Estimation of antimicrobial activities and fatty acid composition of actinobacteria isolated from water surface of underground lakes from Badzheyskaya and Okhotnichya caves in Siberia

Cave description, sampling sites, and isolation of actinobacteria

The karstic cave Badzheyskaya is located near Stepnoy Badzhey village in Krasnoyarsk region (55°14′32″N 93°46′32″E) (Dublyanskiy, 1979; Ananyeva, Ananyev & Zadisensky, 2013). Badzheyskaya cave is the largest enclosure of conglomerates in the world formed in the Quaternary period. Badzheyskaya cave shows a great number of peculiar and various passages, groths and galleries and it has an underground lake and siphons. There is an abundance of clayey substances, peddle stones, and blocks of conglomerates. The cave has sparse speleothems including a small number of moonmilk speleothem, stalactites, and stalagmites. The known dimensions of the Badzheyskaya cave is 6,000 m in length, 170 m deep and 170 m wide. The average annual temperature in the cave fluctuates from 3 to 5 °C (Khizhnyak et al., 2003).

Okhotnichya cave is a karstic cave located in the Irkutsk region (52°8′18″N; 105°27′49″E). The cave length is 5,700 m. The amplitude of the cave is 77 m (Osintsev, 2010). Okhotnichya is the third in length among the known caves in Baikal region (Bazarova et al., 2014). It was formed in the Upper Proterozoic era. The cave is described by a variety of galleries and multifarious formations, namely stalactites, stalagmites, corallite, cave pearls, red-brown clays, and also, a great number of osteological remnants. Moreover, there are three small ponds and a longstanding glacier in the cave. The average annual temperature is 1.26 °C. Also, bats were found in the cave (Klementyev, Korshunov & Osintsev, 2007). Table 1 gives a brief comparative description of the studied caves.

For actinobacteria isolation, 10 mL of water from surface of underground lakes in Badzheyskaya and Okhotnichya caves were collected in triplicate by sterile syringes in 2014. The obtained samples were transported to the laboratory in thermostatic conditions (3–5 °C), where we added equal volume of sterile 40% glycerol to each. The obtained samples were stored at −20 °C before isolating the strains.

Actinobacteria strains were isolated by triplicate plating 100 uL of each water samples on solid nutrient media. To isolate the actinobacteria strains, we used MS mannitol soy flour agar (soy flour –20 g, D-mannitol –20 g, agar –20 g, tap water –1 L, pH 7.2) (Kieser et al., 2000) supplemented with the antibiotics cycloheximide (50 ug/mL) and phosphomycin (100 ug/mL). Aliquots of collected samples (500 uL) were preheated for 5 min at 50 °C to activate spore germination and inactivate vegetative cells of other bacteria. The plates were incubated for 30 days at 28 °C and assessed for appearance of actinobacterial colony every day. Actinobacteria-like strains were selected based on colony morphology: solid density of colonies, growth inside of the agar media and steady border of the colonies (Kieser et al., 2000). The colonies were transferred from the primary plates to the fresh MS plates. Pure cultures were obtained for all colonies identified as actinobacteria on the primary plates. Several isolated strains were deposited in the Russian Collection of Agricultural Microorganisms (RCAM), St. Petersburg, Russia (Act 471/12 of 15.12.2017).

16S rRNA gene sequencing and phylogenetic analysis

For isolation of total DNA, strains were grown in 10 mL of TSB medium at 28 °C for 3 days at 180 rpm. Total DNA was isolated using the salting out procedures as described in (Kieser et al., 2000). To identify the isolates, the 16S rRNA gene was amplified by PCR with the actinobacteria-specific and universal primers. Actinobacteria-specific primers were: F-Act-235(CGC GGC CTA TCA GCT TGT TG) and R-Act-878(CCG TAC TCC CCA GGC GGG G) (Stach et al., 2003). Universal eubacterial primers included: 8F (AGA GTT TGA TCC TGG CTC AG) and 1492R (TAC GGY TAC CTT GTT ACG ACT T) (Shieh, Martin & Millar, 1998). The PCR reaction was performed using the ScreenMix 5X PCR kit (Kat. PK041L, Evrogen, Russia). PCR was performed in a TGradient Thermocycler (Biometra, Göttingen, Germany) in the volume of 25 uL. The PCR parameters were as follows: initial denaturation at 95 °C for 5 min, followed by 25 cycles of 95 °C for 40 s, 49–52 °C for 25 s, and 72 °C for 110 s, and final elongation at 72 °C for 5 min.

The PCR products were purified using QIAquick Gel Extraction Kit (Qiagen, Venlo, The Netherlands) and sequenced with the use of actinobacteria-specific or universal primers. Mixture of PCR product with amplification primers were sent to the Syntol company (Moscow, Russia) to sequencing of PCR products by Sanger methods (Sanger, Nicklen & Coulson, 1977). Forward and reverse sequences were assembled with Bioedit software (version 7.2.5). The obtained sequences were deposited in the GenBank with the following numbers: MG971344–MG971360 (Table 2) and aligned with the bacterial 16S rRNA gene sequences from the EZtaxon database (Kim et al., 2012; Table S1).

For phylogenetic analysis, the sequences were aligned using the MEGA software (version 7.0) (Kumar, Stecher & Tamura, 2016). The evolutionary history was inferred using the maximum parsimony method. The percentage of replicate trees, in which the associated taxa clustered together in the bootstrap test (1,000 replicates), is shown next to branches (Felsenstein, 1985).

Cultivation and extraction

The isolated strains were cultivated in 30 mL of production medium in 250 mL shake flasks with baffles for 5 days at 28 °C at 180 rpm shaking rate. Four different liquid media were chosen to estimate the metabolite production and fatty acids content. All chemicals used in this research were manufactured by Sigma-Aldrich (St. Louis, MO, USA), MP-biomedicals (Illkirch, France), and Bacto (France). These media are: NL19 (soy flour – 20 g, D-mannitol – 20 g, tap water – 1 L, pH 7.2), ISP2 (yeast extract – 4 g, malt extract – 30 g, starch – 4 g, tap water – 1 L, pH 7.3), SGG (starch soluble – 10 g, glucose – 10 g, glycerol – 10 g, cornsteep powder– 2.5 g, bacto peptone – 5 g, yeast extract – 2 g, NaCl – 1 g, CaCO₃ – 3 g, tap water – 1 L, pH 7.3). To compare production efficiency of secondary metabolites of strains cultivated in above rich media, we used minimal medium (MM) that contained only glucose as a carbon source. Composition of this medium is as follows: L-asparagine – 0.5 g, K₂HPO4 – 0.5 g, MgSO₄ × 7H₂O – 0.2 g, FeSO₄ × 7H₂O – 0.01 g, glucose – 10 g, distilled water – 1 L, pH 7.0– 7.2).

Estimation of fatty acids composition

We used gas chromatography to analyze fatty acid composition of the total fatty acid extracts of isolated strains. Fatty acid methyl esters (FAMEs) were identified using a “Chromatek-Crystall-5000.2” with a 2D sample injector (Chromatek, Yoshkar-Ola, Russia) gas chromatograph with a flame-ionization detector and a Zebron ZB-FFAP capillary gas chromatographic column. An isothermal column configuration was used. The temperature of detector and evaporator was 240 °C. The internal standard was C 22:0 FA. Chromatek-Analytik-5000.2 software was used for data recording and integration. FAMEs were identified with standard mixtures Supelco 37 FAME mix (Sigma Aldrich, St. Louis, MO, USA) and by comparing the equivalent lengths of carbon chains and table constants according to (Cabrini et al., 1992; Gago et al., 2011).

Antimicrobial activity assay of extracts from isolated strains

Antimicrobial activities of the extracted metabolites were tested using the disk diffusion method (Burdass, Grainger & Hurst, 2001). 100uL of bacterial and fungal 12-h test cultures were plated and dried on solid LB (for bacteria) and YPD (for fungi) media. Thirty uL of each crude extract dissolved in methanol was loaded on 5 mm diameter paper discs, and the disks were dried naturally. Paper disks loaded with 30uL of pure methanol were used as a negative control. Then, the disks were placed on solid LB or YPD agar media. The plates were incubated 12–24 h at 37 °C for bacteria and 30 °C for fungi (Pinheiro et al., 2018). Several bacterial and fungal test cultures, such as Bacillus subtilis ATCC 66337, Staphylococcus carnosus ATCC 51365, Pseudomonas putida KT 2440, Escherichia coli ATCC25922, Saccharomyces cerevisiae BY4742 and Candida albicans DSM1665 were chosen to test antibiological properties. The test cultures were obtained from Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig , Germany). The activity against C. albicans was estimated in intergovernmental veterinary laboratory of the federal service for veterinary and phytosanitary supervision (Irkutsk). The zones of inhibition around paper disks were measured manually with accuracy ±1 mm.

Isolation and phylogenetic analysis of actinobacteria from water surface of underground lakes in Siberia

A total of 17 culturable actinobacteria strains were isolated from water surface of underground lakes in Badzheyskaya and Okhotnichya caves based on their morphological characteristics. Twelve out of 17 strains were isolated from Badzheyskaya cave, and the other five strains were isolated from Okhotnichya cave (Table 2).

The 16S rRNA gene sequence-based phylogenetic analysis revealed that 10 out of 11 strains isolated from Badzheyskaya cave belonged to the genus Streptomyces. Also, we isolated one strain of the Nocardia genus from this cave. Five other strains were isolated from Okhotnichya cave and they belonged to Nocardia and Nocardiopsis genus. Thus, in Badzheyskaya cave, a group of actinobacteria that belonged to Streptomycetaceae family was found as a dominant group of culturable actinobacteria, unlike in Okhotnichya cave, where the dominant group of culturable actinobacteria was presented by representatives of Nocardiaceae and Nocardiopsaceae families. As Fig. 1 shows, the obtained actinobacterial isolates are clustered with reference sequences of related species. Some of our strains (Streptomyces sp. IB2014I88-6HS and Streptomyces sp. IB2014I88-7) showed a close similarity to actinobacteria previously found in caves, such as Streptomyces lunaelactis strain (Table 1, Table S1, Fig. 1). This species was isolated from a moonmilk speleothem collected in Grotte des Collemboles’ (Comblain-au-Pont, Belgium) and described as a novel producer of ferroverdin A (Maciejewska et al., 2015). Another strain—Streptomyces sp. IB2014I88-4HS—showed a close similarity with Streptomyces scabiei. The latter is known as a phytopathogen (Bignell, Fyans & Cheng, 2014). All close representatives of Nocardia sp. were presented by nonpathogenic forms. A species close to the isolated strains of Nocardiopsis was Nocardiopsis dassonvillei. The latter is reported to be a rare infection agent for humans. This agent has been implicated in cutaneous, pulmonary, eye, nasal and disseminated infections (Bennur et al., 2015; Shivaprakash et al., 2012).

Also, we compared the sequences of the isolated strains with other representatives of Streptomyces, Nocardia and Nocardiopsis genera from other caves and water substrates. As Figs. S1–S3 show, our strains related to Nocardia and Nocardiopsis genera did not form tight different clades and were characterized by low similarity to other species registered in Ez Taxon database (Table S1). Representatives of Streptomyces strains isolated from Siberian caves form clades with other representatives of strains previously found in water sources (in case of Streptomyces sp. IB2014I88-6). Also, some of isolated strains form subclades in the tree. However, in general the absence of tight clades of isolated strains could be explained by ecology of isolated actinobacteria and their common distribution in environment (Schaechter, 2009).

Analysis of biological activity of isolated strains

Antibiotic activities of the isolated strains are presented in Table 3 and Tables S2–S5. Ten (59%) out of 17 tested isolates showed antibiotic activity against at least one tested bacterial or fungal culture. The other seven (41%) strains (Streptomyces sp. IB2014I88-3HS, Nocardia sp.IB2014I88-1HS, Nocardia sp. IB2014I79-1, Nocardia sp. IB2014I79-2HS, Nocardia sp. IB2014I79-3HS, Nocardia sp. IB2014I79-4, and Nocardiopsis sp. IB2014I79-5) did not inhibit growth of any tested microorganisms under the employed conditions of cultivation. Among the seventeen isolates, only three strains (Streptomyces sp.IB2014I88-4, Streptomyces sp.IB2014I88-2HS and Streptomyces sp. IB 2014I88-1) grown in SGG and ISP2 media appeared to have a broad spectrum of antibiotic activity against all test organisms.

Eight (80%) out of ten mentioned active strains cultivated in the tested media inhibited growth of both Gram-positive and Gram-negative bacteria. Crude extracts obtained from the strains Streptomyces sp. IB2014I88-1, Streptomyces sp. IB2014I88-2HS, Streptomyces sp. IB2014I88-4 and Streptomyces sp. IB2014I88-7 were found to inhibit all Gram-positive and Gram-negative bacteria when they were grown in ISP2 medium. Five strains (Streptomyces sp. IB2014I88-2HS, Streptomyces sp. IB2014I88-2, Streptomyces sp. IB2014I88-4, Streptomyces sp. IB2014I88-7 and Streptomyces sp. IB2014I88-8) demonstrated inhibitory effects against B. subtilis, S. carnosus, E. coli and P. putida after cultivation in SGG medium. In addition, two strains—Streptomyces sp. IB2014I88-4HS and Streptomyces sp. IB2014I88-6—were active against all bacteria while cultivated in NL19 medium and MM medium, respectively.

One extract of the strain Streptomyces sp. IB2014I88-1 obtained from SGG medium was able to inhibit growth of all tested Gram-positive microorganisms but did not inhibit growth of Gram-negative bacteria. Growth of B. subtilis was hindered by three strains, including Streptomyces sp. IB2014I88-1 and Streptomyces sp. IB2014I88-4HS grown in MM medium and Streptomyces sp. IB2014I88-6HS grown in NL19 medium. We did not find specific ability of strains to inhibit growth of Gram-negative bacteria.

Eight out of ten mentioned active strains cultivated in the tested media inhibited growth of fungi. Four of them, including Streptomyces sp. IB2014I88-2HS, Streptomyces sp. IB2014I88-3, Streptomyces sp. IB2014I88-4HS and Streptomyces sp. IB2014I88-4 were active to inhibit growth of both S. cerevisiae and C. albicans. Streptomyces sp. IB2014I88-3 strain was able to inhibit growth of fungi but did not inhibit growth of bacteria under all tested conditions.

Along with cultivation of strains in nutrient-rich liquid media, growth of strains in poor MM media led to synthesis of fungicidal compounds. MM medium extracts obtained from three Streptomyces strains (Streptomyces sp. IB2014I88-3, Streptomyces sp. IB2014I88-7 and Streptomyces sp. IB2014I88-4) showed activity against yeasts. Also, some specific activity intended to hinder growth of C. albicans was found. Thus, strains Streptomyces sp. IB2014I88-1, Streptomyces sp. IB2014I88-2 and Streptomyces sp. IB2014I88-7 inhibited growth of pathogenic C. albicans but did not inhibit growth of S. cerevisiae. Also, a multiple activity of strains against fungi was observed in minimal nutrient media.

Estimation of fatty acid composition in isolated strains

In this study we estimated 60 parameters for each extract. Here, we present the summary data. When assessing the fatty acid (FA) profile, we found that polyunsaturated fatty acids (PUFA) were dominant quantitatively in the extracts of Nocardia and Streptomyces grown in different media. Saturated fatty acids (SFA) were the second most abundant type in the fatty acids profile. It was due to palmitic acid. Also, a few monounsaturated fatty acids (MUFA) were detected (Figs. 2–7; Table S6).

It was determined that fatty acid profiles of Nocardia and Streptomyces strains were significantly different. Primarily, it is linked to the cell wall composition of those microorganisms and their biosynthetic capabilities. Significant differences of total PUFA due to dominant class (n − 4) PUFA and minor (n − 6) PUFA were shown for extracts from cell biomass. Total amount of (n − 4) PUFA in cell biomass of Nocardia genera was 30.74–51.07%, while total amount of (n − 6) PUFA was 0.51–4.16%. Regarding representatives of Streptomyces genera, it was demonstrated that total amount of (n − 4) and (n − 6) was lower and varied in the range 23.12–41.33% for (n − 4) and in the range 0.44–1.83% for (n − 6). It was established that PUFA prevailed in the FA profile of the investigated bacteria. Besides, the fatty acids (n − 4) family prevailed in the PUFA because of C22:5 (n − 4). In terms of amount, SFAs took the second place (Fig. 2).

FA profile of cultural liquids of Nocardia and Streptomyces was characterized by SFA dominance. The SFA level for Nocardia was 40.75–63.85%, while for Streptomyces it was 17.22–62.69%. The second most abundant class was MUFA. MUFA levels in representatives of Nocardia sp. varied in the range 13.75–47.01% of overall fatty acid content. For Streptomyces spp. strains, it varied in the range 3.71–47.01% of the overall fatty acid content. Also, it should be noted that we detected significant amount of MUFA –C18:1 (n − 9), oleic acid, and C16:1(n − 7), palmitoleic acid. Figure 3 shows FA profile of cultural liquid of the isolated Nocardia and Streptomyces strains. Differences between those two genera were determined in terms of (n − 9) and (n − 3) PUFA (Fig. 3).

Assessing the fatty acid content in cultural liquid of Nocardia strain cultivated in different media, we found transgression of FA profiles. Thus, the FA profile obtained in case of bacterial growth in NL-19 medium significantly differs in terms of SFA and short chain fatty acids (SCFA) (Fig. 4).

FA profile of Nocardial cell walls grown on NL-19 medium was also different from FA profile of bacteria grown in other media (Fig. 5). Increased level of dominant (n − 4) PUFA as well as (n − 3) PUFA and (n − 6) PUFA was detected in that medium. At the same time, after analysis of FA levels of cultural liquid of Streptomyces it was established that the FA profile was greatly different as compared with samples grown in MM medium (Fig. 6).

Analysis of medium effects on the FA profile of cell biomass in Streptomyces revealed that bacteria growth on MM and ISP2 media is characterized by common FA profile in comparison to FA profile of bacteria cultured in NL-19 and SGG media. The latter pair has the minimal transgression (Fig. 7).

Thus, it was shown that despite the lack of antimicrobial and fungicidal activity of Nocardia extracts, those strains were exceptional in terms of their FA spectrum. Nocardia isolated from underground lakes of the caves contained high levels of PUFA from different classes, mainly (n − 4) and SFA, in cultural liquid. PUFA was dominant in cell biomass.

Even though many fatty acids possess antimicrobial activity, we cannot link antibiotic activity of the strains with the particular type of fatty acids. The activity of Streptomyces strains might be linked to both major and minor fatty acids including MUFA (Agoramoorthy et al., 2007; Gołębiowski et al., 2014). The most abundant monounsaturated fatty acids are palmitoleic and oleic acids. They are precursors of polyunsaturated fatty acids of the (n − 7) and (n − 9) families, respectively.

We found that oleic acid 18:1 (n − 9) prevailed in MUFA. The level of oleic acid ranged 11.89–19.88% of total FA in medium MM in the strains Streptomyces sp. IB2014I88-2, Streptomyces sp. IB2014I88-1, and Streptomyces sp. IB2014I88-3. It was the highest level of FA in comparison with other media. At the same time, concentration of oleic acid 18:1 (n − 9) was 18.58% of total FA in strain Nocardia sp. IB2014I79-3HS grown in SGG medium.

It should be noted that the cultural liquid of strains Streptomyces sp. IB2014I88-6HS and Streptomyces sp. IB2014I88-4HS grown on SGG medium contained another MUFA –palmitoleic acid, C16:1 (n − 7) in levels 22.04% and 19.91% of total FA, respectively. The high content (21.45%) of that fatty acid was noted for the Streptomyces sp. IB2014I88-4HS strain grown in MM medium.

During the present study it was shown that the obtained strains extracellularly synthesize linolenic acid C18:3 (n − 3) that is essential for humans. For the strains Streptomyces sp. IB2014I88-1HS, Streptomyces sp. IB2014I88-2HS, Nocardia sp. IB2014I79-1HS, Nocardia sp. IB2014I79-2HS and Nocardia sp. IB2014I79-3HS grown in NL-19 medium, the linolenic acid level was 23.70–26.19% of total FA; it was the highest level in comparison with other media. Besides, the level of linoleic acid in the strains Streptomyces sp. IB2014I88-2, Streptomyces sp. IB2014I88-3, Nocardia sp. IB2014I79-1, Nocardia sp. IB2014I79-4 grown in the same medium was 13.51–25.31% of total FA in comparison with other PUFA.