Isolation and Identification of actinomycetes
In our study on actinomycetes, we identified 14 isolates with actinomycetes morphology. These isolates underwent screening for antimicrobial activity (primary screening). Among them, one sample, strain EA7, stood out due to its distinct antimicrobial activity and was therefore selected for further identification and investigation of bioactive compounds. Strain EA7 exhibited broad-spectrum antimicrobial activity and is considered a rare actinomycete.
Morphological, Biochemical, and Physiological Characteristics of Strain EA7
The macroscopic appearance of the colonies on ISP2 solid medium showed a dry, chalky appearance (Figure 1a), while the microscopic observation revealed a filamentous structure for strain EA7 (Figure 1b). Biochemical analysis indicated that the strain is catalase-positive and capable of hydrolyzing gelatin and casein, but negative for the hydrolysis of starch and urea. The Simon citrate, oxidase, and SIM tests turned out negative. Strain EA7 can utilize various carbon sources, including glucose, xylose, mannitol, raffinose, and arabinose. It thrives within a temperature range of 27 to 37 °C, at a pH range of 7 to 8, and can tolerate up to 7% sodium chloride (Table 1).
Antibiotic Susceptibility Testing of Strain EA7
Among the 14 actinomycete isolates, only strain EA7 displayed high resistance to eight antibiotics tested in this study, whereas the remaining isolates exhibited low resistance with a maximum halo diameter of 20 mm (Table 1, Figure 2).
Isolation and Identification of Pathogenic Bacteria
In total, six pathogenic bacteria were identified, including S. aureus, a Gram-positive, cocci-shaped bacterium that is catalase-positive, oxidase-negative, coagulase-positive, capable of growth on mannitol salt agar medium. Additionally, P. aeruginosa, a Gram-negative, rod-shaped bacterium was identified, which is catalase-positive, oxidase-positive, non-fermenting, motile, capable of citrate utilization, and grows at 42 °C. Clinical samples of P. aeruginosa, S. aureus, and standard strains were found to be resistant to all antibiotics.
Antimicrobial Activity of Strain EA7 (Primary Screening)
Strain EA7 exhibited a 20 mm diameter growth inhibition zone against S. aureus. However, no growth inhibition halo was observed against P. aeruginosa (Figure 3).
Genomic and Phylogenetic Analyses of Strain EA7
The analysis of the 16S rRNA gene using BLAST software revealed a 99.63% sequence match with Amycolatopsis roodepoortensis. This sequence was deposited in GenBank as Amycolatopsis roodepoortensis strain EA7 (GenBank accession number: OR680714). The results of PCR and phylogenetic analysis of strain EA7 using the neighbor-joining method, compared to other species of Amycolatopsis are shown in Figures 4 and 5, respectively.
Determination of Antimicrobial Activity of the Fermentation Broth of Strain EA7
The antimicrobial activity of strain EA7's fermentation broth was assessed using the agar well diffusion method (Figure 6). Notably, on the 7th day of fermentation, strain EA7 exhibited significant antimicrobial effects against S. aureus (halo diameter of 23mm) compared to P. aeruginosa. The broad-spectrum antimicrobial activity of strain EA7's fermentation culture indicated the production of active antibiotics and bioactive compounds in ISP2 broth nutrient medium.
Antimicrobial Activity of Ethyl Acetate and Methanol Extracts
Agar disc diffusion method
The agar disc diffusion method was employed to assess the antimicrobial activity of ethyl acetate and methanol extracts. The highest inhibition zone diameters observed were 25mm and 11mm against Gram-positive bacteria (S. aureus) for ethyl acetate and methanol extracts, respectively, while the inhibitory effects on Gram-negative bacteria (P. aeruginosa) were relatively lower (Figure 7).
Determination of Minimum Inhibitory and Minimum Bactericidal Concentration Assay
The minimum inhibitory concentration (MIC) of ethyl acetate extract for S. aureus, P. aeruginosa clinical sample, S. aureus PTCC 1112, and P. aeruginosa PTCC 1565 strains was reported as follows: 625 μg/mL, 1250 μg/mL, 312.5 μg/mL, and 1250 μg/mL, respectively. The minimum bactericidal concentration (MBC) values of ethyl acetate extract for S. aureus and P. aeruginosa (both clinical and standard samples) were 2500 μg/mL and 5000 μg/mL. On the other hand, the minimum inhibitory concentration of methanolic extract for S. aureus, P. aeruginosa clinical sample, S. aureus PTCC 1112, and P. aeruginosa PTCC 1565 strain standard was reported as: 1250 μg/mL, 2500 μg/mL, 625 μg/mL, and 2500 μg/mL, respectively. Additionally, the MBC from the methanolic extract for S. aureus was obtained at 5000 µg/mL, while it was not detected for P. aeruginosa (Table 2).
GC-MS Analysis of Ethyl Acetate Extract of Strain EA7
A total of 55 chemical compounds were identified by comparing their mass spectra with the Wiley Registry 8e library, based on the evaluation of retention time, molecular formula, molecular weight, percentages of area, and quality in the ethyl acetate extract of strain EA7 . The results of the GC-MS analysis revealed that the strain EA7 extract contains 24 compound with a quality of 70% or higher(Table 3). Acetic acid, 2-methylpropyl ester (15.8%) as the Major compound was identified in the strain EA7 extract. Other identified compounds included 7,9-di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione (13.6%), isopropyl myristate (11.4%), 9-octadecenoic acid, ethyl ester (10.4%), and 2,3-Butanediol (8.2%). The GC-MS chromatogram displaying the biologically active compounds of strain EA7 is illustrated in figure 8.
LC-MS analysis of the methanolic extract of Strain EA7
Tetracycline, Dihydroxybenzamide, Penipacid E, Valgamycin V, Dipyrimicin A, phenol, 2, 2'-methylenebis[6-(1,1-dimethylethyl)-4-methyl], Tetrangomycin, Amycolamycin A,and Amycolactam were identified in the methanolic extract of strain EA7. The secondary metabolites were known in the methanolic extract of strain EA7 with m/z values is shown in Table 4. Compound 1: appeared at a retention time of 2.15 min, The mass ion peak at m/z 323.091 [M +H]+with predicted MF C19H14O5 was explained as a Tetrangomycin antibiotic. Compound 2: appeared at a retention time of 2.89 min, The mass ion peak at m/z 231.567 [M +H]+ with predicted MF C12H10N2O3 was explained as a Penipacid E antibiotic. Compound 3: appeared at a retention time of 3.68 min, The mass ion peak at m/z 446.O55 [M +H]+ with predicted MF C22H24N2O8 was explained as a tetracycline antibiotic. Compound 4: appeared at a retention time of 4.18 min, The mass ion peak at m/z 138.027 [M +H]+ with predicted MF C7H7NO2 was explained as a Dihydroxybenzzamide compund. Compound 5: appeared at a retention time of 4.18 min, The mass ion peak at m/z 315.206 [M +H]+ with predicted MF C18H22N2O3 was explained as a Amycolactam antibiotic. Compound 6: appeared at a retention time of 5.91 min, The mass ion peak at m/z 609.313 [M +H]+ with predicted MF C29H48N4O8 was explained as a Valgamicin V antibiotic. Compound 7: appeared at a retention time of 8.23 min, The mass ion peak at m/z 246.956 [M +H]+ with predicted MF C12H10N2O4 was explained as a Dipyrimicin A antibiotic.Compound 8: appeared at a retention time of 14.76 min, The mass ion peak at m/z 341.921 [M +H]+ with predicted MF C23H32O2 was explained as a Phenol, 2,2′-methylenebis[6-(1,1-dimethyl ethyl)-4-methyl ] compound . Compound 9: appeared at a retention time of 14.83 min, The mass ion peak at m/z 783.164 [M +H]+ with predicted MF C39H40CINO14 was explained as a Amycolamycin A antibiotic. The LC-MS chromatogram of bioactive compounds (secondary metabolites) in the methanolic extract of strain EA7 is shown in Figure 9.