Metabolite profiling of Streptomyces sp. Al-Dhabi-100 isolated from the marine environment in Saudi Arabia with anti-bacterial, anti-tubercular and anti-oxidant potentials

2.1 Chemicals and reagents

Antibiotics such as streptomycin, actidione and nalidixcic acid was purchased from Himedia, India. Components used for the microbiological media preparations were obtained from Sigma. Different organic solvents such as hexane, ethyl acetate, chloroform and methanol were procured from Somatco, Saudi Arabia. The distilled water used for the experiments were RO treated and sterilized.

2.2 Pathogenic strains and its cultivation

For antimicrobial study different pathogenic strains such as E. faecalis, B. subtilis, S. aureus, S. epidermidis, K. pneumoniae, E. coli, and P. aeruginosa were gifted from the College of medicine King Saud University medical hospital. The pathogenic strains were stored as the lyophilized form and the strains were activated by thawing down and transferred into the sterile 20 ml nutrient broth containing flask with additional 10% tryptone and incubated under shaking condition for 24 h at 37 °C. After occurrence of growth in the form of cell suspension, the cells were streaked on the nutrient agar plates and incubated for 24 h at 37 °C. The purity of the pathogenic strains were evaluated and further stored in the refrigerator for the routine lab work and cell suspensions were mixed with 20% sterile glycerol and stored in the deep freezer for the prolonged uses.

2.3 Isolation of marine actinomycetes for antimicrobial activities

For the isolation of marine actinomycetes, marine sediment, marine soil samples, marine water, marine rock powder, marine fishes and marine decayed plants were collected from the different locations of ocean areas in Jazan region, Saudi Arabia. Soon after collecting the samples using sterile spatula, the samples were transferred into the sterile storage container and kept in the ice box till reaching the laboratory (Al-Dhabi et al., 2019a).

2.4 Determination of antimicrobial activity of the actinomycetes

Antimicrobial activities of the actinomycetes were determined by cross streak perpendicular method. Briefly, the spore suspension of the actinomycetes were prepared using 0.9% ice cold saline water and streaked on the center of the MNG agar medium and kept at 30 °C for 14 days. After incubation, the freshly cultivated different pathogenic bacteria were streaked perpendicular to the actinomycetes and determined for its inhibition property my measuring the zone of inhibition. Once isolates with promising antimicrobial activity was selected and studied further by characterization using biochemical, 16S rRNA identification and mass cultivation and chemical compound extraction.

2.5 Mass cultivation of marine actinomycetes and organic solvent extraction

Mass cultivation of the marine actinomycetes was performed for the bulk level extraction of crude compounds. Briefly, MNG broth supplemented with 40% marine water and 10% glucose was used as the base for the fermentation medium. About 1000 ml of the fermentation broths were transferred into 2000 ml Erlen Mayer flask, after adjusting the pH to 7.0. the flasks were sterilized. Separately, the spore suspensions of the active marine actinomycetes were prepared using 0.9% saline water and 10 percentage of the spore suspensions were transferred into the flask as the starting inoculums. After that the flasks were incubated at 30 °C for 25 days. The excretion of the antimicrobial substances was visually identified by changing the color from yellow to dark black. After incubation, the cells were separated by centrifugation at 10,000 RPM for 15 min. The collected supernatant was tested for antimicrobial activity by cup plate diffusion method to ensure the antimicrobial property of the supernatant. Further, the supernatant was mixed with 1:3 ratio of ethyl acetate. The crude antimicrobial components were concentrated using the vacuum evaporator with the operating conditions at 35 °C at low pressure. The concentrated substances also checked for its antimicrobial activity by disc diffusion method. Further, silica gel column was packed using 45 cm length and 5 cm width glass column. The crude extracts were eluted using different proportion of the organic solvents such as hexane, ethyl acetate and methanol. The collected six fractions were checked for its purity using thin layer chromatography and the spots with similar rf values were pooled together and evaluated for their antimicrobial activity by disc diffusion method. One fraction with better antimicrobial activity against the tested bacterial pathogens was further tested for MIC values, Anti-tubercular activity, antioxidant activities and chemical characterization.

2.6 MIC and MBC values of the fraction

Antibacterial activity of the fraction was accessed by broth micro dilution method (Arasu et al., 2017).

2.7 Cell wall disruption study

The cell wall disruption study of the fraction was tested by transferring 50 µg/ml concentration (Al-Dhabi et al., 2018).

2.8 Anti-tubercular activity of the fraction

Luciferase Reporter Phage (LRP) assays was followed for the determination of the antituberculosis property of the fraction with slight modifications from the method of Sivakumar et al. (2007). Mycobacterium tuberculosis H37Rv strain was cultivated in Middlebrook G7H9 broth with the concentration of #2 MacFarland standards. Briefly, 100 µg/mL of the fraction and 100 µL of freshly prepared M. tuberculosis cell containing 1 ml sterile cryovial were mixed with 300 µL of the G7H9 containing sterile freshly prepared 10% albumin dextrose complex and 0.5% glycerol and incubated at 37 °C for 3 days in an incubator. Separately only sterile water was used as the control. After the proper incubation, approximately 50 μL solution (25 μL of 0.1 M CaCl₂ and 25 μL of titre mycobacteriophage phAETRC202) was carefully transferred into the vial and mixed thoroughly and kept in an incubator at 37 °C for 3 days. After the incubation, 100 μL of the mixed samples were transferred into a specialized luminometer cuvette containing 100 μL of D-luciferin. After the addition of D-luciferin, relative light unit (RLU) was noted in a luminometer (Model – Monolight 2010). The reduction in the RLU value was calculated by following the equation $$\mathit{RLU}reduction\left(\%\right)=RL{U}_{\mathit{Control}}-RL{U}_{\mathit{Test}}/RL{U}_{\mathit{Control}}\times 100$$

The RLU values with less than 50% were considered as the promising anti-tubercular activities.

2.9 Antioxidant activities of the fraction

2.10 Chemical profiling of the fraction by GC–MS

The individual chemical composition of the fraction was assessed by subjecting the fraction to the gas chromatography. Briefly, the methodology for operating the gas chromatography instrument was followed from our previous publications. The chemical components were compared with the library of the mass spectrum and the percentage of the individual components was calculated.

3.1 Isolation and screening of actinomycetes for antimicrobial potential

In the present study strains with different morphological features were isolated from the coastal region of Jazan and screened for the antimicrobial activity. Among the collected stains, the stain Al-Dhabi-100, pronounced significant antimicrobial activities were selected for further characterization and chemical profiling. The biochemical studies confirmed that the strain was Gram positive, spore forming and aerobic in its properties. Also, the strain was able to produce extra cellular enzymes such amylase, protease and cellulase respectively. Growth characteristics of the strain was performed in different cultivation medium suggested that the strain was filamentous in the initial m stage of the growth and able to bear spore generate spores in its outer surfaces. In addition, the strain cannot able to produce diffusible pigment in the medium. The antimicrobial senility and resistance pattern of the strain towards various studies antibiotics were attractive. Especially the strain was resistance to streptomycin confirmed that the strain belonged to the genus Streptomyces. The 16S rRNA gene sequence obtained from the strain showed 99% percentage similarity and identity towards the Streptomyces strains deposited in the gene bank.

3.2 Antibacterial activity of the fraction

MIC and MBC values of the fractions were shown in figure. The results indicated that the fraction showed MIC values of 62.5 µg/ml for E. faecalis, 31.25 µg/ml for B. subtilis, 125 µg/ml for S. aureus, 250 µg/ml for S. epidermidis and 125 µg/ml for K. pneumoniae respectively (Fig. 1). The MBC values were ranged from 62.5 µg/ml to 500 µg/ml respectively for the studied microbial pathogens. The strand antibiotics streptomycin showed 25 µg/ml to 50 µg/ml respectively for the studied microbial pathogens.

Close

Fig. 1

MIC and MBC values of the fraction obtained from the Streptomyces sp strain Al-Dhabi-100.

Export to PPT

3.3 Cell growth inhibition assay

The cell growth inhibition properties of the fractions towards the microbial pathogens were displayed in Fig. 2. The results indicated that E. faecalis, B. subtilis, S. aureus and S. epidermidis 72%,94%,44% and 37% cell killing compared to the control whereas K. pneumoniae, E. coli and P. aeruginosa documented 19.78%, 71.4% and 91.6% respectively. The standard antibiotic suppressed above 95% of the cell growth in the suspension conditions.

Close

Fig. 2

Cell suspension inhibition assay of the fraction obtained from the Streptomyces sp strain Al-Dhabi-100.

Export to PPT

3.4 Anti-tubercular activity of the fraction

The six fractions obtained from the active ethyl acetate extracts were tested for the antimicobacterial studies against the virulent strain M. tuberculosis H37Rv. Results indicated that the six extracts comparatively showed antimicobacterial activities (Fig. 3). Among the fractions, fraction 6 effectively inhibited the growth of the M. tuberculosis H37Rv. The percentage of the RLU reduction was noted in the figure. It is revealed that fraction 6 showed 91.5% of reduction and the other fractions were lesser than the fraction 6. Also the standard antibiotic rifampicin showed 99.6% at 100 µg/ml level.

Close

Fig. 3

Antimicobacterial activity of the fraction obtained from the Streptomyces sp strain Al-Dhabi-100 Blue color; 100 µg/ml concentration, red color 50 µg/ml concentration.

Export to PPT

3.5 Antioxidant activities of the fraction

The antioxidant potentials of the fractions were showed in the Fig. 4. DPPH activity study of the fraction revealed that the antioxidant activities were concentration specific. The fifty percentage antioxidant potentials were noted at 5 µg level fraction. Whereas, the concentration from 1, 2, 3 and 5 µg showed 5 and 11.6, 17.33 and 33% percentage respectively. Similarly hydroxyl radical scavenging activity and nitric oxide scavenging activity of the fraction also documented as concentration specific. The antioxidant potentials were directly proportional to the concentration of the fractions. Even though the standard vitamin showed better antioxidant activity, the fractions were slightly lesser in its antioxidant potentials.

Close

Fig. 4

Antioxidant properties of the fraction obtained from the Streptomyces sp strain Al-Dhabi-100. I; DPPH, II, hydroxyl radical scavenging activity, III; nitric oxide scavenging activity.

Export to PPT

3.6 Chemical profiling of the fraction

Chemical profiling of the fraction guided the presence of various compounds such as benzenebutanoic acid (2.8%), benzestrol (4.44%), 1-(2,6-Dimethyl-4-propoxyphenyl)propan-1-one (13%), phenol, 4-(1,1-dimethylpropyl)- (10.66%), 1-(2,6-Dimethyl-4-propoxyphenyl)propan-1-one (7.0%), ethyl 2-propylphenyl ester (11.9%), Phenol, 2-methyl-4-(1,1,3,3-tetramethylbutyl)- (5.2%), Androst-5,16-diene-3.beta.-ol (0.1%) and .beta.-Carotene-3,3′-diol, (3R,3′R)-all-trans- (0.1%) respectively.