Optimization of the antifungal metabolite production in Streptomyces libani isolated from northern forests soils in Iran

Background and Purpose: Soil bacteria have extreme population diversity among natural sources and are able to produce a wide array of antifungal metabolites. This study aimed to isolate and identify the bioactive metabolite-producing bacteria from forest soils and evaluate their antimicrobial potent against some pathogenic organisms. Materials and Methods: In this study, soil samples were screened for antifungal activity against Aspergillus fumigatus on glucose-yeast extract (GY) agar using a visual agar plate assay method. All growing bacteria were examined for antifungal activity, and antagonistic bacteria were identified based on 16S ribosomal RNA sequence analysis. For optimization of the production of antifungal bioactive metabolites, inhibitory bacteria were cultured on different culture conditions, including media, pH, temperature, and incubation time. Results: In total, 110 bacterial strains were isolated from the forest soils and four species with high antifungal activity were identified as Streptomyces libani, Streptomyces angustmyceticus, Bacillus subtilis, and Sphingopyxis spp. on the basis of 16s ribosomal RNA sequencing. Dichloromethane extract of the starch casein broth culture filtrate of the S. libani (incubated at 30° C for five days) showed strong antifungal activity against A. fumigatus, Aspergillus niger, and Aspergillus flavus. Conclusion: Based on the results, forest soils contain organisms with antifungal activity and could be considered as a good source for novel antifungal metabolites as effective and safe therapeutics.


Introduction
spergillosis is an infection caused by Aspergillus species. Most commonly, it occurs in the form of chronic pulmonary aspergillosis (CPA), aspergilloma, allergic bronchopulmonary aspergillosis (ABPA), and invasive aspergillosis [1]. Invasive form of the disease only occurs in severely immunocompromised patients, such as organ transplant recipients, hematopoietic stem cell transport, and patients taking chemotherapy or taking corticosteroids [2]. Aspergillus fumigatus is the most prevalent etiologic species that is mainly responsible for increasing the incidence of invasive aspergillosis in immunocompromised patients [3]. Invasive aspergillosis has been reported in 2-26% and 1-15% of hematopoietic stem cell and organ transplant recipients, respectively [4]. Synthetic antifungal medications and fungicides, particularly azole group medications, have been used to treat aspergillosis [1,5]. Despite the effectiveness of azoles, polyenes, and echinocandins as therapeutics for clinical forms of aspergillosis, there are still some limitations, including life-threatening side effects, an increase of the antifungal resistant species, and undesirable effects on non-target beneficial microorganisms that are shared in the ecosystem [1,5,6]. Amphotericin B; newly developed azoles, such as voriconazole and posaconazole; and the echinocandins are active against Aspergillus spp. However, the resistance of Aspergillus isolates to amphotericin B, itraconazole, voriconazole, and caspofungin have also been reported in some patients [1,5,6]. Management of invasive aspergillosis continues to be challenging, and the mortality rate remains unacceptably high; therefore, there is a need for the development of new, safe, and more effective antifungal antibiotics that will reduce the damage to human health and the ecosystem around them.
In recent years, natural products have widely been considered for the development of new antimicrobial agents with the potential for the treatment of fungal infections [7]. According to the Antimicrobial Peptide Database, 959 antifungal peptides have been isolated and chemically synthesized from various sources, such A Curr Med Mycol, 2020, 6(4): 20-26 21 as soil bacteria [8]. Soil bacteria have received major consideration due to their extreme population diversity and production of a variety of antifungal compounds. They mainly belong to the genera Streptomyces, Bacillus, and Pseudomonas [9][10][11][12][13]. According to different studies, the genus Streptomyces produces approximately 75-80% of the total antibiotics produced by microbes, such as nystatin, amphotericin B, natamycin, bafilomycin A1, concanamycin, and 3phenylpropionic acid [14][15][16]. This study aimed to screen and identify the antifungal metabolitesproducing bacteria isolated from Northern forest soils in Iran and evaluate their antimicrobial potent against some pathogenic organisms.

Fungal and bacterial strains
The

Isolation of bacteria with antifungal activity from bioactive soil samples
Soil samples with antifungal activity in initial screening were cultured on GCA and incubated at 30º C for seven days. Inhibitory effect of bacterial isolates against A. fumigatus growth was tested by visual agar plate assay according to Ranjbariyan et al. [13]. The antifungal activity of each bacterium was analyzed by determining the fungal growth zone of inhibition. The bacteria with the highest activity were stored in 20% glycerol (20%, v/v) at -20° C.

Molecular identification of antifungal-producing bacteria (DNA extraction, PCR, and sequencing)
The DNA extraction was performed according to Kumar et al. with slight modifications [19]. The selected antifungal-producing bacteria were inoculated in 100 ml starch casein broth for four days at 28º C, pH 7 in 500 ml flasks on a rotary shaker (120 rpm). The media were centrifuged (8000 rpm for 6 min) and biomass was harvested, washed twice with sterile distilled water, and suspended in 800 µl lysis buffer (100 mM Tris-Hcl, pH=7, 20 mM EDTA, 250 mM NaCl, 2% SDS, and 1mg/ml lysozyme). Purity of genomic DNA was checked by gel electrophoresis 1% agarose. The 16S ribosomal DNA sequences were amplified using 0.5 µl from universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-CGGTTACCTTGTTACGACTT-3′), 12.5 µl master mix, 1 µl DNA, and 10.5 µl distilled water in a thermal cycler (TC-E-96G, China). The conditions were as follows: initial denaturation at 95° C for five min, followed by 35 cycles of denaturation at 94° C for 30 s, annealing at 58° C for 30 s, extension at 72° C for 70 s, and a final extension at 72°C for five min; eventually, the PCR product was kept at 4°C. The amplified products were confirmed by electrophoresis on 1% agarose gel [20].
The PCR products were sequenced using the forward primer which was used for amplification by ABI prism Big Dye Terminator v3.1 Cycle Sequencing Kit (manufactured in Applied Biosystems, USA). The highest sequence similarity of the 16s ribosomal RNA sequence of isolates with the reference species of bacteria was confirmed by using the NCBI BLAST tool (http://WWW.ncbi.nlm.nih.gov/ BLAST).

Optimization of antifungal compounds produced by inhibitory bacteria
In order to optimize the cultural conditions and to identify the best culture broth for the production of inhibitory bioactive metabolite against the different

Evaluation of the antifungal activity of optimized culture filtrate
The antifungal activity of all culture filtrates was examined against A. fumigatus by the agar well diffusion method [22]. Fungal strains (2×10 5 spores/ml) were swab cultured on the SDA plates. Wells with diameters of 6 mm were made in the center of SDA plates, filled with 100 µl of bacteria culture filtrates, and incubated at 30º C for 5 days. The SCB, ISP-2B, and GYB media without organism were used as control. Zones of fungal growth inhibition around the central well were measured at the end of the incubation period. It should be noted that all experiments were performed in triplicate sets.

Solvent extraction of antifungal compounds from Streptomyces libani
Antifungal metabolites were extracted using several organic solvents, including ethyl acetate (EtOAc), acetone (AC), dichloromethane (DCM), ethanol (EtOH), and methanol (MeOH). For this purpose, the solvents were separately added to the culture filtrate in the ratio of 1:1 (v/v), shaken vigorously for 45 min, and concentrated using a rotary evaporator (IKA WERKE, Germany) at 45° C [22,23]. The residues were dissolved in 1 ml of MeOH and examined against A. fumigatus, A. flavus, A. niger, C. albicans, C. glabrata, E. coli, and S. aureus using the agar well diffusion method as described above while methanol was used as the only solvent control.

Statistical analysis
The collected data were analyzed in GraphPad Prism software (version 6) using ANOVA at a significance level of less than 0.05.

Soil sampling and isolation of bacteria with antifungal activity from selected soil samples
Out of the 45 soil samples screened for antifungal activity against A. fumigatus., 30 soil samples (66.66%) were found to have an inhibitory effect against A. fumigatus. Figure 1A shows the inhibitory (a) and non-inhibitory (b, c, and d) soils in the initial screening against A. fumigatus. Soils that contained inhibitor organisms were isolated on the basis of their morphological characteristics on GCA plates and gram staining. A total number of 110 bacteria isolated from 30 samples of inhibitory soils were classified into four groups, including high (4 isolates, 3.6%), moderate (40 isolates, 36.36%), low (21 isolates, 19.09%), and negative (45 isolates, 40.9%) activity, based on the visual agar plate assay ( Table 1). As shown in Figure  1B, a clear zone of fungal growth inhibition is obvious for one bacterium (d), while the other three tested bacteria (a, b, and c) were not able to inhibit fungal growth. Finally, four isolates named G-92, H-57, I-65, and I-71 with strong inhibitory activity against A. fumigatus growth were selected for further studies.

Identification of antagonistic bacteria
The genomic DNA of the bacterial isolates with antifungal activity were successfully amplified by PCR using primers 27F and 1492R. After the electrophoresis of the PCR product on the agarose gel 1%, a fragment of 1500 bp was observed. On the basis of 16s ribosomal RNA sequencing results, they were identified as

Optimization production of antifungal compounds
The results obtained from the optimization of production conditions of the antifungal compound based on the type of culture medium, pH, temperature, and incubation time showed that the maximum production of antifungal compounds was in the SCB medium with pH 7 at 30º C for 5 days (Figures 2, 3, and 4). The highest zone of inhibition against A. fumigatus (46 mm) was related to the culture filtrate of S. libani I-65.

Determination of the antimicrobial spectrum of solvent extracts
The antifungal activity of S. libani solvent extracts was tested by the agar well diffusion method. All the extracts exhibited varying degrees of inhibition zone against Aspergillus species (within the range of 6-38 mm). The DCM extract showed the strongest antifungal activity which had a statistically significant difference with all the other solvent extracts (P<0.05). The MeOH and EtOH extracts showed the same level of inhibition against fungal growth (P>0.05) and the AC extract showed minimum antifungal activity (P<0.05). The DCM, EtOAc, MeOH, and EtOH extracts of S. libani SCB culture filtrate inhibited the mycelial growth and

Discussion
Aspergillosis is an opportunistic fungal infection that has a high mortality rate among fungal diseases in the world. Increase of triazole resistance in clinical isolates of A. fumigatus and their limited effect and toxicity are the cause of mortality and morbidity in aspergillosis [24,25]. In these cases, the mortality rate was reported to be 88% [24,25]. Discovery of new medications with more efficiency and less toxicity and resistance is necessary for the treatment of mycotic diseases, especially aspergillosis.
The soil-inhabiting bacteria, particularly Streptomyces genus, are able to produce diverse bioactive compounds and are used for producing metabolites, such as peptides, macrolides, aminoglycosides, polyenes, polyethers, tetracyclines, and βˍlactams with antimicrobial activity [16,26,27]. In several studies, the production of antifungal metabolites, such as bafilomycin A1, oligomycin, geldanamycin, nikkomycin, and reveromycin A and B, has been reported by various strains to belong to the genus of Streptomyces [27][28][29]. Most of these compounds have polyketide and peptide structures and have inhibitory activity against filamentous fungi. Therefore, this study aimed to isolate and identify the antifungal-producing bacteria with emphasis on soil Streptomyces since many Streptomyces populations inhabit soils.
In the present study, 110 bacterial isolates were isolated from 45 soils from the northern forests of Iran.  [30]. Preliminary screening of antifungal-producing bacteria revealed that four isolates (3.63%) had high inhibitory activity against A. fumigatus. The antagonistic bacteria were identified as Streptomyces libani, Streptomyces angustmyceticus, Bacillus subtilis, and Sphingopyxis sp. As these bacteria have been reported to produce antifungal metabolites, various culture conditions were tested to optimize the production of antifungal metabolites by actinomycetes [28,30,31,32].
In this study, the most antifungal activity was observed in SCB culture medium with pH 7, at 30º C for five days. It has been reported by several researchers that starch and casein are the best sources of carbon and nitrogen for the production of antibiotics [20,33]. Reddy et al. observed that peptone followed by casein can be the best nitrogen source for the production of the antimicrobial metabolite from Streptomyces rochei [32]. In addition, Aliero et al. showed yeast extract starch broth was a good medium for the production of antimicrobial metabolites from an actinomycete isolate KBMWDSb6 [30].
Temperature and pH are important factors for the production of the antifungal metabolite by species of Streptomyces genus [20,30]. Reddy et al. found that maximum antifungal metabolite was produced against C. albicans at a pH of 7.5 at 32°C for five days [32]. Kumar  reported that S. libani has the ability to produce oligomycin in soluble starch broth medium (SSB), at pH 7, 30º C for 12 days. This compound had an inhibitory effect on the mycelia growth of Cladosporium cucumerinum, Colletotrichum lagenarium, and Botrytis cinerea from 3-5 µg/ml of minimum inhibitory concentration [26].
Antifungal compounds have been extracted from Streptomyces culture filtrate by organic solvent. In this study, the DCM extract of the S. libani SCB culture showed significant antifungal activity against Aspergillus species. Moreover, the maximum zone of inhibition was observed at 38 mm. The EtOAc extract shows less activity than the DCM extract, and EtOH and MeOH extracts showed minimum antifungal activity. However, Bssn et al. reported that EtOAc extract of Streptomyces lavendulocolor VHB-9 YMD has significant antifungal activity against A. niger, A. flavus, Fusarium solani, P. Citrinum, and C. albicans. The bioactive compounds produced by S. lavendulocolor VHB-9 were Bis (7-methyloctyl) phthalate and (Z)-3aminoacrylic acid [34]. Aouiche et al. found that Streptomyces spp. PAL114 ISP-2 DCM extract has strong antifungal activity against A. carbonarius, A. flavus, Fusarium culmorum, Penicillium glabrum, and Candida sp. The inhibitory compounds were identified as saquayamycins A and C [35]. Khebizi et al. reported that DCM solvent of the S. gancidicus beef extract glucose broth (BEG) culture has good antifungal activity against Fusarium and Penicillium species and two active metabolites were identified as oligomycins E and A [26].

Conclusion
Bioactive metabolites of microbial origin are now widely considered as novel therapeutics in the treatment of fungal infections. The S. libani that was isolated from forest soils of Northern regions of Iran was able to produce antifungal compounds against Aspergillus species, particularly the pathogenic species A. fumigatus. It is recommended to purify and identify the bioactive antifungal compounds from S. libani, which was introduced in this study, as potential sources for designing novel antifungal therapeutics.