Production, Purification, Characterization of Antibiotic from Locally Isolated of Actinomycetes spp. and Studying its Cytotoxicity and Antitumor Activity

Recently, the increase of the pathogens resistant to recent antimicrobial agents has become a health problem. Accordingly, the current study aimed at the isolation of local actinomycetes spp. that is capable of producing novel bioactive compounds, as well as investigating their biological (antimicrobial and anticancer) activities, in addition to, characterizing and partial purifiying the antimicrobials  metabolites. Forty soil samples were collected in Garmian area,during the period from 2 nd  to 15 th Feb. 2015. Out of the forty soil sources were screened, only (65%) of them harbored potential actinomycetes, and 55 suspected colonies with different morphotypes were isolated.All isolates were tested for antimicrobial activities by primary screening against reference strains. Among all suspected actinomycetes candidates, only six isolates KH14, KH16, KH18, KA19, KA38 and KA39 were able to inhibit the growth at least two of tested microorganisms. Optimizing media composition for enhancing the production of antimicrobial metabolites, five different broth media ISP1, ISP2, ISP4, ISP5 and Glycerol yeast extract broth were used and ISP2 revealed the best results (inhibition zone) among all media used.Furthermore, five different organic solvents were used to obtain the maximum extraction of extracellular antimicrobial metabolites. Results exhibited the extraction by n- butanol, methanol, ethyl acetate, n-hexane and chloroform, for recording zones of inhibition as 46.6%, 33.3%, 20%,13.3% and 6.6% respectively against the tested microorganisms. Comparisons of antimicrobial activities between extra and intracellular crude extracts were carried out. In general, the extracellular exerted more potent antimicrobial activity than the intracellular.Ten different synthetic antibiotic discs were used to compare their
activities with the extracellular crude extract. Interestingly, the extract, when compared their activities, show a higher inhibitory activity against the pathogens than synthetics discs activity. The results of the optimization experiments, such as growth kinetic, pH and salt tolerance NaCl revealed that the 3 days of incubation at pH 8 and 5% of NaCl tolerance especially for KH14 isolate.Minimum inhibitory concentration against Staphylococcus aureus, Escherichia coli and Candida albicans shows that 64, 500 and 500µl/ml respectively. The extracellular crude extract exhibited a strong inhibitory activity against Grampositive bacteria and a moderate inhibitory activity against both Gram-negative bacteria and yeast. In addition to the antimicrobial actions, DPPH assay has been performed to determine the antioxidant activity. The extracellular crude extract from KH14 isolates showed IC about5.24 mg/ml which scavenged 91% of formed DPPH free radicals. Determining the nature of the antimicrobial compounds was analyzed by TLC, which indicated the presence of four  separate spots, only one of them revealed the antimicrobial activities, as well as their position was determined by bioautography, with different solvents (mobile phase) exhibited different R values. HPLC-MS analysis of the scraped active spot and ethyl acetate extract indicated the presence of many compounds. In addition, the results showed that
most of them have a similar mass, which becomes noticeable in chromatogram.UV absorbance designated the presences of only two peaks at(221.85)and (264.85)nm from the scraped spot, while three distinguished peaks were shown at 222.85, 264.85 and 442.85 nm from ethyl acetate extract. Furthermore, the similarity between the two extracts on the basis of UV absorbance was achieved
by the second peak of both extracts which is 264.85nm, and this was considered as an antimicrobial absorbency. GC-MS analysis results for ethyl acetate extract of KH14 displayed 12 peaks, their molecular weight and molecular formulas were determined depending on NIST library.The antitumor activities were determined by MTT assay, which indicated that KH14 possesses a powerful effect against used cancerous cell lines, especially against MCF-7 by inhibiting 75.84% (400 µg concentration) of cancer cells after 72 hours of incubation, while against the rest of other cell lines,PC3and A375with moderate activities. High content screen assay was carried out for six cellular multi-parameters, against MCF-7 cell line. Besides, the comparison was carried out between the vehicle and extracellular crude extract of KH14.Statistical analyses within different (P values) revealed significant differences between vehicle samples with extracellular crude extract concentrations exactly in case of cell viability, nuclear morphology, cell membrane permeability and mitochondrial membrane potential with different concentrations, while both total nuclear intensity and cytochrome c did not show any significant differences with the negative control (vehicle).