Bioactive compounds of the Actinomycetes isolated from Garmsar Saline River, Iran

doi:10.21203/rs.3.rs-4184934/v1

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Bioactive compounds of the Actinomycetes isolated from Garmsar Saline River, Iran

https://doi.org/10.21203/rs.3.rs-4184934/v1

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Saline environments are unique ecosystems that harbor novel bacterial species with the potential to produce valuable secondary metabolites. In the present study, 26 actinomycetes strains were recovered predominantly from the rhizosphere (81%), in addition to sediments and water samples collected from four sites along the Garmsar Saline River in Iran. According to phylogenetic analysis, the isolated strains were identified as belonging to two different genera: Streptomyces, and Nocardiopsis. Several of the isolated actinomycetes exhibited valuable biotechnological potential, producing a diversity of enzymes including amylase, gelatinase, protease, lipase, lecithinase, and laccase. Inhibitory effects on the growth of human tumor cells were also observed; the extract of strain M15 displayed notable cytotoxicity against MCF7 human breast cancer cells with an IC50 value of 32.81 µg/ml. Also, 59% of the strains had antibacterial activity against some pathogenic and non-pathogenic test strains. Ethyl acetate extracts of the selected actinomycetes were screened, and strain M16 was found to exhibit the most potent inhibitory effect on Proteus mirabilis. GC/MS analysis of M16’s extract revealed it mainly consisted of 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (16.61%), and 9-octadecenamide (12.77%). Based on 16S rRNA sequence analysis, Strain M16 showed the highest similarity to Nocardia aegyptia. The study concludes that actinomycetes from the Garmsar Saline River possess considerable potential for biotechnological application.

Anticancer

Antimicrobial

Dual culture

enzyme

Halophiles

Screening

Actinomycetes have been isolated from terrestrial and aquatic environments for decades constituting a main branch of microbial diversity with significant industrial applications. They are renowned for producing valuable metabolites such as enzymes, antimicrobial, anticancer, antioxidant agents, and plant growth-promoting substances (Ezeobiora et al., 2022).

Notably, 61% of all modern-day bioactive microbial metabolites have been isolated from streptomycetes (Vigneshwari et al., 2021). The escalating resistance of pathogenic bacteria to antibiotics underscores the need to explore new species of actinomycetes for developing novel bioactive scaffolds, natural compounds, and microbial metabolites (Selim et al., 2021). Unexplored ecosystems present a promising reservoir of novel actinomycetes that may be capable of producing new secondary metabolites (Farda et al., 2022; Trivedi and Thumar, 2021).

Recent research highlights the value of isolating halophilic or halotolerant bacteria from saline ecosystems, such as aquatic habitats, salt lakes, and saline soils. Studies on the diversity of halophilic and halotolerant actinomycetes have unveiled considerable novelties and diversities within these groups (Malviya et al., 2014), including their prolific production of antimicrobial metabolites (Tsavkelova et al., 2005).

The diversity of culture media can greatly impact the isolation of different actinomycete strains, potentially increasing the yield several-fold. This diversity stems from the varying chemical compositions of different ecosystems and the unique nutritional requirements of each bacterial strain (Oskay, 2009). Furthermore, as the genus Streptomyces typically grows faster than other actinomycete genera, the employment of various pretreatments can promote the isolation of non-Streptomyces genera (Ezeobiora et al., 2022).

Garmsar Salin River is an unexplored ecosystem in Semnan province, Iran, with no reports on its microbial diversity. This river originates from the southern slopes of Alborz Mountain and flows into the Desert plain. The river is sweet by itself, but after passing through salt marshes, it becomes saline. The river is characterized by its shallow and broad nature (Safari et al., 2021). To date, no studies have been conducted on the diversity of halophilic actinomycetes in this area. In this study, we examine actinomycetes isolated from the Garmsar River utilizing various treatments and culture media to isolate an extensive array of actinomycete strains. Additionally, we investigate their biochemical characteristics and potential for secondary metabolite production, including enzyme activity as well as anticancer and antimicrobial activities.

Sample collection, Media Cultures, and Strain Isolation

The Garmsar Saline River is an intact ecosystem, is located at 35° 28′ N latitude and 52° 14′E longitude. Samples were collected from from four predetermined locations indicated on the map (Fig. 1). These samples encompassed river water (Fig. 1, location 1), Sediments from two different locations from depths of 5 cm (Fig. 1, location 2 and 4) and the rhizosphere of flora adjacent to the river bank(Fig. 1, location 3). The pH range of the samples varied from 6.9–8.2.

Soils and sediment samples were air-dried and ground. Several pretreatments were applied to the samples. including: T1. dry heat (100°C, 60 min) and T2. wet heat (50°C, 15 min) as physical treatments and T3. 0.05% SDS and 6% yeast extract for 30 min as a chemical treatment (Jayashree et al., 1991). Serial dilutions of the pretreated samples were prepared with a 2% (w/v) sodium chloride solution down to 10^− 4. Finally, these samples, along with the water sample, were cultured on two different selective media.

The rich media consisted of 1. Starch Casein Agar (SGA) (g/L); starch, 10; casein, 0.3; K2HPO4, 2; KNO3, 2; NaCl, 2; MgSO4·7H2O, 0.05; CaCO3, 0.02; agar, 15; and 2. ISP-2 agar (g/L): yeast extract, 4; malt extract, 10; glucose, 4; agar, 15. Additionally, the oligotrophic medium was soil extracts (SE) agar: twice sterilized precipitated soli suspension 400, yeast extract 10, agar 15 (Hong et al., 2009). The pH of these culture media was adjusted to 8.0. Each medium was supplemented with 30 mg/L nystatin and 10 mg/L nalidixic acid. Different sodium chloride concentrations (0, 5, and 10% w/v) were incorporated into the media. Cultures were incubated at 28°C for 1 to 8 weeks. Actinomycete-like colonies growing on the plates were inoculated onto SC agar plates and incubated for 7 d at 28°C. Purified cultures were preserved in 30% glycerol at – 20°C (Hong et al., 2009).

To distinguish between halophiles and halotolerant isolates, a Modified Sea Water (SW) agar medium was used. This medium contained 0.5% (w/v) yeast extract, 15 g/L agar, and varied NaCl concentrations ranging from 0 to 15% (w/v), using water from the lake to make up 1 L of the medium. Moderate halophiles demonstrated optimal growth at NaCl concentrations of 3.5–15%, whereas isolates capable of growing in both high and no salinity conditions were considered halotolerant (Kushner and Kamekura 1988).

Screening for the production of extracellular hydrolytic enzyme activity

The investigation focused on screening for the production of extracellular hydrolases, including amylase, protease, lipase, peroxidase, and lecithinase. All media were supplemented with either 0% or 5% (w/v) sodium chloride, and their pH was adjusted to 8.0.

The enzyme activities of the actinomycete strains are detailed as below:

Amylase activity was screened using starch agar medium. A solution comprising 0.3% I2 and 0.6% KI indicated amylolytic activity in the cultures. A clear zone surrounding the colonies confirmed starch hydrolysis. To determine proteolytic activity, skim milk agar containing 10% (w/v) skim milk was employed. A clear zone observed around the growth confirmed the presence of proteolytic activity. Lipase activity was assessed using nutrient agar supplemented with 1% (v/v) Tween 20. The appearance of a precipitation zone around the colonies indicated lipase production. For lecithinase production, egg yolk agar was utilized. A white opaque precipitation zone surrounding the colonies signified positive lecithinase activity (Tille and Forbes 2014). laccase enzyme production was determined using SC agar supplemented with 0.02% (v/v) guaiacol. A reddish-brown zone around the colonies was indicative of laccase activity (Musengi et al., 2020).

Cytotoxicity screening of actinomycete strains

Actinomycetes were investigated for their cytotoxic activity against the MCF7 human breast cancer cell line. The strains were cultured in SC broth at 28°C, 220 rpm, and 7 days. Using ethyl acetate as a solvent to extract possible cytotoxic metabolites of the filtrate-fermented medium. Cell viability was assessed with an MTT reduction assay (Loosdrecht et al, 1994). MCF7 cells were seeded in 96-well culture plates at 0.1 × 10⁵ cells/well in (DMEM/F12, Bioidea, Iran) medium and allowed to attach for 24h. The cells were treated with different concentrations of ethyl acetate extracts (0, 25, 50, 75, 100, 150, 200, 500 µg/ml) for 48 h. Then the medium was removed and the cells were incubated with 5 mg/ml of 3- [4, 5 dimethylthiazol-2-yl]-2, 5- diphenyltetrazolium bromide (MTT) (Sigma, USA). After incubation for 3–4 h at 37°C and 5% CO2, the amount of formazan deposits after dissolving in DMSO was quantified at 570 nm (Van Lossdrecht et al., 1994). Normal MCF10 cells were used as a control. All the experiments were conducted in triplicate. The statistical significance of the data was analyzed with one-way ANOVA (P < 0.05.). The cytotoxic activity was expressed as mean IC50 ± standard error (SE). The IC50 values of the samples were calculated using the software GraphPad PRISM version 6 (GraphPad Software, San Diago, CA).

Evaluation of antimicrobial activity using a modified spektra‑plak method

The antimicrobial activity of isolated actinomycetes was evaluated with a modified spektra-plak method against different indicator microorganisms, including Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591, Escherichia coli ATCC 29998, Kocuria rhizophila ATCC 9341, Salmonella typhi ATCC 6534, Bacillus cereus ATCC 11778, Pseudomonas aeruginosa ATCC 2853, obtained from the Persian Type Culture Collection (PTCC). Indicator bacteria were cultured in Nutrient Broth (NB) for 24 h at 37°C. Mueller Hinton Agar (MHA) was used for the antibacterial assay. A small circle of the actinomycete strains were cultured at the center of the plate and incubated at 28°C for 4 d. The plates containing grown isolates were overlayed with one ml of NB containing 0.3% (w/v) agar and one of the tested strains. The inoculation sizes for the indicator bacterial strains were 10⁴ CFU/ml. The formation of the inhibition zone showed the antagonism relationship and the size of that was measured (Salehghamari and Najafi 2016; Madigan et al., 1997). All experiments were performed in triplicate.

Production, extraction, and identification of antibacterial metabolite

Some of the actinomycete isolates which showed inhibition zone larger than 25mm diameter against Proteus mirabilis were incubated in Starch casein broth (SC agar) (g/L); Soluble starch, 20; K2HPO4, 0.5; KNO3, 1; MgSO4·7H2O, 0.5; at 28°C, 220 rpm for 4 d. Ethyl acetate (1:2 v/v) was used as a solvent for antimicrobial metabolite extraction for two hours. The solvent layer which may include the bioactive metabolite, was evaporated using a rotary evaporator at 40°C. The crude extract was tested for antimicrobial activity against P. mirabilis on Mueller Hinton agar overnight at 37°C and 28°C respectively. The presence of an inhibition zone is considered indicative of bioactive metabolite existence (Sengupta et al., 2015).

To identify the antimicrobial metabolite, the ethyl acetate extract of the selected isolate was subjected to a gas chromatograph (Agilent 7890A) equipped with an HP-5MS column (diameter 0.25 mm, length 30.0 m, film thickness 0.25µm) mass spectrometer (Agilent 5975, EI 70 eV). The initial temperature was set at 50°C and increased to 230°C at a rate of 10°C/min, then to 270°C at a rate of 30°C/min and kept for 10.667 min. The total run time was 30 min. The injector temperature was set at 250°C and a flow rate of 1 ml/min.

Dual culture of actinomycete strains

Starch casein agar was used to test actinomycete's effects on each other’s antibiotic synthesis against MRSA. Two isolates were cultured 0.5 cm away from each other, and they were also cultured alone as controls at 28°C for 5 d (Seyedsayamdost et al., 2012; Salehghamari et al., 2017). When the control colonies started development, seven ml of LB containing 0.3% (w/v) agar and 10⁶ CFU/ml of an overnight culture of MRSA were poured over the interaction assay and control plates. The plates were incubated overnight at 37°C and checked for the creation of the inhibition zone. This experiment was performed in triplicates.

Molecular identification and phylogenetic relationship analysis

Actinomycete strains were cultured in SC broth and incubated at 28°C, 220 rpm for 4 d. The cells were harvested and their genomes were extracted as described by Kieser et al. (2000). Their 16S rDNA was amplified using PCR with pfu DNA polymerase and the primers 9F (5’ AAG AGT TTG ATC ATG GCT CAG 3’) and 1542R (5’ AGG AGG TGA TCC AAC CGC 3’). The reaction mixture (100 µl) contained 1 µl of genomic DNA, 0.5 µl of pfu DNA polymerase, 0.2 mM of dNTP, and 0.5 mM of each primer in a final volume of 30 µl. The reaction was started with an initial denaturation at 95°C for 300 seconds followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 54.5°C for 60 seconds and extension at 72°C for 90 seconds, with a final extension at 72°C for 300 seconds.

Agarose gel electrophoresis was used to analyze the PCR products. They were submitted for purification and sequencing to Macrogen Inc. (Seoul, Korea). The identification of the phylogenetic relationship of the isolates and the calculation of pairwise 16S rDNA sequence similarities were determined using the Eztaxon server (http://www.ezbiocloud.net/eztaxon). Sequences were aligned using CLUSTAL X software (version 2.0, Conway Institute, USA), and a phylogenetic analysis was performed by the neighbor-joining method using MEGA software (version 6.0, Biodesign Institute, USA). Mycobacterium avium MG13T was used as the out-group. The bootstrap analysis was used to evaluate the tree topology by performing 1,000 replicates (Salehghamari et al., 2017). Partial 16S rRNA gene sequences (> 1000 nt) of the isolates M3, M4, M5, M8, M9, M10, M12, M16, M19, M25 were deposited in the GenBank database under the accession numbers OR603161, OR889128, OR889129, OR603162, OR616766, OR603163, OR739420, OR616767, OR739423, and OR739424 respectively.

Actinomycetes screening media and treatments

A total of 26 actinomycete strains were isolated from the environmental samples: 81% from the rhizosphere, 13% from lake sediments, and the remaining 6% from water. Of these, 87% formed gypsum-like colonies, while the remainder produced leather-like colonies. Among the isolates, 29% grow optimally on 10% (w/v) NaCl, and 51% on 5% (w/v) NaCl. Other strains grew optimally without NaCl, but could tolerate its presence in the medium.

To isolate as diverse a population of actinomycetes as possible, three different physical and chemical pretreatments were applied to the environmental samples. Following these pretreatments, 27% of the isolates exhibited unique colony morphologies, such as leather-like colonies with dark yellow, black, and brown pigments; these were obtained using the chemical treatment SDS. Physical treatments, specifically wet and dry heat, resulted in the isolation of most strains, 47% and 26% respectively (Fig. 2).

To screen the actinomycete isolates, three different culture media were utilized. The highest number of strains was purified from SC agar medium (65%), followed by ISP2 (21%). Soil extract agar, as an oligotrophic medium, isolated the fewest actinomycete strains (14%) (Fig. 2). All isolates produced aerial mycelium on SC agar medium in various colors: white (59%), yellow (22%), brown (15%), and black (4%).

Ten strains were selected for 16S rRNA sequence analysis based on characteristics such as aerial mycelial color and colony morphology: three isolates each from the white, yellow, and brown groups, and one isolate with black coloration (Fig. 3 and Fig. S1). Phylogenetic analysis revealed that these strains were 98–100% similar to their nearest matching type strains. Eight strains belonged to the genus Streptomyces, while two were identified as members of the genus Nocardiopsis. For instance, isolate M25, the most anti-microbial producer against MRSA displayed high sequence similarity to Streptomyces microflavus NBRC 13062. Similarly, isolate M3 showed 99.84% similarity to S. microflavus NBRC 13062, while isolate M8 presented a 100% match with Streptomyces tendae ATCC 19812. Likewise, isolates M4 and M5 both showed 100% similarity to S. tendae. Other isolates, M9 and M16, exhibited 100% and 98.95% similarity, respectively, to Nocardiopsis aegyptia DSM 44442. The partial 16S rRNA gene sequences of isolates M10 and M12 demonstrated high similarities of 100% and 99.67% to Streptomyces sparsus YIM 90018 and Streptomyces violaceochromogenes NBRC 13100, respectively. Isolate M19 showed a 100% match with Streptomyces thinghirensis DSM 41919.

Screening of the extracellular enzyme production in actinomycetes

Screening for the production of extracellular enzymes revealed that 95% of the isolates produced the amylase enzyme (Fig. 4). Similarly, 62%, 58%, 50%, 46%, and 32% of the strains generated gelatinase, protease, lipase, lecithinase, and laccase, respectively. None of the strains produced urease. Many strains were capable of producing more than one enzyme; for instance, 21% and 29% of the strains could produce five and four enzymes, respectively. Strains of the genus Nocardiopsis consistently produced three of the tested enzymes.

Cytotoxic activity of the secondary metabolites extracted from the actinomycetes

The cytotoxic effects of actinomycetes extracts were evaluated on MCF7 cells treated with 0–500 µg/ml extract for 48 hours. The MTT assay determined cell viability, which demonstrated a concentration-dependent decrease in cell viability upon exposure to the extracts (Fig. 5). The IC50 doses for extracts from actinomycetes M10, M11, M14, and M15 were calculated as 289.6, 248.5, 182.5, and 32.81 µg/ml, respectively.

Antagonistic activity of the isolated halophilic actinomycetes

The antibacterial metabolite production against six tested bacteria was investigated highlighting that 59% of the strains produced at least one antimicrobial compound (Fig. 6). The most significant antibacterial production was observed against K. rhizophila (59%). No strain showed anti-bacterial activity against S. typhi.

Isolates M5 and N. aegyptiae M16 which had the largest inhibition zones against P. mirabilis on agar plates were selected for metabolite extraction. The extract from strains M16 exhibited the most substantial antibacterial activity against the pathogen. GC-MS analysis identified 1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester (16.61%) with an RT of 15.81, and 9-octadecenamide (12.77%) with an RT of 22.81, respectively (Fig. S2).

Binary actinomycete interactions against MRSA

The dual culture method investigated the induction of anti-MRSA metabolite production in actinomycete strains with no initial antimicrobial activity against MRSA (Fig. 7). Only one strain, S. violaceochromogenes M12, was induced to produce an anti-MRSA metabolite by the strain M2.

Actinomycetes play a crucial role in the production of various bioactive secondary metabolites. Research has shown that hypersaline soil, rhizosphere soil, marine sediments, and deserts are rich sources of diverse secondary metabolites due to the presence of Actinomycetes species, especially those found in near-extreme conditions, which are highly valued for their potential in novel secondary metabolite production (Khan et al., 2022). Efforts have been made to explore new habitats for screening actinomycete strains capable of producing novel secondary metabolites (Al-Shaibani et al., 2021). However, the study of halophilic actinomycete biodiversity in Iran’s saline soils is relatively limited.

In this report, we investigate the biodiversity of actinomycetes in the Garmsar Saline River using various culture media and pretreatments. We found that different culture media lead to varying diversity in the isolated actinomycetes. Notably, the use of Starch Casein Agar as an isolation medium, coupled with wet heat pretreatment, yielded the highest number of isolates (Fig. 2). Thermal pretreatment has been acknowledged for its significant role in screening rare actinobacteria (Subramani and Sipkema, 2019). Ezeobiora et al. (2022) reported that dry heat pretreatment conducted before plating the sample is essential for isolating uncommon actinobacteria. Furthermore, the starch casein agar culture medium is highly effective in reducing Gram-negative bacterial isolation, thereby increasing actinomycete yields (Bundale and Pathak, 2022; Oskay, 2009). According to Rifaat (2003), most actinomycete strains belonging to the genera Streptomyces and Micromonospora were retrieved using a starch-containing agar medium.

Actinomycetes are classified into various genera based on their morphological, chemical, and genetic attributes. Streptomyces, discovered as the most common genus within the Actinomycetales order, is often identified alongside Nocardia sp. and Micromonospora sp. from hypersaline environments (Selim et al., 2021; Singh et al., 2020). In our research, the majority of the identified isolates belonged to the Streptomyces genus, with only 20% belonging to Nocardiopsis. In contrast, a study conducted on the biodiverse actinomycetes from Hainan Island, China, reported Streptomyces as the dominant genus followed by Micromonospora (Ye et al., 2023).

Streptomyces species are known for their production of valuable enzymes such as amylase, protease, and lipase, with various industrial applications. The isolates collected in our study mainly produced amylase, gelatinase, and protease. Research by Rohban et al. (2009) on halophilic non-actinomycetes from Howz Soltan Lake in Iran identified lipase and amylase as the most frequently produced enzymes, followed by proteases. Notably, halophilic bacteria from both Iranian lake and river environments produced two of the top three enzymes in abundance, possibly due to the similar sources of carbon and nitrogen available to the microbes. However, contrasting studies have shown that actinomycetes from Laohu Ditch showed high production rates of catalase, lipase, urease, gelatinase, and amylase (Ma et al., 2020), while amylase, proteases, and lipases were most abundantly produced by halophilic bacteria in a saline lake in Spain (Sanchez pro et al., 2003).

Streptomyces are also known for synthesizing cytotoxic compounds with anticancer properties, effectively combating various malignant cell lines (Lee et al., 2023; Manhas et al., 2022). In our current study, the prominent isolate S. microflavus M15 showed considerable cytotoxicity towards human tumor cell lines without being toxic to normal cells (P < 0.05), alongside its selective activity against MCF7 cells (Fig. 5).

Actinomycetes are known to produce various secondary metabolites with different biological effects. The Actinomycetal order is notably responsible for producing bioactive compounds that can inhibit or suppress microbial growth. Specifically, the Streptomyces genus contributes approximately 80% of the naturally sourced antibiotics available for clinical use (Donald et al., 2022). Further observations suggest that most discovered antibiotics are more effective against gram-positive than gram-negative bacteria, likely due to the protective outer membrane of the latter (Chanthasena and Nantapong 2016; Sapkota et al., 2020). Our study found that 59% of the isolates exhibited antibacterial activity against gram-positive bacteria, while only 13% were effective against gram-negative bacteria.

GC-MS analysis of the extract from N. aegyptiae M16, which showed pronounced activity against P. mirabilis, identified 1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester, and 9-octadecenamide as the principal metabolites. The former compound has a formula of C16H22O4, a molecular weight of 278.35 g/mol, and is noted for its antimicrobial activity in some bacterial extracts (Koilybayeva et al., 2023). It is interesting to note that this compound has not been previously reported in the Nocardiopsis genus. The latter compound, oleamide, is the amide derived from the fatty acid oleic acid, with the formula C18H35NO and a molecular weight of 281.477 g/mol. It is postulated that the presence of oleamide in the surface extracts of some plant leaves would exert antimicrobial activities (Manyawi et al., 2023). Although oleamide has been found to have antibacterial effects in certain fungi and bacterial strains, it remains unreported in the Nocardiopsis genus.

In this study, a dual culture technique was employed to examine the induction of anti-MRSA compound production by actinomycetes when placed in co-culture with other non-producing strains. Only a pairing between two of the strains successfully induced bioactive metabolite production.

The actinomycetes discovered in this study represent a promising reservoir for novel bioactive metabolites. Their chemicals, encompassing enzymes, antitumor agents, and antibiotics, are poised to offer significant benefits across diverse industrial sectors. This research emphasizes the critical need to extensively explore all ecological niches to uncover unique actinomycete varieties capable of producing distinct metabolites."

Data availability statements

Data supporting fig. 3 are publicly available in Gene bank database as these records:

https://www.ncbi.nlm.nih.gov/nuccore/2582020780

https://www.ncbi.nlm.nih.gov/nuccore/OR889128

https://www.ncbi.nlm.nih.gov/nuccore/OR889129

https://www.ncbi.nlm.nih.gov/nuccore/OR603162

https://www.ncbi.nlm.nih.gov/nuccore/OR616766

https://www.ncbi.nlm.nih.gov/nuccore/OR603163

https://www.ncbi.nlm.nih.gov/nuccore/OR739420

https://www.ncbi.nlm.nih.gov/nuccore/OR616767

https://www.ncbi.nlm.nih.gov/nuccore/OR739423

https://www.ncbi.nlm.nih.gov/nuccore/OR739424

Data supporting Fig 4, 5 and 6 are publicly available in the figshare repository, as part of this record:

https://drive.google.com/file/d/1BGuT8LwWs1SNg6va-OJSPcGrcE_NaCIL/view?usp=sharing

The authors declare that there is no funding, grant, or other support.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

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Bioactive compounds of the Actinomycetes isolated from Garmsar Saline River, Iran

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Abstract

Figures

Introduction

Materials and methods

Sample collection, Media Cultures, and Strain Isolation

Screening for the production of extracellular hydrolytic enzyme activity

Cytotoxicity screening of actinomycete strains

Evaluation of antimicrobial activity using a modified spektra‑plak method

Production, extraction, and identification of antibacterial metabolite

Dual culture of actinomycete strains

Molecular identification and phylogenetic relationship analysis

Results

Actinomycetes screening media and treatments

Screening of the extracellular enzyme production in actinomycetes

Cytotoxic activity of the secondary metabolites extracted from the actinomycetes

Antagonistic activity of the isolated halophilic actinomycetes

Binary actinomycete interactions against MRSA

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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