonium chloride was combined, there was no synergistic effect with myrtenol on planktonic cells of S. aureus and an additive effect was shown, the synergism of an antiseptic and myrtenol (-) was shown for 1 strain, and on 4 strains for myrtenol (+). For biofilms, synergism was shown on 6 of 12 strains. Against C. albicans, synergism between fluconazole and myrtenol (+) was shown on 7 strains out of 11. For myrtenol (-), synergism was observed in 4 cases. For biofilm suppression, synergism between fluconazole and myrtenol (-) is shown on 6 strains. In the case of myrtenol (+), synergism was observed in 4 cases out of 11. Mirtenol (-) showed synergy with benzalkonium chloride on five strains, and myrtenol (+) - on 9 strains. With regard to biofilms, most of the strains (7 out of 11) were more sensitive to the combination of an antiseptic with myrtenol (+), while the use of myrtenol (-) with benzene chloride was synergistic only on 4 strains. The ability of myrtenol to increase the effectiveness of benzalkonium chloride against the fungal-bacterial community was shown. The combination of benzalkonium chloride with myrtenol, a significant increase in the effectiveness of the antimicrobial drug (by 8-16 times) was observed, and the complete death of planctonic cells of C. albicans was observed at its concentration of 0.5 μg / ml, the death of S. aureus was observed at an antiseptic concentration of 0.5 μg / ml in combination with myrtenol (+) and 1 μg / ml in combination with myrtenol (-). With regard to the cells in the mixed biofilm, an increase was observed; the combined use of benzalkonium chloride with terpenes led to the death of only S. aureus cells. The effect of terpenes on the integrity of the bacterial membrane was evaluated. In the presence of myrtenol (-) and myrtenol (+), as in the case of benzalkonium chloride, a dose-dependent drop in the membrane potential of S. aureus cells was observed, determined by the intensity of DioC2 fluorescence in cells, which indicates membrane damage. The drop in the potential of C. albicans cells in the presence of myrtenol (-) was comparable to that of fluconazole. At the same time, the introduction of myrtenol (+) at the maximum concentration contributes to a significant decrease in the membrane potential, which indicates serious damage to the cell membrane. Probably, it is precisely because of such a strong effect on cell membranes that myrtenol (+) demonstrated a high percentage of synergism with antimicrobial drugs against C. albicans. The half-penetration time of the substance into the cell was t½ = 24 ± 1.3 min and t½ = 26 ± 1.5 min for myrtenol (-) and myrtenol (+), respectively. In the case of C. albicans cells, the half-penetration time of myrtenol (+) is 18 minutes, and that of myrtenol (-) is 24 minutes. These data are consistent with previously obtained results on the synergistic nature of the interaction of myrtenol (+) with antimicrobial drugs. Using NMR spectroscopy, the interaction between the model cell membrane and compounds 30-32 (obtained at the previous stage of work) was investigated. Thus, compounds 29 and 31 bind to the surface of DPC micelles, while compounds 30 and 32 do not bind to DPC micelles. It should be emphasized separately that in both cases these were terpenesulfides.

 

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Annotation of the results obtained in 2022
1. It has been shown that the introduction of the culture liquid of S. aureus to P. aeruginosa at a concentration of 1/16-1/4 v/v increases the effectiveness of aminoglycoside antibiotics against planktonic cells of P. aeruginosa by 2 times, and ciprofloxacin and gentamicin against cells in a biofilm - 4 times. The maximum effect was observed for amikacin, where the effectiveness of the antibiotic increased by 32 times. The molecular mechanism of this phenomenon is related with the increase in the amount of β-polysaccharides and proteins, which contributes to the formation of a porous structure of the biofilm, as a result of which the permeability for antimicrobials is significantly increased. 2. A comprehensive analysis of the microbiota of throat swabs and hematology of apparently healthy people and patients diagnosed with bronchial asthma was carried out. In patients with bronchial asthma, the indices of microbial diversity are significantly reduced and the content of bacteria of the families Micrococcaceae, Porphyromonadaceae, Prevotellaceae, Fusobacteriaceae, Neisseriaceae is different. Also in the group with bronchial asthma, an increase in clinically significant genera Streptococcus, Staphylococcus, Klebsiella, Actinomyces, Fusobacterium, Prevotella, Porhyromonas, Treponema and a decrease in Actinomyces, Fusobacterium, Neisseria, Treponema were found. Further, on the basis of partial correlations between factors (“Bronchial asthma” and in its absence, and with data from metagenomic analysis of oral microflora, biodiversity indices, microbiological culture and hematology), the main relationships between individual factors were summarized in a common graphical scheme representing the Bayesian network. A relationship has been identified between an increase in neutrophils and bacteria: Peptostreptococcaceae; Fusobacteriaceae; Prevotellaceae; Campylobacteraceae. Our data indicate that the microfungal factor Candida albicans has also been associated with asthma indirectly via the monocyte factor. The most significant relationships between microscopic fungi and bacteria were also compiled. So, between the factor-Candida parapsilosis and the bacteria Micrococcaceae, Carnobacteriaceae Veillonellaceae, there are positive relationships and negative ones with Coriobacteriacea; Porphyromonadaceae; Lactobacillaceae; Peptostreptococcaceae. Multiple indicators of the microbiome are associated with the number of leukocytes, monocytes, basophils, and neutrophils. One possibility for this observation is that the microbiome may influence disease through mechanisms mediated by systemic immune responses. Otherwise, inflammatory processes cause changes in the microbiome. 3. 10 new derivatives of terpenoids were synthesized. Compounds E2, E4 and E11 in relation to S. aureus cells had an MIC and MBC of 2 μg/ml, which exceeds the MIC and MBC of miramistin by 2 and 4 times. Compounds E1, E2 and E4 resulted in suppression of biofilm formation by S. aureus cells at 4 μg/ml. Thus, compounds E1, E2, E4 and E11 can serve as promising compounds for combating staphylococcal infections. With regard to P. aeruginosa cells, compounds E7 and E11 have an activity comparable to miramistin. Compound E8 also had an MIC up to 8 times lower than that of miramistin. Thus, compounds E7, E8 and E11 can serve as promising compounds for combating infections associated with P. aeruginosa. Compounds E1, E4, E11 had MICs of 12, 94 and 47 μg/ml against Candida albicans, Aspergillus niger, Fusarium solani, respectively, which is 4 times lower than the MIC of fluconazole. For compounds E5 and E7, the MICs were comparable to those of fluconazole. Compound E8 had a fungicidal and fungistatic effect on all types of fungi. Thus, compounds E8 and E11 are promising drugs. 4. For new terpenoids, conjugates with fluorophores were obtained and the degree of their permeability into fungal and bacterial cells and into the matrix of mono- and two-species bacterial biofilms was assessed. Compound D4 penetrated into S. aureus, but not into P. aeruginosa cells as part of biofilms. However, it quickly penetrated Candida and Fusarium cells and bound to the cytoplasmic membranes of the organelles. Substance D5 had a low ability to penetrate Candida cells and S. aureus biofilms, while being part of the S. aureus-P. aeruginosa, this compound penetrated both S. aureus and P. aeruginosa. Probably, as part of a mixed community, S. aureus becomes more susceptible to the action of this compound. D5 also penetrated into filamentous fungi by binding to the internal membranes. Compounds with reference numbers D8 and D9 had a high penetrating ability into the matrix of S. aureus biofilms, but not into cells. In the case of P. aeruginosa and S. aureus-P. aeruginosa, the D8 compound showed a similar effect, while for D9 it penetrated only into the depth of the mixed community. Substances D10, D13, and D15 had a high penetrating ability against Staphylococcus aureus cells both in mono- and two-species biofilms, while D10 and D15 did not penetrate P. aeruginosa cells. D10 strongly bound to the cell wall of Candida yeast without penetrating into the cell, and weakly bound to the cell wall of the filamentous fungus. Compounds D9 and D15 showed increased penetration compared to control compounds. Similar results were obtained for mono- and two-species biofilms of S. aureus and K. pneumoniae. 5. During this stage, a sulfur-containing terpenoid based on borneol was synthesized - bornan sulfide, designated KS1, which has both antimycotic activity and a synergistic effect with fluconazole against micromycetes. The synthesis scheme and formula have not yet been disclosed, since a patent search is currently underway and a patent application is being prepared for its method of use as an antibacterial and antimycotic drug. In vitro, KS1 was antimycotic at concentrations lower than standard antimycotics. The compound was found to be able to synergize with fluconazole, increasing drug susceptibility in resistant strains. Using the KS1 conjugate cross-linked with the BODIPY fluorophore, it was shown that KS1 is able to penetrate into the cell without binding to the cytoplasmic membrane. The results of the performed NMR spectroscopy showed that the compound KS1 interacts with DPC micelles mainly with its bornane part of the molecule. Similar experiments with mirtenol showed that mirtenol molecules, unlike bornane sulfide, "fall through" into the micelles, while bornane sulfide has largely membranotropic properties.

 

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