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Документ Synergetic antimicrobial activity of a mixture of essential oils and Acinetobacter calcoaceticus IMV B-7241 surfactants synthesized in the presence of the eukaryotic inducer(2023) Pirog, Tatiana; Kliuchka, Igor; Kliuchka (Nykytyuk), LiliaThe research was devoted to study the synergetic antimicrobial effect and role in the destruction of biofilms under the action of a mixture of tea tree or cinnamon essential oil with surfactant Acinetobacter calcoaceticus IMV B-7241, synthesized in the presence of a yeast inducerДокумент Synergism of antimicrobial activity of antibiotics with biocides of natural origin(2024) Pirog, Tatiana; Kliuchka, IgorCurrently, antibiotic therapy remains the primary method for treating infectious diseases in humans. Nevertheless, its effectiveness is rapidly decreasing due to the widespread emergence of resistant pathogens, necessitating the exploration of new treatment options. One potential approach involves the use of antibiotics in combination with other natural compounds. The aim of the review was to summarize the literature data on the synergy of the antimicrobial action of combinations of antibiotics with various biocides against Gram-positive and Gram-negative pathogenic microorganisms. The analysis of literature data has shown that promising compounds for use in combinations with antibiotics include essential oils, other plant components, antimicrobial peptides (both natural and synthetic), and microbial surfactants. In the majority of studies, the researchers calculated the fractional inhibitory concentration index, confirming the synergistic antimicrobial activity of antibiotics and the mentioned compounds. The use of natural biocides in combination with commercial antibiotics, particularly against Gram-negative (including methicillin-resistant) Staphylococcus species and Gram-positive microorganisms (Escherichia coli, Pseudomonas aureginosa, Klebsiella pneumoniae, Proteus mirabilis, Acinetobacter baumannii), enabled to consider these mixtures not only as effective antimicrobial agents but as one of the ways to reduce the effective concentration of antibiotics as well. It should be noted that in the presented studies, the researchers only observed the synergy of antimicrobial activity between a combination of antibiotics and other biocides, without emphasizing the potential mechanisms of interaction between the components of the complex. This likely depended on various factors, including the qualitative composition of natural compounds. Therefore, it was important to continue research not only on the synergy of antimicrobial activity in compound mixtures but also on the underlying mechanisms of their interaction. This would provide insights to enhance their effectiveness in combating resistant microorganismsДокумент Influence of biological inductors on the synthesis and biological activity of microbial metabolites(2023) Pirog, Tatiana; Ivanov, MykytaThe increasing antibiotic resistance is a severe concern for humanity. Co-cultivation of microorganisms is a promising method for obtaining new secondary antimicrobial metabolites. An effective strategy for co-cultivation of microorganisms involves the usage of certain biological inductors. The aim of this review is to summarize existing scientific research in the literature related to the influence of physiologically different types of biological inductors on the synthesis and biological activity of microbial secondary metabolites. An analysis of the literature has shown that in such studies, either live or inactivated cells of the inductor are added to the culture medium at significantly lower concentrations compared to the producer cells of the final metabolites, or the supernatant (filtrate) after cultivation of a competitive microorganism is used as an inductor. According to the literature and our own experimental studies, the using inductors is an effective approach not only for intensifying the synthesis of bacteriocins, surfactants, and antibiotics, but also for increasing their biological activity. Additionally, it often leads to the production of novel antimicrobial compounds that are not typical for the producer. However, the mechanisms of effect of inductors on the synthesis of biologically active secondary metabolites require further research, as the literature suggests that their introduction into the cultivation medium of producer does not always lead to an intensification of the synthesis of the final product. Moreover, the biological activity of secondary metabolites depends on the cultivation conditions of the producer, including the presence of biological inductors in the culture medium. Therefore, it is essential to conduct further research on the interaction between producers and competitive microorganisms to regulate the biological activity of the synthesised metabolites. In addition, there is a necessity to search for more cost-effective substrates for the biosynthesis of secondary metabolites, optimize the composition of the culture medium and expand the range of both pro- and eukaryotic inductorsДокумент Ethapolan synthesis by Acinetobacter sp. IMV B-7005 on the mixture of С2-С6-substrates and waste sunflower oil(2021) Voronenko, Andryi; Pirog, TatianaThe cultivation conditions of Acinetobacter sp. IMV B-7005, which would provide maximum synthesis of the microbial exopolysaccharide (EPS) ethapolan on the mixture of C2-C6–substrates (molasses, acetate, or ethanol) and waste sunflower oil was studied.Документ Industrial wastes as substrates for synthesis of surfactants with antiadhesive activity by Rhodococcus erythropolis IMV Ac-5017(2022) Pirog, Tatiana; Kliuchka, Igor; Kliuchka (Nykytyuk), LiliaMicrobial surfactants can affect the degree of microorganism’s adhesion to different surfaces and lead to biodegradation of the already formed biofilms. They are also non-toxic to the environment. These properties allow to use the microbial surfactants in medicine and food industry.Документ Biological activity of Acinetobacter calcoaceticus IMV B-7241 surfactants synthesized in the presence of competitive bacteria Bacillus subtilis BT-2(2023) Pirog, Tatiana; Ivanov, Mykyta; Shevchuk, TetianaCurrently, the effectiveness of technologies of microbial surfactant, which are characterized by a complex of practically valuable physicochemical and biological properties, is lower than that of synthetic analogues. To reduce the cost of these products of microbial synthesis, industrial waste is used as substrates for their biosynthesis. In previous studies, it was established that surfactants synthesized by Acinetobacter calcoaceticus IMV B-7241 on crude glycerol had lower antimicrobial activity compared to those obtained on purified glycerol. The main approaches to the regulation of the biological activity of microbial surfactants are their post-fermentation chemical modification, as well as the improvement of producer strains by methods of metabolic and genetic engineering. In recent years, the great amount of studies have appeared on the co-cultivation of producers of antimicrobial compounds with competitive microorganisms (biological inductors), in response to the presence of which the antimicrobial activity of the final product increases. Aim. To study the effect of live and inactivated cells of Bacillus subtilis BT-2, as well as the corresponding supernatant, on the antimicrobial, anti-adhesive activity and the ability to destroy biofilms of A. calcoaceticus IМV B-7241 surfactants, synthesized in a medium with glycerol of different degrees of purification. Methods. The IMV B-7241 strain was grown in the liquid mineral medium with purified and crude glycerol, into which live and inactivated B. subtilis BT-2 cells, as well as the supernatant after growing the BT-2 strain (2.5−10%, v/v) were added. Surfactants were extracted from the supernatant of the culture liquid with Folch's mixture. Anti-adhesive activity and the degree of destruction of biofilms were determined by the spectrophotometric method, antimicrobial activity − by the indicator of the minimum inhibitory concentration. The activity of enzymes of surface-active aminolipids biosynthesis (NADP+-dependent glutamate dehydrogenase) and glycolipids (phosphoenolpyruvate (PEP)-carboxylase, PEP-synthetase, PEP-carboxykinase, trehalose-phosphate synthase) was analyzed in cell-free extracts obtained after сells sonication. Results. It was established that the introduction of inactivated B. subtilis BT-2 cells and supernatant into the medium with both substrates did not affect the indicators of the surfactants synthesis, while in the presence of live cells of the BT-2 strain in the medium with purified glycerol, a decrease in the concentration of the final product by 1.5 times, and in the culture medium with crude glycerol - an increase of 1.4 times were observed compared to the indicators without the inductor. The study of the antimicrobial activity of surfactants showed that the most effective of the used inductors (live, inactivated cells, supernatant) were live cells of B. subtilis BT-2. The introduction of BT-2 strain live cells into the culture medium with both substrates was accompanied by the formation of surfactants, the minimum inhibitory concentrations of which in relation to bacterial (Bacillus subtilis BT-2, Staphylococcus aureus BMS-1, Proteus vulgaris PA-12, Enterobacter cloacae С-8 ) and yeast (Candida albicans D-6, Candida tropicalis PE-2) test-cultures were 3-23 times lower than established for those synthesized on the medium without this inductor. Anti-adhesive activity of surfactants obtained on purified and crude glycerol in the presence of all types of inductors was higher than those synthesized in the culture medium without inductors (cells adhesion of bacterial and yeast test-cultures on polyvinyl chloride was 13−70 and 33−96%, respectively). Introduction into A. calcoaceticus IMV B-7241 medium cultivation of both live and inactivated B. subtilis BT-2 cells, as well as the supernatant, was accompanied by the synthesis of surfactants in the presence of which the disruption of bacterial biofilms was on average 10-20 % higher compared to using surfactants synthesized without an inductor. In the presence of B. subtilis BT-2 in the medium, in the cells of the IMV B-7241 strain the activity of NADP+-dependent glutamate dehydrogenase (a key enzyme of aminolipids biosynthesis) increased by 1.5-2 times, while the activity of glycolipids enzymes biosynthesis remained practically at the same level as without an inductor. Such data indicated that the higher biological activity of surfactants obtained by A. calcoaceticus IMV B-7241 in the presence of biological inductors might be due to an increase in the content of aminolipids in their composition. Conclusions. As a result of research, it was established the possibility of regulating the antimicrobial and anti-adhesive activity, as well as the ability to disrupt biofilms of A. calcoaceticus IМV B-7241 surfactants by introducing into the culture medium of competitive bacteria B. subtilis BT-2. It is important that under such cultivation conditions the antimicrobial activity of surfactants synthesized on toxic crude glycerol significantly increased.Документ Synergistic effect of surfactants of Nocardia vaccinii IMV B-7405 and essential oils on Candida genus yeast(2022) Pirog, Tatiana; Kliuchka (Nykytyuk), Lilia; Shevchuk, Tetiana; Muchnyk, FainaThe increase in the number of resistant strains of Candida genus representatives, capable of forming biofi lms on various surfaces, stimulates the search for new, alternative methods of combating them, one of which is the use of compounds of natural origin, such as essential oils. However, at the same time, their concentration should be minimal, which is due to the ability of essential oils to cause severe damage of the human’s central nervous system and aspiration pneumonia. This leads to the necessity of searching for new methods to reduce the concentration of essential oils and at the same time to preserve their properties, in particular, by their use in a mixture with other antimicrobial agents, which can be microbial surfactants. Previously, it was found that the degree of yeast biofi lm destruction under the action of Nocardia vaccinii IMV B-7405 surfactants dependson the nature of the growth substrate and is the highest in the presence of preparations synthesized on purified glycerol. Aim. To study the synergism of antifungal activity and the role in the destruction of biofilms of a mixture of Nocardia vaccinii IMV B-7405 surfactants synthesized on glycerol of different quality and essential oils. Methods. N. vaccinii IMV B-7405 was grown in a medium containing purified glycerol or waste from biodiesel production at a concentration of 2% (v/v) as carbon sources. The surfactants were extracted from the supernatant of cultural liquid by a modified Folch mixture. The antimicrobial activity of essential oils, surfactants, and their mixtures was determined by the index of the minimum inhibitory concentration. To assess the synergistic effect of a mixture of surfactants with essential oils, the fractional inhibitory concentration index was used. The degree of biofi lm destruction (%) was determined as the difference between the cell adhesion in untreated and treated with surfactants, essential oil, or their mixture wells of the polystyrene microplates. Results. It was found that the surfactants synthesized by N. vaccinii IMV B-7405 on both purifi ed glycerol and waste from biodiesel production showed synergistic antifungal activity in mixtures with cinnamon and lemongrass essential oils. Thus, the minimum inhibitory concentrations against Candida albicans D-6, Candida utilis BVS-65, and Candida tropicalis RE-2 of a mixture of surfactants synthesized on purified glycerol with cinnamon and lemongrass essential oils were 1.8—7.5 and 3.7— 15 μg/mL, respectively, and were lower than in the case of using surfactants (30-60 μg/mL), cinnamon or lemongrass essential oil (156—312 μg/mL) alone. The use of a mixture of surfactants obtained on waste from biodiesel production and cinnamon or lemongrass essential oils made it possible to reduce the minimum inhibitory concentrations of the latter against studied yeast test cultures by 14—56 times. At the same time, the index of fractional inhibitory concentration did not exceed 0.5, which indicates the synergism of the antifungal activity of the mixture of these compounds. The destruction of Candida yeast biofi lms under the action of surfactants synthesized on both purified glycerol and waste from biodiesel production in a mixture with cinnamon or lemongrass essential oils reached 60—67 and 67—77%, respectively, which is an average of 25—35% higher compared to the use of each monopreparation separately. Conclusions. The results presented in this paper confirm the previously obtained data that N. vaccinii IMV B-7405 surfactants, synthesized on both traditional substrates and toxic industrial wastes, have antimicrobial and antiadhesive synergistic action with essential oils, which allows us to consider them as potential components of the so-called «antifungal locks» in the fight against of Candida genus representatives.Документ Ways of auxin biosynthesis in microorganisms(2022) Pirog, Tatiana; Piatetska, Daria; Klymenko, Natalia; Iutynska, GalynaAmong plant hormones, auxins, in particular indole-3-acetic acid (IAA), are the most studied and researched. Almost all groups of soil microorganisms, both plant-associated and non-plant-associated bacteria, fungi, and phytopathogenic microorganisms are capable of producing auxins. Th e development of preparations for crop production is directly related to the production of bacterial strains with high auxin-synthesizing potential, which is possible only with a full understanding of the ways of regulation and synthesis of auxins in bacteria. Th e synthesis of auxins in microorganisms can take place in two ways: by the gradual conversion of tryptophan to IAA (tryptophan-dependent pathway) or by the use of other intermediates (tryptophan-independent pathway). Th e latter is poorly clarifi ed, and in the literature available today, there is only a small amount of information on the functioning of this pathway in microorganisms. Th e review presents literature data on the ways of auxin biosynthesis in diff erent groups of microorganisms, as well as approaches to the intensifi cation of indole-3-acetic acid synthesis. Th e formation of IAA from tryptophan can be carried out in the following ways: through indole-3-pyruvate, through indole-3-acetamide, and through indole-3-acetonitrile. Th e vast majority of available publications are related to the assimilation of tryptophan through the formation of indole-3-pyruvate as this pathway is the most common among microorganisms. Th us, it functions in rhizospheric, symbiotic, endophytic, and free-living bacteria. Th e concentration of synthesized IAA among natural strains is in the range from 260 to 1130 μg/mL. Microorganisms in which the indole-3-acetamide pathway functions are characterized by lower auxin-synthesizing ability compared to those that assimilate tryptophan through indole-3-pyruvate. Th ese include bacteria of the genera Streptomyces, Pseudomonas, and Bradyrhizobium and fungi of the genus Fusarium. The level of synthesis of IAA in such microorganisms is from 1.17•10−4 to 255.6 μg/mL. To date, only two strains that assimilate tryptophan via the indole-3-acetonitrile pathway and form up to 31.5 μg/mL IAA have been described in the available literature. To intensify the synthesis of indole-3-acetic acid, researchers use two main approaches: the fi rst consists in introducing into the culture medium of exogenous precursors of biosynthesis (usually tryptophan, less oft en indole-3-pyruvate, indole-3-acetamide, and indole-3-acetonitrile); the second — in increasing the expression of the corresponding genes and creating recombinant strains-supersynthetics of IAA. Th e largest number of publications is devoted to increasing the synthesis of IAA in the presence of biosynthesis precursors. Depending on the type of bacteria, the composition of the nutrient medium, and the amount of exogenously introduced precursor, the synthesis of the fi nal product was increased by 1.2—27 times compared to that before the intensifi cation. Th us, in the presence of 11 g/L tryptophan, Enterobacter sp. DMKU-RP206 synthesized 5.56 g/L, while in a medium without the precursor, it yielded only 0.45 g/L IAA. Recombinant strains Corynebacterium glutamicum ATCC 13032 and Escherichia coli MG165 formed 7.1 and 7.3 g/L IAA, respectively, when tryptophan (10 g/L) was added to the culture medium. Th e level of auxin synthesis in microorganisms may be increased under stress conditions (temperature, pH, biotic and abiotic stress factors), but in this case, the IAA concentration does not exceed 100 mg/L, and therefore this method of intensifi cation cannot compete with the others above.Документ Effect on phytopathogenic microorganisms of surfactants of microbial origin(2021) Pirog, Tatiana; Piatetska, Daria; Yarova, Hanna; Iutynska, GalynaBiodegradable non-toxic surfactants of microbial origin are multifunctional preparations, which due to antimicrobial activity are promising for use in crop production to control phytopathogenic microorganisms. Studies on the prospects of using microbial surfactants to control the number of phytopathogenic microorganisms are conducted in three directions: laboratory studies of antimicrobial activity of surfactants in vitro, determination of the effect of surfactants on phytopathogens in vegetative experiments in the process of plants growing in a laboratory or greenhouse, post-harvest treatment of fruits and vegetables with solutions of microbial surfactants to extend their shelf life. The review presents literature data on antimicrobial activity of surfactants against phytopathogenic bacteria and fungi in vitro. Antimicrobial activity of surfactants is evaluated by three main parameters: minimum inhibitory concentration, zones of growth retardation of test cultures on agar media and inhibition of growth of test cultures on agar or liquid media. The vast majority of available publications relate to the antifungal activity of surfactant lipopeptides and rhamnolipids, while data on the effect of these microbial surfactants on phytopathogenic bacteria (representatives of the genera Ralstonia, Xanthomonas, Pseudomonas, Agrobacterium, Pectobacterium) are few. The researchers determined the antimicrobial activity of either total lipopeptides extracted with organic solvents from the culture broth supernatant, or individual lipopeptides (iturin, surfactin, fengycin, etc.) isolated from a complex of surfactants, or culture broth supernatant. Lipopeptides synthesized by members of the genus Bacillus exhibit antimicrobial activity on phytopathogenic fungi of the genera Alternaria, Verticillium, Aspergillus, Aureobasidium, Botrytis, Rhizoctonia, Fusarium, Penicillium, Phytophora, Sclerotinia, Curvularia, Colletotrichum, etc. in sufficiently high concentrations. Thus, the minimum inhibitory concentrations of lipopeptides against phytopathogenic fungi are orders of magnitude higher (in average 0.04–8.0 mg/mL, or 40–8000 μg/mL) than against phytopathogenic bacteria (3–75 μg/mL). However, the antifungal activity of lipopeptidecontaining supernatants is not inferior by the efficiency to the activity of lipopeptides isolated from them, and therefore, to control the number of phytopathogenic fungi in crop production, the use of lipopeptidecontaining supernatants is more appropriate. Rhamnolipids synthesized by bacteria of the genus Pseudomonas are more effective antimicrobial agents comparing to lipopeptides: the minimum inhibitory concentrations of rhamnolipids against phytopathogenic fungi are 4–276 μg/mL, which is an order of magnitude lower than lipopeptides. In contrast to the data on the antifungal activity of rhamnolipids against phytopathogens, there are only a few reports in the literature on the effect of these surfactants on phytopathogenic bacteria, whilst the minimal inhibitory concentrations are quite high (up to 5000 μg/mL). The advantage of rhamnolipids as antimicrobial agents compared to lipopeptides is the high level of synthesis on cheap and available in large quantities industrial waste. Currently in the literature there is little information about the effect of surface-active sophorolipids of microbial origin on phytopathogenic fungi, and all these works are mainly about the antifungal activity of sophorolipids. We note that in contrast to surfactant lipopeptides and rhamnolipids, the effective concentration of most sophorolipids, which provides the highest antimicrobial activity against phytopathogens, is higher and reaches 10,000 μg/mL.Документ Destruction of biofilms on silicone tubes under the action of a mixture of Nocardia vaccinii IMV B-7405 surfactants with other biocides(2021) Pirog, Tatiana; Kliuchka (Nykytyuk), Lilia; Shevchuk, Tetiana; Iutynska, GalynaThe formation of pathogenic microorganisms biofilms on the central venous catheter is the cause of catheter-associated infections. An alternative method of combating biofilms is the use of “antibacterial” and “antifungal” locks, which are solutions of antibiotics or antifungal drugs in a mixture with other natural compounds, which can be microbial surface-active substances (surfactants) or essential oils. Aim. To investigate the role of Nocardia vaccinii IMV B-7405 surfactants mixture with other antimicrobial compounds in the destruction of biofilm on silicone tubes. Methods. N. vaccinii IMV B-7405 was grown in medium containing as carbon source purified glycerol and waste from biodiesel production, refined sunflower oil, oil after frying French-fried potatoes, Potato wedges and meat. The surfactants were extracted from supernatant of cultural liquid by modified Folch mixture. 2 mL of surfactant solutions, antifungal drugs (nystatin, fluconazole) or tea tree essential oil of the same concentration (5–640 μg/mL) were added to test tubes with silicone tubes (with pre-formed biofilm from test culture). To study the synergistic effect of the biofilms destruction, a mixture of surfactant solutions and antifungal substances (or essential oil) of the same concentration in a ratio of 1:1 (1 mL of each solution) was added to the test tubes. Sterile tap water (2 mL) was added to control test tubes instead of surfactants preparations, antifungal substances or essential oil. The degree of biofilm destruction (%) was determined as the difference between the adhesion of cells on the inner side of silicone tubes, untreated and treated with surfactants, antifungal drugs, essential oil, or their mixture. Results. It was found that surfactants synthesized by N. vaccinii IMV B-7405 on all substrates showed synergism of yeast and bacterial biofilms destruction on silicone tubes in a mixture with nystatin, fluconazole and tea essential oil in the whole range of investigated concentrations (5–640 μg/mL), but the highest effect was achieved at a concentration of 20–40 μg/mL. Thus, the degree of Candida albicans D-6, Candida utilis BVS-65 and Candida tropicalis PE-2 biofilms destruction under the action of a mixture of surfactants synthesized on waste from the biodiesel production and waste oil, with antifungal drugs was 45.8–71.8 % and was higher than with only surfactants (21.2–41.6 %), nystatin (22.4–24.1 %) or fluconazole (28.1–31.3 %). The destruction of Candida genus yeast biofilms under the action of surfactants synthesized on oil-containing substrates in a mixture with both nystatin and fluconazole reached 50.1–71.2 %, which is 10–30 % higher compared to the use of surfactants alone or only antifungal agents. The degree of Pseudomonas sp. MI-2, Escherichia coli IEM-1, Staphylococcus aureus BMS-1, Bacillus subtilis BT-2 (spores) biofilms destruction on silicone tubes treated with a mixture of tea tree essential oil and surfactants synthesized on all oil-containing substrates was 10–29 % higher than in the case of using only solutions of surfactants (11.5–45.4 %) or essential oil (21.4–34.5 %) for the tubes treatment. Conclusions. The data obtained make it possible to consider surfactants synthesized by N. vaccinii IMV B-7405 on a wide range of cheap and accessible substrates as promising components of “antibacterial” and “antifungal” locks in combination with essential oils and antifungal agents.
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