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Antimicrobial Potential of the Alkalophilic Fungus Sodiomyces alkalinus and Selection of Strains–Producers of New Antimicotic Compound

Antimicrobial Potential of the Alkalophilic Fungus Sodiomyces alkalinus and Selection of... ISSN 0003-6838, Applied Biochemistry and Microbiology, 2021, Vol. 57, No. 1, pp. 86–93. © The Author(s) 2021. This article is an open access publication. Russian Text © The Author(s), 2021, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2021, Vol. 57, No. 1, pp. 59–67. Antimicrobial Potential of the Alkalophilic Fungus Sodiomyces alkalinus and Selection of Strains–Producers of New Antimicotic Compound a, a, b a, d c A. E. Kuvarina *, M. L. Georgieva , E. A. Rogozhin , A. B. Kulko , a a, I. A. Gavryushina , and V. S. Sadykova ** Gause Institute of New Antibiotics, Moscow, 119021 Russia Moscow State University, Moscow, 119234 Russia Moscow Municipal Scientific Practical Center of Tuberculosis Control, Health Department of Moscow, Moscow 107076 Russia Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Moscow, 117997 Russia *e-mail: nastena.lysenko@mail.ru **e-mail: sadykova_09@mail.ru Received July 10, 2020; revised August 31, 2020; accepted September 2, 2020 Abstract—The ability of alkalophilic micromycetes of the species Sodiomyces alkalinus to produce antimicro- bial compounds was studied. As a result of the determination of the spectrum and yield of antibiotic com- pounds, a promising producer of the antimycotics Sodiomyces alkalinus was selected from the most active strains 8KS17-10. The producer exhibited antifungal activity against opportunistic fungi, as well as patho- genic clinical isolates of molds and yeasts—pathogens of systemic mycoses. The isolated active compound can be attributed to the group of antimicrobial glycopeptides based on the totality of the identified structural features (molecular weight, absorption ratio at certain wavelengths). Keywords: antimicrobial glycopeptides, Sodiomyces, alkaliphiles, micromycetes, antibiotics DOI: 10.1134/S0003683821010142 INTRODUCTION seas and oceans; etc. Survival under such conditions is associated with the synthesis of various metabolites The spread of antibiotic-resistant strains of patho- with structures that differ from those previously stud- genic microorganisms is a global challenge to the ied. Over the past 10–15 years, more than 20 000 such health systems of all countries. The widespread use of compounds produced by extremophilic microorgan- antibiotics in recent decades has led to a situation in isms have been isolated and characterized [2, 3]. which 30% of cases of infectious diseases cannot be Despite the diff iculties in the detection and cultivation treated with known drugs, including the latest genera- of extremophilic fungi, in particular, alkalophilic tions of antibiotics. Complex radical measures, fungi, screening studies indicate their enormous including the search for fundamentally new com- potential as sources of new bioactive compounds [4]. pounds with antimicrobial activity, are needed to restrain the expansion of resistant strains observed Thus, the alkalotolerant fungus Paecilomyces lilaci- around the world. nus demonstrates the synthesis of peptide antibiotics 1907-II and 1907-VIII with antibacterial and antifun- Fungi are one of the main groups of living organ- gal activities [5]. The alkalophilic fungus Aspergillus isms that are considered to be producers of antibiotics. flavus produces kojic acid and fomaligol A, which are However, only a small proportion of them has been active against gram-positive and gram-negative bacte- studied to date for the synthesis of secondary antimi- ria [6, 7]. A new lipopeptaibol, emericellipsin A, was crobial products. discovered and described in strains of the alkalophilic Traditionally, antibiotic-producing fungi have been fungus Emericellopsis alkalina isolated from alkaline isolated from soil samples. Nevertheless, this source saline soils. It has antifungal, antibacterial (including has been exhausted for the most part, and unconven- against gram-negative bacteria), and antitumor activi- tional biotopes with recently discovered organisms ties [8]. have come to the fore in the search for new and more effective antimicrobial compounds [1]. These bio- Most of the currently known alkalophilic and topes include arid soils; caves; areas with high or low alkalotolerant fungal taxa belong to ascomycetes temperatures and high salinity and alkalinity; deep from the Plectosphaerellaceae. Members of the 86 ANTIMICROBIAL POTENTIAL OF THE ALKALOPHILIC FUNGUS 87 recently described genus Sodiomyces (Ascomycota, tion (g/L): Na CO , 24; NaHCO , 12; NaCl, 6; 2 3 3 Plectosphaerellaceae), for which the obligate-alkalo- KNO , 1; K HPO , 1; malt extract (15° Balling), 3 2 4 philic type of adaptation was conf irmed, are especially 200 mL; yeast extract, 1; and distilled water, 800 mL interesting. Species of this genus serve as models in [15]. Stationary fungi were grown in 500-mL f lasks for studies of the ecophysiology of fungi, the study of the 14 days. After cultivation, the culture liquid was sepa- biochemical basis of adaptation to the pH factor [9, rated via f iltration through membrane f ilters on a Seitz 10], the transfer of bacterial genes into fungal genes, funnel under a vacuum. and the evolution of enzymes [11]. It was shown that The antimicrobial activity for three strains was the genome S. alkalinus contains sequences encoding compared for different storage methods. We compared the main enzymes necessary for the biosynthesis of the same strains; some were stored in glycerin at beta-lactam antibiotics. It is also known that beta-lac- ‒70°C in a kelvinator, and others were stored on an tams rapidly degrade at a high pH [12, 13]. It remains alkaline medium in test tubes in a refrigerator at 6°C. an open question whether the fungus synthesizes these Both variants were stored under these conditions for at substances in natural conditions, where the variety of least 2 years. various groups of prokaryotes is high and some alkali- To isolate antibiotic substances, the culture fluid resistant fungi are abundantly represented, or whether (CF) of the producer was extracted with ethyl acetate in it produces other antimicrobial compounds under the ratio of organic solvent, CL 5 : 1. The obtained alkaline conditions [11]. extracts were evaporated in a vacuum on a rotary evap- The goal of this work is to assess the antimicrobial orator Rotavapor-RBuchi (Büchi, Switzerland) at 42°C activity of alkalophilic isolates of the Sodiomyces alka- to a dry state; the dry residue was dissolved in aqueous linus, to select producers of peptide antibiotics, and to 70% ethanol, and alcohol concentrates were obtained. identify the most active of them. The antimicrobial activity was determined in the initial culture liquid and in alcoholic concentrates of extracts of CL and mycelium on sterile paper disks EXPERIMENTAL (filter paper F GOST 12026-76, Russia) soaked in The objects of study were 25 alkalophilic strains of extracts and dried under sterile conditions. The sensi- the recently described species Sodiomyces alkalinus tivity of the test organism was controlled with standard (Bilanenko & M.Ivanova) A.A. Grum-Grzhim., discs containing amphotericin B for fungi (40 μg/disk) A.J.M. Debets & Bilanenko (https://www.ncbi.nlm. and ampicillin for bacteria (10 μg/disk) (Research nih.gov/nuccore/?term=Sodiomyces+alkalinus), iso- Institute of Pasteur, Russia). lated from alkaline saline soils in different geographic The spectrum of antimicrobial activity of the cul- regions [14]. The cultures were obtained from the ture liquid, extracts, and individual compounds was Fungi of Extreme Conditions collection of the determined on test cultures of mycelial and yeast Department of Mycology and Algology, Biology microscopic fungi and on bacteria from the collection Faculty, Lomonosov Moscow State University (Rus- of cultures of the Gause Institute of New Antibiotics sia). Some isolates were deposited in the All-Russia (Moscow, Russia). Opportunistic mold and yeast test collection of microorganisms (VKM, Pushchino, cultures of the fungal species Aspergillus fumigatus Russia) and the Fungal Biodiversity Center (CBS, KPB F-37, A. niger INA 00760, and Candida albicans Utrecht, Netherlands). The strain used in this work, ATCC 2091 and test cultures of gram-positive strains F11 (= CBS 110278 = VKM F-3762), is type for this Bacillus subtilis ATCC 6633, Staphylococcus aureus species, and its complete genome is annotated [11]. FDA 209P and gram-negative bacteria – Escherichia The antimicrobial activity of the strains was coli ATCC 25922 were used. assessed in the first stages of work via diffusion in agar The spectrum of antimycotic action of the antimi- on test cultures of opportunistic microorganisms crobial peptide was also evaluated on clinical isolates of Aspergillus niger INA 00760 and Bacillus subtilis ATCC molds and yeasts—pathogens of opportunistic pneu- 6633. Cultures were considered to be highly active if momycosis of the bronchi and lungs—in tuberculosis the zone of growth retardation of the test organism was patients with multiresistance to the antibiotics-azoles 25 mm or more; moderately active cultures had a used in clinical practice, from the collection of the growth retardation zone of 10–25 mm, and weakly mycological laboratory of the Moscow City Scientific active cultures had a zone of less than 10 mm. and Practical Center for Tuberculosis Control (Russia): The ability to synthesize antimicrobial substances Candida albicans 1582m 2016, C. glabrata 1402m 2016, was evaluated with growth on a specialized alkaline C. krusei 1447m 2016, C. parapsilopsis 571m, medium that, according to previously obtained data, is C. tropicalis 156m 2017, Cryptococcus neoformans 297 m optimal for the growth and development of this fungus 2017, Aspergillus fumigatus 390m, and A. niger 219. [11, 14]. Further separation of the active fractions (after For the selected eight strains, the formation of anti- extraction) was carried out via analytical reversed-phase microbial substances was studied upon cultivation in a high-performance liquid chromatography (RP-HPLC) liquid alkaline medium with the following composi- with an XBridge 5 μm 130 А column with a size of APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 57 No. 1 2021 88 KUVARINA et al. 250 × 4.6 mm (Waters, Ireland) in a growing linear nus. Previous studies of the features of ecophysiology gradient of the acetonitrile concentration as a mobile have shown that S. alkalinus is an obligate alkaliphile, phase (eluent A, 0.1% trifluoroacetic acid (TFA) in i.e., it is incapable of growth at an acidic pH. At pH water MQ; eluent B, 80% acetonitrile in 0.1% aqueous values of 6–7, its growth rate significantly decreases, TFA) at a f low rate of 950 μL/min. Ultragradient ace- asexual and sexual sporulation is poorly developed or tonitrile (Panreac, Spain) and TFA (Sigma-Aldrich, absent, the aerial mycelium is poorly expressed, and United States) were used for RP-HPLC. The sub- the hyphae often have an ugly appearance with stances to be separated were determined at the wave- numerous swellings. An alkaline medium specially length of 214 nm in the concentration gradient of elu- designed for alkalophilic fungi made it possible to ent B: 16–28% in 12 minutes; 28–55% in 27 minutes; maintain high pH values (10.5), thereby simulating the 55–75% in 20 min; and 75–85% in 10 min. This was conditions of natural alkaline biotopes from which followed by isocratic elution for 25 min. In order to isolates of this unique fungus were isolated. The max- scale up the production of individual components of imal S. alkalinus growth rate was noted on an alkaline the alcoholic concentrate of the culture broth extract medium (as compared to media with acidic and neu- of the producer, a similar separation was carried out tral pHs), and all its characteristic morphological and via semipreparative RP-HPLC on an XBridge 10 μm cultural features were fully manifested [14]. Optimal 130 А column (250x10 mm). The absorbance (D) was conditions for many S. alkalinus enzymes (cellulases, determined at a wavelength of 214 nm and a mobile hemicellulases, proteases) were observed at pH levels phase f low rate of 4 mL/min. The fractions obtained of 8 and 10 [11]. For isolates of another alkalophilic during RP-HPLC, which correspond to individual fungus also isolated from biotopes with an alkaline peaks, were collected manually, and the excess of medium, Emericellopsis alkalina, it was previously organic solvent (acetonitrile) was then removed via shown that the greatest antifungal activity was mani- evaporation in a SpeedVac vacuum centrifuge (Savant, fested in a medium with a high pH value [15, 16]. United States) and lyophilized (Labconco, United Evaluation of the antimicrobial activity of 25 Sodio- States) to remove residual amounts of TFA. The spec- myces alkalinus strains in relation to test fungi and bac- trum of antimicrobial action of the substances con- teria showed that about half of the studied strains had tained in the fractions was determined via disk diffu- moderate or high activity against A. niger INA 00760 sion, as described above. and B. subtilis ATCC 6633, while the proportion of The molecular weights of the active compounds in highly active isolates with antifungal activity was 20% the isolated fraction were determined on an AutoSpeed (Table 1). The revealed antimicrobial spectrum may matrix-assisted laser desorption/ionization (MALDI) be a ref lection of the habitat in natural saline biotopes, time-of-f light (TOF) mass spectrometer (Bruker Dal- where numerous bacteria developed, and several species tonis, Germany) equipped with a 355-nm UV laser of alkali-resistant micromycetes are abundant. The fore- (Nd: YAG) in positive-ion mode with a ref lectron. On most of these is E. alkalina, the isolates of which also the target, 1 μL of a sample solution and 1 μL of a solu- show pronounced antifungal activity [15, 16]. tion of 2,5-dihydroxybenzoic acid with a concentration Based on the results of primary screening on solid of 10 mg/mL in 20% acetonitrile with 0.5% TFA acid media, eight active S. alkalinus strains were selected, were mixed. The resulting mixture was dried in air. including the type strain (F11) used in further studies The absorption spectra were recorded with a UV-1800 with culturing on liquid media. spectrophotometer (Shimadzu, Japan) in 2-mL Of the eight selected strains, four (5KS17-8, quartz cuvettes with an optical path length of 1 cm. 8KS17-10, 11KS17-1, F11) showed significant antifun- The minimum inhibitory concentration (MIC) was gal activity of the culture f luid and its extracts grown in determined at 24 h for the yeast fungi C. albicans and liquid medium against the opportunistic fungus A. niger at 48 hours for the molds A. niger and A. fumigatus. INA 00760, and one strain (1KS13-4) showed the same The MIC was defined as the minimum concentration against the yeast C. albicans ATCC 2091 (Table 2). Two of a substance that completely suppresses the growth from the eight strains,1KS13-4 and F11 (Table 2), of the test culture. were highly active against the gram-positive bacteria B. subtilis ATCC 6633. No activity was detected The experiments were carried out in three to five against the gram-positive bacteria Staphylococcus replicates. Statistical processing of the results and aureus FDA 209P and the gram-negative bacteria assessment of the reliability of differences in mean val- Escherichia coli ATCC 25922. ues were carried out according to the Student’s t-test for a probability level of at least 95% with the Micro- Three strains, 8KS17-10, 11KS17-1, and F11, were soft Excel 2007 and Statistica 10.0 software. selected for further study of an antibiotic complex exhibiting antifungal activity; the activity of their ethyl acetate extracts of culture f luids was higher than that of RESULTS AND DISCUSSION amphotericin B at a concentration of 40 μg/disk. Anal- An alkaline medium was used to study the antimi- ysis of the antibiotic activity of these strains showed that crobial activity of the fungal strains Sodiomyces alkali- culture storage in a kelvinator at –70°C in glycerin or in APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 57 No. 1 2021 ANTIMICROBIAL POTENTIAL OF THE ALKALOPHILIC FUNGUS 89 (а) 0 10 2030405060min Fig. 1. RP-HPLC profile of extracts of the culture liquid after affinity chromatography on a Heparin-Sepharose column; a dominant peak is marked by a star. Axis: X—time, min, Y—is a digital signal axis (mAU, milli Absorbance Units): (a) 8KS17-10, (b) 11KS17-1, (c) F11. a refrigerator on agar medium in a test tube at 6°C did nents of the active concentrate was obtained for all not have a significant effect on antibiotic synthesis. studied strains, with three prevailing fractions (Fig. 1) having varying degrees of hydrophobicity. Subsequently, a scheme was developed for the sep- aration of an antibiotic complex of three strains via Testing of the obtained main individual com- RP-HPLC. As a result, a similar prof ile of the compo- pounds of the complex for the presence of antimicro- Table 1. Ratio of the studied Sodiomyces alkalinus strains with antifungal and antibacterial activity S. alkalinus strains (25 strains, 100%) Test organism weak moderately active highly active A. niger INA 00760 12 (52%) 8 (28%) 5 (20%) B. subtilis ATCC 6633 11 (44%) 11 (44%) 3 (12%) APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 57 No. 1 2021 Intensity 3.494 4.654 5.214 6.875 7.455 8.658 10.316 10.881 11.924 13.155 13.800 14.664 15.730 16.451 17.008 18.638 19.147 20.838 21.229 21.861 22.556 23.462 23.844 24.262 24.804 25.329 25.727 26.573 27.205 27.782 28.406 29.093 28.605 30.123 31.746 32.422 33.560* 34.927 35.727 36.227 36.779 37.730 43.377 44.125 44.933 90 KUVARINA et al. (b) 0 10 2030405060 min Fig. 1. (Contd.) bial properties found a pronounced activity in only The primary structural characteristics were car- one dominant peak, Sod 1 which eluted from the col- ried out by MALDI-mass-spectrometric analysis. umn at 34.2 min. The zone of growth inhibition of The average molecular weight of the compound is A. niger INA 00760 for the Sod 1 compound was 22 mm. 7918.4 Da (Fig. 3). The nature of the distribution of m/z signals with a step of more than 100 Da indicates For the active compound, a spectrum was the fragmentation of the molecule through hydrolysis obtained absorption in the wavelength range of 210– of glycosidic bonds, which determines the difference 340 nm. It has only one characteristic maximum in the region of short-wave ultraviolet radiation (about in molecular weight with aglycone (7586.34 Da) of 205 nm), which indicates the presence of peptide approximately 332 Da, which presumably corresponds bonds in its structure (Fig. 2). to the presence of sugar residues. APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 57 No. 1 2021 Intensity 3.248 4.278 4.975 5.500 6.063 6.548 6.912 7.453 8.611 9.388 10.168 10.760 11.293 11.880 13.006 13.928 15.619 16.398 16.949 17.425 18.565 19.363 19.812 20.638 21.121 21.756 22.802 23.304 23.757 24.194 25.268 26.608 27.615 28.285 29.049 30.677 31.248 31.831 32.387 33.170 34.171* 36.554 37.147 37.698 39.310 40.274 41.158 43.344 ANTIMICROBIAL POTENTIAL OF THE ALKALOPHILIC FUNGUS 91 (c) 0 10 2030405060 min Fig. 1. (Contd.) Table 2. Antimicrobial activity of culture liquid and extracts of the culture liquid of Sodiomyces alkalinus (in mm zones of inhibition of test-organism growth) Zone of inhibition, culture liquid, mm Zone of inhibition, extracts of the culture liquid, mm Strain, no. B. subtilis A. niger C. albicans A. fumigatus B. subtilis A. niger C. albicans A. fumigatus ATCC 6633 INA 00760 ATCC 2091 KBP F-37 ATCC 6633 INA 00760 ATCC 2091 KBP F-37 5KS17-8 18 14 0 0 0 17 8 8 8KS17-10 33 9 12 0 0 40 9 12 9KS17-1 15 11 0 0 0 12 0 9 11KS17-1 17 12 0 0 13 40 17 12 2KS10-1 23 16 0 0 13 8 0 8 3KS11-1 0 12 12 0 0 9 9 10 1KS13-4 30 13 0 0 25 10 18 8 F11 (type 10 13 0 0 22 18 0 13 strain) APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 57 No. 1 2021 Intensity 3.219 4.122 5.379 5.807 6.513 6.870 7.248 8.613 9.277 9.584 10.131 10.857 11.639 12.758 14.377 14.781 15.534 16.377 16.955 17.686 18.162 18.570 19.282 19.815 20.540 21.108 21.661 22.223 22.785 23.375 23.756 25.165 24.647 25.747 26.281 26.600 27.273 27.607 28.319 30.971 31.822 32.356 32.938 34.109 35.438* 36.064 36.597 37.642 38.346 39.373 43.330 44.921 92 KUVARINA et al. D a concentration of 16 μg/mL, it inhibits C. albicans 2.0 1582m, A. niger 219, and A. fumigatus 390 m. 1.8 1.6 CONCLUSIONS 1.4 Thus, the ability to produce antimycotic com- 1.2 pounds with high and moderate activity was estab- 1.0 lished in one third of all tested extremophilic isolates 0.8 of S. alkalinus, which may indicate the prospects for the search for antimycotic producers in representatives 0.6 of this species. Three strains of this species showing 0.4 pronounced antifungal activity against yeast fungi, 0.2 including clinical isolates (the causative agents of 0.5 invasive mycoses) were preliminarily selected. They 200 220 240 260 280 300 can be recommended in the future as producers of nm promising new drugs for the treatment of severe myco- Fig. 2. UV absorption profile of the Sod 1 in the range ses in cancer patients (presumably, the absence of 200–340 nm. undesirable phenomena and side toxic effects). The pronounced antifungal action against C. neoformans For the Sod 1 compound, the MIC was determined may also hold promise for the etiotropic treatment of in relation to opportunistic–pathogenic mold and cryptococcosis, for which amphotericin B is now used yeast fungi and bacteria, as well as clinical isolates— in therapy with significant side effects. the causative agents of invasive aspergillosis, candi- demia, and cryptococcosis (Table 3). Based on the set of revealed structural features (molecular weight, fragmentation pattern), the isolated It was found that the MIC of the Sod 1 compound active compound can be preliminarily assigned to the for Cryptococcus neoformans 297m 2017 is 1 μg/mL; at 18 000 3794.290 17 000 100.00% 16 000 15 000 7588.959 14 000 81.38% 13 000 7587.337 12 000 68.17% 3795.012 59.59% 10 000 7591.40 4 3796.024 40.98% 39.08% 7589.579 33.07% 3793.001 1441.615 26.33% 24.47% 7592.512 1506.536 3844.440 20.33% 18.35% 18.23% 3000 7702.937 1550.568 3851.664 13.40% 13.07% 12.57% 7803.864 1985.608 1985.498 3902.421 8.13% 7.76% 2604.714 7.43% 6.54% 4545.120 5000.541 5819.531 6302.188 6705.792 5.39% 7919.399 8328.798 9025.336 9793.470 3.62% 3.07% 3.19% 2.75% 3.14% 2.88% 3.02% 2.24% 2.13% –1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 10 000 Molecular weight, Da Fig. 3. MALDI mass spectrometric analysis of the Sod 1 compound. APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 57 No. 1 2021 Number of molecules ANTIMICROBIAL POTENTIAL OF THE ALKALOPHILIC FUNGUS 93 Table 3. Antifungal activity of the Sod 1 compound against clinical pathogenic molds and yeasts with multiple resistance to azoles Suppression zone diameter, mm Test organism Sod 1 (40 mcg/disk) Amphotericin B Fluconasol Vorikonasol C. albicans 1582m 2016 18 ± 0.1 10 ± 0.6 0 0 C. glabrata 1402m 2016 16 ± 0.3 15 ± 0.1 0 0 C. krusei 1447m 2016 12.5 ± 0.2 0 0 0 C. tropicalis 156m 2017 14 ± 0.1 0 0 0 C. parapsilosis 571m 14 ± 0.2 18 ± 0.3 0 0 Cryptococcus neoformans 297m 2017 30 ± 0.1 18 ± 0.6 0 0 A. fumigatus 390m 12 ± 0.5 9 ± 0.6 0 0 A. niger 219 14 ± 0.1 15 ± 0.8 0 0 4. Ibrar, M., Ullah, M.W., Manan, S., Farooq, U., group of glicosilated antimicrobial peptides with strong Rafiq, M., and Hasan, F., Appl. Microbiol. Biotechnol., antiffungal activity. 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Rogozhin, E.A., Sadykova, V.S., Baranova, A.A., the spectrum of antibiotic activity and MIC of individual Vasilchenko, A.S., Lushpa, V.A., Mineev, K.S., Geor- compounds were supported by the Russian Foundation for gieva, M.L., Kulko, A.B., Krasheninnikov, M.E., Ly- Basic Research (project no. 19-34-90088 (I.A. Gavryush- undup, A.V., Vasilchenko, A.V., and Andreev, Y.A., Molecules, 2018, vol. 23, no. 2785, pp. 1–12. ina and V.S. Sadykova). HPLC analysis, isolation of indi- vidual metabolites, and primary structural characterization 9. Bondarenko, S.A., Ianutsevich, E.A., Danilova, O.A., were carried out with the support of the Russian Science Grum-Grzhimaylo, A.A., Kotlova, E.R., Kamzolki- na, O.V., Bilanenko, E.N., and Tereshina, V.M., Ex- Foundation (project no. 18-74-10073 (E.A. Rogozhin)). tremophiles, 2017, vol. 21, no. 4, pp. 743–754. 10. Bondarenko, S.A., Yanutsevich, E.A., Sinitsyna, N.A., COMPLIANCE WITH ETHICAL STANDARDS Georgieva, M.L., Bilanenko, E.N., and Tereshina, V.M., Microbiology (Moscow), 2018, vol. 87, no. 1, pp. 12–22. The authors declare that they have no conf lict of interest. This article does not contain any studies involving animals or 11. Grum-Grzhimaylo, A.A., Falkoski, D.L., Heuvel, J., human participants performed by any of the authors. Valero-Jiménez, C.A., Min, B., Choi, I.-G., Lipzen, A., Daum, C.G., Aanen, D.K., Tsang, A., Henrissat, B., Bilanenko, E.N., de Vries, R.P., van Kan, J.A.L., Grig- OPEN ACCESS oriev, I.G., and Debets, A.J.M., Mol. Ecol., 2018, vol. 27, no. 23, pp. 4808–4819. This article is distributed under the terms of the Creative 12. Deshpande, G.R., Dhekne, V.V., Kulkarni, S.B., Commons Attribution 4.0 International Public License Biswas, S.S., Deo, M.D., and Ayyangar, N.R., Hindu- (http://creativecommons.org/licenses/by/4.0/), which per- stan Antibiot. 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Antimicrobial Potential of the Alkalophilic Fungus Sodiomyces alkalinus and Selection of Strains–Producers of New Antimicotic Compound

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Copyright © The Author(s) 2021. ISSN 0003-6838, Applied Biochemistry and Microbiology, 2021, Vol. 57, No. 1, pp. 86–93. © The Author(s) 2021. This article is an open access publication. Russian Text © The Author(s), 2021, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2021, Vol. 57, No. 1, pp. 59–67.
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ISSN 0003-6838, Applied Biochemistry and Microbiology, 2021, Vol. 57, No. 1, pp. 86–93. © The Author(s) 2021. This article is an open access publication. Russian Text © The Author(s), 2021, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2021, Vol. 57, No. 1, pp. 59–67. Antimicrobial Potential of the Alkalophilic Fungus Sodiomyces alkalinus and Selection of Strains–Producers of New Antimicotic Compound a, a, b a, d c A. E. Kuvarina *, M. L. Georgieva , E. A. Rogozhin , A. B. Kulko , a a, I. A. Gavryushina , and V. S. Sadykova ** Gause Institute of New Antibiotics, Moscow, 119021 Russia Moscow State University, Moscow, 119234 Russia Moscow Municipal Scientific Practical Center of Tuberculosis Control, Health Department of Moscow, Moscow 107076 Russia Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Moscow, 117997 Russia *e-mail: nastena.lysenko@mail.ru **e-mail: sadykova_09@mail.ru Received July 10, 2020; revised August 31, 2020; accepted September 2, 2020 Abstract—The ability of alkalophilic micromycetes of the species Sodiomyces alkalinus to produce antimicro- bial compounds was studied. As a result of the determination of the spectrum and yield of antibiotic com- pounds, a promising producer of the antimycotics Sodiomyces alkalinus was selected from the most active strains 8KS17-10. The producer exhibited antifungal activity against opportunistic fungi, as well as patho- genic clinical isolates of molds and yeasts—pathogens of systemic mycoses. The isolated active compound can be attributed to the group of antimicrobial glycopeptides based on the totality of the identified structural features (molecular weight, absorption ratio at certain wavelengths). Keywords: antimicrobial glycopeptides, Sodiomyces, alkaliphiles, micromycetes, antibiotics DOI: 10.1134/S0003683821010142 INTRODUCTION seas and oceans; etc. Survival under such conditions is associated with the synthesis of various metabolites The spread of antibiotic-resistant strains of patho- with structures that differ from those previously stud- genic microorganisms is a global challenge to the ied. Over the past 10–15 years, more than 20 000 such health systems of all countries. The widespread use of compounds produced by extremophilic microorgan- antibiotics in recent decades has led to a situation in isms have been isolated and characterized [2, 3]. which 30% of cases of infectious diseases cannot be Despite the diff iculties in the detection and cultivation treated with known drugs, including the latest genera- of extremophilic fungi, in particular, alkalophilic tions of antibiotics. Complex radical measures, fungi, screening studies indicate their enormous including the search for fundamentally new com- potential as sources of new bioactive compounds [4]. pounds with antimicrobial activity, are needed to restrain the expansion of resistant strains observed Thus, the alkalotolerant fungus Paecilomyces lilaci- around the world. nus demonstrates the synthesis of peptide antibiotics 1907-II and 1907-VIII with antibacterial and antifun- Fungi are one of the main groups of living organ- gal activities [5]. The alkalophilic fungus Aspergillus isms that are considered to be producers of antibiotics. flavus produces kojic acid and fomaligol A, which are However, only a small proportion of them has been active against gram-positive and gram-negative bacte- studied to date for the synthesis of secondary antimi- ria [6, 7]. A new lipopeptaibol, emericellipsin A, was crobial products. discovered and described in strains of the alkalophilic Traditionally, antibiotic-producing fungi have been fungus Emericellopsis alkalina isolated from alkaline isolated from soil samples. Nevertheless, this source saline soils. It has antifungal, antibacterial (including has been exhausted for the most part, and unconven- against gram-negative bacteria), and antitumor activi- tional biotopes with recently discovered organisms ties [8]. have come to the fore in the search for new and more effective antimicrobial compounds [1]. These bio- Most of the currently known alkalophilic and topes include arid soils; caves; areas with high or low alkalotolerant fungal taxa belong to ascomycetes temperatures and high salinity and alkalinity; deep from the Plectosphaerellaceae. Members of the 86 ANTIMICROBIAL POTENTIAL OF THE ALKALOPHILIC FUNGUS 87 recently described genus Sodiomyces (Ascomycota, tion (g/L): Na CO , 24; NaHCO , 12; NaCl, 6; 2 3 3 Plectosphaerellaceae), for which the obligate-alkalo- KNO , 1; K HPO , 1; malt extract (15° Balling), 3 2 4 philic type of adaptation was conf irmed, are especially 200 mL; yeast extract, 1; and distilled water, 800 mL interesting. Species of this genus serve as models in [15]. Stationary fungi were grown in 500-mL f lasks for studies of the ecophysiology of fungi, the study of the 14 days. After cultivation, the culture liquid was sepa- biochemical basis of adaptation to the pH factor [9, rated via f iltration through membrane f ilters on a Seitz 10], the transfer of bacterial genes into fungal genes, funnel under a vacuum. and the evolution of enzymes [11]. It was shown that The antimicrobial activity for three strains was the genome S. alkalinus contains sequences encoding compared for different storage methods. We compared the main enzymes necessary for the biosynthesis of the same strains; some were stored in glycerin at beta-lactam antibiotics. It is also known that beta-lac- ‒70°C in a kelvinator, and others were stored on an tams rapidly degrade at a high pH [12, 13]. It remains alkaline medium in test tubes in a refrigerator at 6°C. an open question whether the fungus synthesizes these Both variants were stored under these conditions for at substances in natural conditions, where the variety of least 2 years. various groups of prokaryotes is high and some alkali- To isolate antibiotic substances, the culture fluid resistant fungi are abundantly represented, or whether (CF) of the producer was extracted with ethyl acetate in it produces other antimicrobial compounds under the ratio of organic solvent, CL 5 : 1. The obtained alkaline conditions [11]. extracts were evaporated in a vacuum on a rotary evap- The goal of this work is to assess the antimicrobial orator Rotavapor-RBuchi (Büchi, Switzerland) at 42°C activity of alkalophilic isolates of the Sodiomyces alka- to a dry state; the dry residue was dissolved in aqueous linus, to select producers of peptide antibiotics, and to 70% ethanol, and alcohol concentrates were obtained. identify the most active of them. The antimicrobial activity was determined in the initial culture liquid and in alcoholic concentrates of extracts of CL and mycelium on sterile paper disks EXPERIMENTAL (filter paper F GOST 12026-76, Russia) soaked in The objects of study were 25 alkalophilic strains of extracts and dried under sterile conditions. The sensi- the recently described species Sodiomyces alkalinus tivity of the test organism was controlled with standard (Bilanenko & M.Ivanova) A.A. Grum-Grzhim., discs containing amphotericin B for fungi (40 μg/disk) A.J.M. Debets & Bilanenko (https://www.ncbi.nlm. and ampicillin for bacteria (10 μg/disk) (Research nih.gov/nuccore/?term=Sodiomyces+alkalinus), iso- Institute of Pasteur, Russia). lated from alkaline saline soils in different geographic The spectrum of antimicrobial activity of the cul- regions [14]. The cultures were obtained from the ture liquid, extracts, and individual compounds was Fungi of Extreme Conditions collection of the determined on test cultures of mycelial and yeast Department of Mycology and Algology, Biology microscopic fungi and on bacteria from the collection Faculty, Lomonosov Moscow State University (Rus- of cultures of the Gause Institute of New Antibiotics sia). Some isolates were deposited in the All-Russia (Moscow, Russia). Opportunistic mold and yeast test collection of microorganisms (VKM, Pushchino, cultures of the fungal species Aspergillus fumigatus Russia) and the Fungal Biodiversity Center (CBS, KPB F-37, A. niger INA 00760, and Candida albicans Utrecht, Netherlands). The strain used in this work, ATCC 2091 and test cultures of gram-positive strains F11 (= CBS 110278 = VKM F-3762), is type for this Bacillus subtilis ATCC 6633, Staphylococcus aureus species, and its complete genome is annotated [11]. FDA 209P and gram-negative bacteria – Escherichia The antimicrobial activity of the strains was coli ATCC 25922 were used. assessed in the first stages of work via diffusion in agar The spectrum of antimycotic action of the antimi- on test cultures of opportunistic microorganisms crobial peptide was also evaluated on clinical isolates of Aspergillus niger INA 00760 and Bacillus subtilis ATCC molds and yeasts—pathogens of opportunistic pneu- 6633. Cultures were considered to be highly active if momycosis of the bronchi and lungs—in tuberculosis the zone of growth retardation of the test organism was patients with multiresistance to the antibiotics-azoles 25 mm or more; moderately active cultures had a used in clinical practice, from the collection of the growth retardation zone of 10–25 mm, and weakly mycological laboratory of the Moscow City Scientific active cultures had a zone of less than 10 mm. and Practical Center for Tuberculosis Control (Russia): The ability to synthesize antimicrobial substances Candida albicans 1582m 2016, C. glabrata 1402m 2016, was evaluated with growth on a specialized alkaline C. krusei 1447m 2016, C. parapsilopsis 571m, medium that, according to previously obtained data, is C. tropicalis 156m 2017, Cryptococcus neoformans 297 m optimal for the growth and development of this fungus 2017, Aspergillus fumigatus 390m, and A. niger 219. [11, 14]. Further separation of the active fractions (after For the selected eight strains, the formation of anti- extraction) was carried out via analytical reversed-phase microbial substances was studied upon cultivation in a high-performance liquid chromatography (RP-HPLC) liquid alkaline medium with the following composi- with an XBridge 5 μm 130 А column with a size of APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 57 No. 1 2021 88 KUVARINA et al. 250 × 4.6 mm (Waters, Ireland) in a growing linear nus. Previous studies of the features of ecophysiology gradient of the acetonitrile concentration as a mobile have shown that S. alkalinus is an obligate alkaliphile, phase (eluent A, 0.1% trifluoroacetic acid (TFA) in i.e., it is incapable of growth at an acidic pH. At pH water MQ; eluent B, 80% acetonitrile in 0.1% aqueous values of 6–7, its growth rate significantly decreases, TFA) at a f low rate of 950 μL/min. Ultragradient ace- asexual and sexual sporulation is poorly developed or tonitrile (Panreac, Spain) and TFA (Sigma-Aldrich, absent, the aerial mycelium is poorly expressed, and United States) were used for RP-HPLC. The sub- the hyphae often have an ugly appearance with stances to be separated were determined at the wave- numerous swellings. An alkaline medium specially length of 214 nm in the concentration gradient of elu- designed for alkalophilic fungi made it possible to ent B: 16–28% in 12 minutes; 28–55% in 27 minutes; maintain high pH values (10.5), thereby simulating the 55–75% in 20 min; and 75–85% in 10 min. This was conditions of natural alkaline biotopes from which followed by isocratic elution for 25 min. In order to isolates of this unique fungus were isolated. The max- scale up the production of individual components of imal S. alkalinus growth rate was noted on an alkaline the alcoholic concentrate of the culture broth extract medium (as compared to media with acidic and neu- of the producer, a similar separation was carried out tral pHs), and all its characteristic morphological and via semipreparative RP-HPLC on an XBridge 10 μm cultural features were fully manifested [14]. Optimal 130 А column (250x10 mm). The absorbance (D) was conditions for many S. alkalinus enzymes (cellulases, determined at a wavelength of 214 nm and a mobile hemicellulases, proteases) were observed at pH levels phase f low rate of 4 mL/min. The fractions obtained of 8 and 10 [11]. For isolates of another alkalophilic during RP-HPLC, which correspond to individual fungus also isolated from biotopes with an alkaline peaks, were collected manually, and the excess of medium, Emericellopsis alkalina, it was previously organic solvent (acetonitrile) was then removed via shown that the greatest antifungal activity was mani- evaporation in a SpeedVac vacuum centrifuge (Savant, fested in a medium with a high pH value [15, 16]. United States) and lyophilized (Labconco, United Evaluation of the antimicrobial activity of 25 Sodio- States) to remove residual amounts of TFA. The spec- myces alkalinus strains in relation to test fungi and bac- trum of antimicrobial action of the substances con- teria showed that about half of the studied strains had tained in the fractions was determined via disk diffu- moderate or high activity against A. niger INA 00760 sion, as described above. and B. subtilis ATCC 6633, while the proportion of The molecular weights of the active compounds in highly active isolates with antifungal activity was 20% the isolated fraction were determined on an AutoSpeed (Table 1). The revealed antimicrobial spectrum may matrix-assisted laser desorption/ionization (MALDI) be a ref lection of the habitat in natural saline biotopes, time-of-f light (TOF) mass spectrometer (Bruker Dal- where numerous bacteria developed, and several species tonis, Germany) equipped with a 355-nm UV laser of alkali-resistant micromycetes are abundant. The fore- (Nd: YAG) in positive-ion mode with a ref lectron. On most of these is E. alkalina, the isolates of which also the target, 1 μL of a sample solution and 1 μL of a solu- show pronounced antifungal activity [15, 16]. tion of 2,5-dihydroxybenzoic acid with a concentration Based on the results of primary screening on solid of 10 mg/mL in 20% acetonitrile with 0.5% TFA acid media, eight active S. alkalinus strains were selected, were mixed. The resulting mixture was dried in air. including the type strain (F11) used in further studies The absorption spectra were recorded with a UV-1800 with culturing on liquid media. spectrophotometer (Shimadzu, Japan) in 2-mL Of the eight selected strains, four (5KS17-8, quartz cuvettes with an optical path length of 1 cm. 8KS17-10, 11KS17-1, F11) showed significant antifun- The minimum inhibitory concentration (MIC) was gal activity of the culture f luid and its extracts grown in determined at 24 h for the yeast fungi C. albicans and liquid medium against the opportunistic fungus A. niger at 48 hours for the molds A. niger and A. fumigatus. INA 00760, and one strain (1KS13-4) showed the same The MIC was defined as the minimum concentration against the yeast C. albicans ATCC 2091 (Table 2). Two of a substance that completely suppresses the growth from the eight strains,1KS13-4 and F11 (Table 2), of the test culture. were highly active against the gram-positive bacteria B. subtilis ATCC 6633. No activity was detected The experiments were carried out in three to five against the gram-positive bacteria Staphylococcus replicates. Statistical processing of the results and aureus FDA 209P and the gram-negative bacteria assessment of the reliability of differences in mean val- Escherichia coli ATCC 25922. ues were carried out according to the Student’s t-test for a probability level of at least 95% with the Micro- Three strains, 8KS17-10, 11KS17-1, and F11, were soft Excel 2007 and Statistica 10.0 software. selected for further study of an antibiotic complex exhibiting antifungal activity; the activity of their ethyl acetate extracts of culture f luids was higher than that of RESULTS AND DISCUSSION amphotericin B at a concentration of 40 μg/disk. Anal- An alkaline medium was used to study the antimi- ysis of the antibiotic activity of these strains showed that crobial activity of the fungal strains Sodiomyces alkali- culture storage in a kelvinator at –70°C in glycerin or in APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 57 No. 1 2021 ANTIMICROBIAL POTENTIAL OF THE ALKALOPHILIC FUNGUS 89 (а) 0 10 2030405060min Fig. 1. RP-HPLC profile of extracts of the culture liquid after affinity chromatography on a Heparin-Sepharose column; a dominant peak is marked by a star. Axis: X—time, min, Y—is a digital signal axis (mAU, milli Absorbance Units): (a) 8KS17-10, (b) 11KS17-1, (c) F11. a refrigerator on agar medium in a test tube at 6°C did nents of the active concentrate was obtained for all not have a significant effect on antibiotic synthesis. studied strains, with three prevailing fractions (Fig. 1) having varying degrees of hydrophobicity. Subsequently, a scheme was developed for the sep- aration of an antibiotic complex of three strains via Testing of the obtained main individual com- RP-HPLC. As a result, a similar prof ile of the compo- pounds of the complex for the presence of antimicro- Table 1. Ratio of the studied Sodiomyces alkalinus strains with antifungal and antibacterial activity S. alkalinus strains (25 strains, 100%) Test organism weak moderately active highly active A. niger INA 00760 12 (52%) 8 (28%) 5 (20%) B. subtilis ATCC 6633 11 (44%) 11 (44%) 3 (12%) APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 57 No. 1 2021 Intensity 3.494 4.654 5.214 6.875 7.455 8.658 10.316 10.881 11.924 13.155 13.800 14.664 15.730 16.451 17.008 18.638 19.147 20.838 21.229 21.861 22.556 23.462 23.844 24.262 24.804 25.329 25.727 26.573 27.205 27.782 28.406 29.093 28.605 30.123 31.746 32.422 33.560* 34.927 35.727 36.227 36.779 37.730 43.377 44.125 44.933 90 KUVARINA et al. (b) 0 10 2030405060 min Fig. 1. (Contd.) bial properties found a pronounced activity in only The primary structural characteristics were car- one dominant peak, Sod 1 which eluted from the col- ried out by MALDI-mass-spectrometric analysis. umn at 34.2 min. The zone of growth inhibition of The average molecular weight of the compound is A. niger INA 00760 for the Sod 1 compound was 22 mm. 7918.4 Da (Fig. 3). The nature of the distribution of m/z signals with a step of more than 100 Da indicates For the active compound, a spectrum was the fragmentation of the molecule through hydrolysis obtained absorption in the wavelength range of 210– of glycosidic bonds, which determines the difference 340 nm. It has only one characteristic maximum in the region of short-wave ultraviolet radiation (about in molecular weight with aglycone (7586.34 Da) of 205 nm), which indicates the presence of peptide approximately 332 Da, which presumably corresponds bonds in its structure (Fig. 2). to the presence of sugar residues. APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 57 No. 1 2021 Intensity 3.248 4.278 4.975 5.500 6.063 6.548 6.912 7.453 8.611 9.388 10.168 10.760 11.293 11.880 13.006 13.928 15.619 16.398 16.949 17.425 18.565 19.363 19.812 20.638 21.121 21.756 22.802 23.304 23.757 24.194 25.268 26.608 27.615 28.285 29.049 30.677 31.248 31.831 32.387 33.170 34.171* 36.554 37.147 37.698 39.310 40.274 41.158 43.344 ANTIMICROBIAL POTENTIAL OF THE ALKALOPHILIC FUNGUS 91 (c) 0 10 2030405060 min Fig. 1. (Contd.) Table 2. Antimicrobial activity of culture liquid and extracts of the culture liquid of Sodiomyces alkalinus (in mm zones of inhibition of test-organism growth) Zone of inhibition, culture liquid, mm Zone of inhibition, extracts of the culture liquid, mm Strain, no. B. subtilis A. niger C. albicans A. fumigatus B. subtilis A. niger C. albicans A. fumigatus ATCC 6633 INA 00760 ATCC 2091 KBP F-37 ATCC 6633 INA 00760 ATCC 2091 KBP F-37 5KS17-8 18 14 0 0 0 17 8 8 8KS17-10 33 9 12 0 0 40 9 12 9KS17-1 15 11 0 0 0 12 0 9 11KS17-1 17 12 0 0 13 40 17 12 2KS10-1 23 16 0 0 13 8 0 8 3KS11-1 0 12 12 0 0 9 9 10 1KS13-4 30 13 0 0 25 10 18 8 F11 (type 10 13 0 0 22 18 0 13 strain) APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 57 No. 1 2021 Intensity 3.219 4.122 5.379 5.807 6.513 6.870 7.248 8.613 9.277 9.584 10.131 10.857 11.639 12.758 14.377 14.781 15.534 16.377 16.955 17.686 18.162 18.570 19.282 19.815 20.540 21.108 21.661 22.223 22.785 23.375 23.756 25.165 24.647 25.747 26.281 26.600 27.273 27.607 28.319 30.971 31.822 32.356 32.938 34.109 35.438* 36.064 36.597 37.642 38.346 39.373 43.330 44.921 92 KUVARINA et al. D a concentration of 16 μg/mL, it inhibits C. albicans 2.0 1582m, A. niger 219, and A. fumigatus 390 m. 1.8 1.6 CONCLUSIONS 1.4 Thus, the ability to produce antimycotic com- 1.2 pounds with high and moderate activity was estab- 1.0 lished in one third of all tested extremophilic isolates 0.8 of S. alkalinus, which may indicate the prospects for the search for antimycotic producers in representatives 0.6 of this species. Three strains of this species showing 0.4 pronounced antifungal activity against yeast fungi, 0.2 including clinical isolates (the causative agents of 0.5 invasive mycoses) were preliminarily selected. They 200 220 240 260 280 300 can be recommended in the future as producers of nm promising new drugs for the treatment of severe myco- Fig. 2. UV absorption profile of the Sod 1 in the range ses in cancer patients (presumably, the absence of 200–340 nm. undesirable phenomena and side toxic effects). The pronounced antifungal action against C. neoformans For the Sod 1 compound, the MIC was determined may also hold promise for the etiotropic treatment of in relation to opportunistic–pathogenic mold and cryptococcosis, for which amphotericin B is now used yeast fungi and bacteria, as well as clinical isolates— in therapy with significant side effects. the causative agents of invasive aspergillosis, candi- demia, and cryptococcosis (Table 3). Based on the set of revealed structural features (molecular weight, fragmentation pattern), the isolated It was found that the MIC of the Sod 1 compound active compound can be preliminarily assigned to the for Cryptococcus neoformans 297m 2017 is 1 μg/mL; at 18 000 3794.290 17 000 100.00% 16 000 15 000 7588.959 14 000 81.38% 13 000 7587.337 12 000 68.17% 3795.012 59.59% 10 000 7591.40 4 3796.024 40.98% 39.08% 7589.579 33.07% 3793.001 1441.615 26.33% 24.47% 7592.512 1506.536 3844.440 20.33% 18.35% 18.23% 3000 7702.937 1550.568 3851.664 13.40% 13.07% 12.57% 7803.864 1985.608 1985.498 3902.421 8.13% 7.76% 2604.714 7.43% 6.54% 4545.120 5000.541 5819.531 6302.188 6705.792 5.39% 7919.399 8328.798 9025.336 9793.470 3.62% 3.07% 3.19% 2.75% 3.14% 2.88% 3.02% 2.24% 2.13% –1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 10 000 Molecular weight, Da Fig. 3. MALDI mass spectrometric analysis of the Sod 1 compound. APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 57 No. 1 2021 Number of molecules ANTIMICROBIAL POTENTIAL OF THE ALKALOPHILIC FUNGUS 93 Table 3. Antifungal activity of the Sod 1 compound against clinical pathogenic molds and yeasts with multiple resistance to azoles Suppression zone diameter, mm Test organism Sod 1 (40 mcg/disk) Amphotericin B Fluconasol Vorikonasol C. albicans 1582m 2016 18 ± 0.1 10 ± 0.6 0 0 C. glabrata 1402m 2016 16 ± 0.3 15 ± 0.1 0 0 C. krusei 1447m 2016 12.5 ± 0.2 0 0 0 C. tropicalis 156m 2017 14 ± 0.1 0 0 0 C. parapsilosis 571m 14 ± 0.2 18 ± 0.3 0 0 Cryptococcus neoformans 297m 2017 30 ± 0.1 18 ± 0.6 0 0 A. fumigatus 390m 12 ± 0.5 9 ± 0.6 0 0 A. niger 219 14 ± 0.1 15 ± 0.8 0 0 4. Ibrar, M., Ullah, M.W., Manan, S., Farooq, U., group of glicosilated antimicrobial peptides with strong Rafiq, M., and Hasan, F., Appl. Microbiol. Biotechnol., antiffungal activity. Further structural identification will 2020, vol. 104, no. 7, pp. 2777–2801. be carried out with a combination of physicochemical methods: high resolution mass spectrometry and nuclear 5. Sato, M., Beppu, T., and Arima, K., Agric. Biol. Chem., 1980, vol. 44, no. 12, pp. 3037–30 40. magnetic resonance (NMR) spectroscopy. 6. Wu, Y., Shi, Y., Zeng, L., Pan, Y., Huang, X., Bian, L., Zhu, Y., Zhang, R., and Zhang, J., Food Sci. Technol. FUNDING Int., 2019, vol. 25, no. 1, pp. 3–15. The cultivation and identification of isolates were car- 7. Yang, G., Sandjo, L., Yun, K., Leutou, A.S., Kim, G.-D., ried out with the financial support of the Russian Founda- Choi, H.D., Kang, J.S., Hong, J., and Son, B.W., tion for Basic Research (project no. 20-04-00992, Chem. Pharm. Bull., 2011, vol. 59, no. 9, pp. 1174–1177. M.L. Georgieva and A.E. Kuvarina), and Determination of 8. 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Bondarenko, S.A., Yanutsevich, E.A., Sinitsyna, N.A., COMPLIANCE WITH ETHICAL STANDARDS Georgieva, M.L., Bilanenko, E.N., and Tereshina, V.M., Microbiology (Moscow), 2018, vol. 87, no. 1, pp. 12–22. The authors declare that they have no conf lict of interest. This article does not contain any studies involving animals or 11. Grum-Grzhimaylo, A.A., Falkoski, D.L., Heuvel, J., human participants performed by any of the authors. Valero-Jiménez, C.A., Min, B., Choi, I.-G., Lipzen, A., Daum, C.G., Aanen, D.K., Tsang, A., Henrissat, B., Bilanenko, E.N., de Vries, R.P., van Kan, J.A.L., Grig- OPEN ACCESS oriev, I.G., and Debets, A.J.M., Mol. Ecol., 2018, vol. 27, no. 23, pp. 4808–4819. This article is distributed under the terms of the Creative 12. Deshpande, G.R., Dhekne, V.V., Kulkarni, S.B., Commons Attribution 4.0 International Public License Biswas, S.S., Deo, M.D., and Ayyangar, N.R., Hindu- (http://creativecommons.org/licenses/by/4.0/), which per- stan Antibiot. 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