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

The ability of alkalophilic micromycetes of the species Sodiomyces alkalinus to produce antimicrobial compounds was studied. As a result of the determination of the spectrum and yield of antibiotic compounds, 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 pathogenic 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).


INTRODUCTION
The spread of antibiotic-resistant strains of pathogenic microorganisms is a global challenge to the health systems of all countries. The widespread use of antibiotics in recent decades has led to a situation in which 30% of cases of infectious diseases cannot be treated with known drugs, including the latest generations of antibiotics. Complex radical measures, including the search for fundamentally new compounds with antimicrobial activity, are needed to restrain the expansion of resistant strains observed around the world.
Fungi are one of the main groups of living organisms that are considered to be producers of antibiotics. However, only a small proportion of them has been studied to date for the synthesis of secondary antimicrobial products.
Traditionally, antibiotic-producing fungi have been isolated from soil samples. Nevertheless, this source has been exhausted for the most part, and unconventional biotopes with recently discovered organisms have come to the fore in the search for new and more effective antimicrobial compounds [1]. These biotopes include arid soils; caves; areas with high or low temperatures and high salinity and alkalinity; deep seas and oceans; etc. Survival under such conditions is associated with the synthesis of various metabolites with structures that differ from those previously studied. Over the past 10-15 years, more than 20000 such compounds produced by extremophilic microorganisms have been isolated and characterized [2,3]. Despite the difficulties in the detection and cultivation of extremophilic fungi, in particular, alkalophilic fungi, screening studies indicate their enormous potential as sources of new bioactive compounds [4].
Thus, the alkalotolerant fungus Paecilomyces lilacinus demonstrates the synthesis of peptide antibiotics 1907-II and 1907-VIII with antibacterial and antifungal activities [5]. The alkalophilic fungus Aspergillus flavus produces kojic acid and fomaligol A, which are active against gram-positive and gram-negative bacteria [6,7]. A new lipopeptaibol, emericellipsin A, was discovered and described in strains of the alkalophilic fungus Emericellopsis alkalina isolated from alkaline saline soils. It has antifungal, antibacterial (including against gram-negative bacteria), and antitumor activities [8].
Most of the currently known alkalophilic and alkalotolerant fungal taxa belong to ascomycetes from the Plectosphaerellaceae. Members of the recently described genus Sodiomyces (Ascomycota, Plectosphaerellaceae), for which the obligate-alkalophilic type of adaptation was confirmed, are especially interesting. Species of this genus serve as models in studies of the ecophysiology of fungi, the study of the biochemical basis of adaptation to the pH factor [9,10], the transfer of bacterial genes into fungal genes, and the evolution of enzymes [11]. It was shown that the genome S. alkalinus contains sequences encoding the main enzymes necessary for the biosynthesis of beta-lactam antibiotics. It is also known that beta-lactams rapidly degrade at a high pH [12,13]. It remains an open question whether the fungus synthesizes these substances in natural conditions, where the variety of various groups of prokaryotes is high and some alkaliresistant fungi are abundantly represented, or whether it produces other antimicrobial compounds under alkaline conditions [11].
The goal of this work is to assess the antimicrobial activity of alkalophilic isolates of the Sodiomyces alkalinus, to select producers of peptide antibiotics, and to identify the most active of them.

EXPERIMENTAL
The objects of study were 25 alkalophilic strains of the recently described species Sodiomyces alkalinus (Bilanenko & M.Ivanova) A.A. Grum-Grzhim., A.J.M. Debets & Bilanenko (https://www.ncbi.nlm. nih.gov/nuccore/?term=Sodiomyces+alkalinus), isolated from alkaline saline soils in different geographic regions [14]. The cultures were obtained from the Fungi of Extreme Conditions collection of the Department of Mycology and Algology, Biology Faculty, Lomonosov Moscow State University (Russia). Some isolates were deposited in the All-Russia collection of microorganisms (VKM, Pushchino, Russia) and the Fungal Biodiversity Center (CBS, Utrecht, Netherlands). The strain used in this work, F11 (= CBS 110278 = VKM F-3762), is type for this species, and its complete genome is annotated [11].
The antimicrobial activity of the strains was assessed in the first stages of work via diffusion in agar on test cultures of opportunistic microorganisms Aspergillus niger INA 00760 and Bacillus subtilis ATCC 6633. Cultures were considered to be highly active if the zone of growth retardation of the test organism was 25 mm or more; moderately active cultures had a growth retardation zone of 10-25 mm, and weakly active cultures had a zone of less than 10 mm.
The ability to synthesize antimicrobial substances was evaluated with growth on a specialized alkaline medium that, according to previously obtained data, is optimal for the growth and development of this fungus [11,14].
The antimicrobial activity for three strains was compared for different storage methods. We compared the same strains; some were stored in glycerin at -70°C in a kelvinator, and others were stored on an alkaline medium in test tubes in a refrigerator at 6°C. Both variants were stored under these conditions for at least 2 years.
To isolate antibiotic substances, the culture fluid (CF) of the producer was extracted with ethyl acetate in the ratio of organic solvent, CL 5 : 1. The obtained extracts were evaporated in a vacuum on a rotary evaporator Rotavapor-RBuchi (Büchi, Switzerland) at 42°C to a dry state; the dry residue was dissolved in aqueous 70% ethanol, and alcohol concentrates were obtained.
The antimicrobial activity was determined in the initial culture liquid and in alcoholic concentrates of extracts of CL and mycelium on sterile paper disks (filter paper F GOST 12026-76, Russia) soaked in extracts and dried under sterile conditions. The sensitivity of the test organism was controlled with standard discs containing amphotericin B for fungi (40 μg/disk) and ampicillin for bacteria ( The absorption spectra were recorded with a UV-1800 spectrophotometer (Shimadzu, Japan) in 2-mL quartz cuvettes with an optical path length of 1 cm.
The minimum inhibitory concentration (MIC) was determined at 24 h for the yeast fungi C. albicans and at 48 hours for the molds A. niger and A. fumigatus. The MIC was defined as the minimum concentration of a substance that completely suppresses the growth of the test culture.
The experiments were carried out in three to five replicates. Statistical processing of the results and assessment of the reliability of differences in mean values were carried out according to the Student's t-test for a probability level of at least 95% with the Microsoft Excel 2007 and Statistica 10.0 software.

RESULTS AND DISCUSSION
An alkaline medium was used to study the antimicrobial activity of the fungal strains Sodiomyces alkali-nus. Previous studies of the features of ecophysiology have shown that S. alkalinus is an obligate alkaliphile, i.e., it is incapable of growth at an acidic pH. At pH values of 6-7, its growth rate significantly decreases, asexual and sexual sporulation is poorly developed or absent, the aerial mycelium is poorly expressed, and the hyphae often have an ugly appearance with numerous swellings. An alkaline medium specially designed for alkalophilic fungi made it possible to maintain high pH values (10.5), thereby simulating the conditions of natural alkaline biotopes from which isolates of this unique fungus were isolated. The maximal S. alkalinus growth rate was noted on an alkaline medium (as compared to media with acidic and neutral pHs), and all its characteristic morphological and cultural features were fully manifested [14]. Optimal conditions for many S. alkalinus enzymes (cellulases, hemicellulases, proteases) were observed at pH levels of 8 and 10 [11]. For isolates of another alkalophilic fungus also isolated from biotopes with an alkaline medium, Emericellopsis alkalina, it was previously shown that the greatest antifungal activity was manifested in a medium with a high pH value [15,16].
Evaluation of the antimicrobial activity of 25 Sodiomyces alkalinus strains in relation to test fungi and bacteria showed that about half of the studied strains had moderate or high activity against A. niger INA 00760 and B. subtilis ATCC 6633, while the proportion of highly active isolates with antifungal activity was 20% ( Table 1). The revealed antimicrobial spectrum may be a reflection of the habitat in natural saline biotopes, where numerous bacteria developed, and several species of alkali-resistant micromycetes are abundant. The foremost of these is E. alkalina, the isolates of which also show pronounced antifungal activity [15,16].
Based on the results of primary screening on solid media, eight active S. alkalinus strains were selected, including the type strain (F11) used in further studies with culturing on liquid media.
Of the eight selected strains, four (5KS17-8, 8KS17-10, 11KS17-1, F11) showed significant antifungal activity of the culture fluid and its extracts grown in liquid medium against the opportunistic fungus A. niger INA 00760, and one strain (1KS13-4) showed the same against the yeast C. albicans ATCC 2091 (Table 2). Two from the eight strains,1KS13-4 and F11 (Table 2), were highly active against the gram-positive bacteria B. subtilis ATCC 6633. No activity was detected against the gram-positive bacteria Staphylococcus aureus FDA 209P and the gram-negative bacteria Escherichia coli ATCC 25922.
Three strains, 8KS17-10, 11KS17-1, and F11, were selected for further study of an antibiotic complex exhibiting antifungal activity; the activity of their ethyl acetate extracts of culture fluids was higher than that of amphotericin B at a concentration of 40 μg/disk. Analysis of the antibiotic activity of these strains showed that culture storage in a kelvinator at -70°C in glycerin or in   a refrigerator on agar medium in a test tube at 6°C did not have a significant effect on antibiotic synthesis.
Subsequently, a scheme was developed for the separation of an antibiotic complex of three strains via RP-HPLC. As a result, a similar profile of the compo-nents of the active concentrate was obtained for all studied strains, with three prevailing fractions (Fig. 1) having varying degrees of hydrophobicity.
Testing of the obtained main individual compounds of the complex for the presence of antimicro-   For the active compound, a spectrum was obtained absorption in the wavelength range of 210-340 nm. It has only one characteristic maximum in the region of short-wave ultraviolet radiation (about 205 nm), which indicates the presence of peptide bonds in its structure (Fig. 2).
The primary structural characteristics were carried out by MALDI-mass-spectrometric analysis. The average molecular weight of the compound is 7918.4 Da (Fig. 3). The nature of the distribution of m/z signals with a step of more than 100 Da indicates the fragmentation of the molecule through hydrolysis of glycosidic bonds, which determines the difference in molecular weight with aglycone (7586.34 Da) of approximately 332 Da, which presumably corresponds to the presence of sugar residues.   For the Sod 1 compound, the MIC was determined in relation to opportunistic-pathogenic mold and yeast fungi and bacteria, as well as clinical isolatesthe causative agents of invasive aspergillosis, candidemia, and cryptococcosis (Table 3).
It was found that the MIC of the Sod 1 compound for Cryptococcus neoformans 297m 2017 is 1 μg/mL; at a concentration of 16 μg/mL, it inhibits C. albicans 1582m, A. niger 219, and A. fumigatus 390 m.

CONCLUSIONS
Thus, the ability to produce antimycotic compounds with high and moderate activity was established in one third of all tested extremophilic isolates of S. alkalinus, which may indicate the prospects for the search for antimycotic producers in representatives of this species. Three strains of this species showing pronounced antifungal activity against yeast fungi, including clinical isolates (the causative agents of invasive mycoses) were preliminarily selected. They can be recommended in the future as producers of promising new drugs for the treatment of severe mycoses in cancer patients (presumably, the absence of undesirable phenomena and side toxic effects). The pronounced antifungal action against C. neoformans may also hold promise for the etiotropic treatment of cryptococcosis, for which amphotericin B is now used in therapy with significant side effects.
Based on the set of revealed structural features (molecular weight, fragmentation pattern), the isolated active compound can be preliminarily assigned to the  Number of molecules group of glicosilated antimicrobial peptides with strong antiffungal activity. Further structural identification will be carried out with a combination of physicochemical methods: high resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.