الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 167 |  |  |
| | | from | 167 | | |
Abstract
The need for novel chemical compounds to treat human ailments is growing. The discovery of new cases of potentially fatal infections, the steady recurrence of diseases, and the quick evolution of bacteria resistant to current treatments have all contributed to the advancement of drug research. There are three methods for identifying novel compounds of pharmacological significance: Combinatorial chemistry is the process of creating a library of chemicals and evaluating them against the biological target in order to identify the most effective ones; rational drug design, which targets the drug specifically at the microbial cell; and natural product discovery, which isolating bioactive compounds from biological sources. Pharma companies have recently shown more interest in the first two paths. These routes make use of the most recent developments in drug-docking instruments, three-dimensional X-ray crystallography, and other computer-aided techniques that drastically reduce the time it takes to produce a molecule from compound synthesis to commercial delivery. These methods do have certain drawbacks, though, such as the high expense of producing and finding new chemicals. As a result, attempts are being undertaken to reexamine the possibility of using natural products as the basis for innovative medications.
A number of bacterial strains are becoming more resistant to common antibiotics, including ESKAPE (six highly virulent and antibiotic-resistant bacterial pathogens), which includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter aerogenes. K. pneumoniae is a significant contributor to hospital-acquired pneumonia, septicemias, and soft tissue infections. This has made the finding and creation of new types of anti-Klebsiella agents essential. Also, there is a pressing need to find natural antifungal agents since Candida albicans is a fungal pathogen that causes infections linked to a high mortality rate.
The obtained results can be concluded as the following:
• 20 fungal endophyte labeled A1–A5, B1–B7, C, D, E, F, L, O, S, and X were isolated from Ziziphus spina-christi (L.) Desf. leaves. These fungi were identified morphologically and photographed under light microscope for the most promising isolates. The twenty fungal endophytes belong to six different genera and ten different species.
• The agar plug method was used to examine the fungal endophytes’ antimicrobial activity against six bacterial and fungal strains. Eight endophytic isolates in all (40%) showed activity against at least one of the tested pathogens. The acquired results showed that four isolates were the most effective anti-infective fungus, including A2/ZSEFL2, D/ZSEFL14, E/ZSEFL15 and S/ZSEFL19. The four isolates were chosen to be compared in a disc diffusion assay (second screening), in addition to six more active isolates (B5, B6, C, F, O and X) that were confirmed by the Antimicrobial Index (AI) in solid (PDA) and liquid (PDB) potato dextrose medium.
• On the other hand, 12 (60%) of the endophytic fungi (A3/ZSEFL3, A4/ZSEFL4, A5/ZSEFL5, B2/ZSEFL7, B3/ZSEFL8, B4/ZSEFL9, B5/ZSEFL10, B6/ZSEFL11, B7/ZSEFL12, L/ZSEFL17, O/ZSEFL18, X/ZSEFL20) showed negative activity against the tested microbes.
• Disc diffusion assays for solid medium extracts of the ten selected fungal endophytes represented a higher potential as antifungal (against Candida albicans) than the potentiality of liquid extracts. In relation to liquid medium, extracts of the ten fungal endophytic isolates showed more antibacterial than antifungal activities.
• By using liquid fermentation growth, the four isolates that were the most active were examined for their bioactive production of fungal endophytic metabolites under various microbiological cultural conditions. Compared to the other three culture media (PDB-y, rice, and Cz), PDB proved to be the best culture medium in production of antimicrobial metabolites for the four tested fungal endophytes.
• The generation of bioactive metabolites was improved by raising the incubation temperatures from 25 to 30 oC. However, the lowest IZ was found at either lower temperature of 20°C or higher temperature of 40°C with low AI.
• Generally, the optimum pH for either antifungal or antibacterial metabolite production of all tested fungal endophytes was between 5 and 6. The majority of fungal endophytes’ metabolites at pH 4 and pH 8 exhibited decreased or no action against the examined microorganisms.
• When the conditions were changed from stationary to shaking, the bioactive secondary metabolites of the four examined fungal endophytes generally reduced their antimicrobial efficacy.
• By employing the ethyl acetate extracts of the most active fungal endophytes obtained from PDB, PDB-y, REB, , and Cz culture mediums by micro-dilution assay, MIC and MBC/MFC were calculated. As can be seen in results, data showed that different isolate extracts and media types had MICs that varied between 0.19 and 6.25 mg/mL against the studied bacterial and fungal human pathogens.
• MBC of the four effective fungal extracts was employed to evaluate their bactericidal and bacteriostatic characteristics. MBCs of extracts were measured. Fungal endophyte extracts showed potential bactericidal activity against all tested pathogens.
• Antibiofilm assay was performed using ethyl acetate extracts of the four selected fungal endophytes (Alternaria alternate A2/ZSEFL2, Epicoccum nigrum E/ZSEFL15, Ulocladium graphium B6/ZSEFL11, and Penicillium crustosum S/ZSEFL19) grown in PDB media by measuring OD absorbency (tested organisms) using ELISA reader against 9 uropathogenic strains. Alternaria alternata (A2) extract.showed.considerable.activity.against.Gram-positive.tested.bacteria (91 and 88% against S. aureus and S. epidermidis respectively), while a moderate inhibition (54-69%) was observed against some tested Gram-negative bacteria (E. coli, K. pneumoniae, P. mirabilis and P. aeruginosa).
• Eight isolates (80%) of the total tested isolated fungal endophytes (ten isolates) showed positive results for peroxidase, whereas five isolates (50%) showed positive results for laccase in the test of enzyme activity qualitatively. Potential enzyme activities of laccase and peroxidase were assayed quantitatively using ABTS as a substrate. The results revealed that the four most promissing fungal endophytes (A2/ZSEFL2, Alternaria alternata; D/ZSEFL14, Aspergillus niger; E/ZSEFL15, Epicoccum nigrum and S/ZSEFL19, Penicillium crustosum) in antimicrobial screening experiments demonstrated high values of peroxidase and laccase activities in their extracellular culture filtrates compared with the other six isolates.
• Results of the total antioxidant revealed that the isolates D and E were the most potent and gave the highest values of TAC, whereas A2 and S came in at the next level.
• The thin layer chromatography technique was carried out for visualisation of the metabolites presented in each ethyl acetate extract recovered from the culturing of endophytic isolated strains on different media (PDB, PDB-y, Rice, Cz) to verify the most appropriate medium for the production of enormous metabolites. Generally, the TLC techniques herein displayed that the PDB culture medium was the most convenient medium for all strains to produce numerous metabolites either in the extract of culture filtrate or mycelium, and the PDB-y medium came in next according to the visualized band appearance.