الفهرس 
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Abstract
5. Summary
The crisis of antimicrobial resistance is globally widespread even though pharmacological industries have produced a number of new antibiotics in the last decades. Therefore, actions must be taken to face this crisis by developing new natural drugs.
The objectives of this study were to investigate the antibacterial activity of natural bioactive products from both essential oils and biometabolites of endophytic microorganisms of some Egyptian medicinal plants to treat infections caused by multidrug resistant Gram negative bacteria (MDRGNB), so the extracted essential oils were assessed for the antibacterial activity against MDRGNB and the fungal, bacterial and actinomycetes endophytes were isolated from some medicinal plants. The efficiency of fermentation filtrates of the isolated endophytic microorganisms was screened against MDRGNB. The most potent endophytic isolate was identified. The efficiency of the antibacterial activity of biometabolites by the most potent endophytic isolate was optimized two steps; first step by one-factor-at-a-time approach and the second step by statistical analysis of all factors by Design-Expert® software by Placket-Burmann design followed by Central Composite design of Response Surface Methodology.
After the optimization experiments, the most potent endophytic isolate had to undergo the fermentation process in large scale under the optimized growth conditions.
To achieve the objectives of this study, the following steps were performed:
A) Multidrug resistant Gram negative bacteria
• Isolation, identification of six different clinical isolates of MDRGNB and testing the antibiotic susceptibility.
B) Medicinal plants
• Collection of eight Egyptian medicinal plants belonging to family Labiatae from the different habitats.
1- Essential oils
• Extraction of essential oils from fresh aerial parts of the collected medicinal plants.
• Testing the antibacterial activity of extracted essential oils against MDRGNB and determination of minimum inhibitory concentration (MIC) for the most effective essential oils.
• Identification of the components of the essential oils by using Gas chromatography mass spectrometry (GC-MS) analysis.
2- Endophytic microorganisms
• Isolation of fungal, bacterial and actinomycetes endophytes from surface sterilized aerial parts of collected medicinal plants and purification of these isolates.
• Screening the efficiency of antibacterial activity of fermentation filtrates of all isolated endophytes against two isolates of MDRGNB.
• Selection and identification of the most potent endophytic isolate.
• Optimization of physical and nutritional factors to maximize the efficiency of antibacterial activity of biometabolites of the most potent endophytic isolate by two ways:
- Optimization by using one-factor-at-a-time (OFAT) approach.
- Optimization by using all factors by statistical analysis by Design-Expert® software by Plackett-Burman design (PBD) followed by Central Composite design (CCD) of Response Surface Methodology.
• Fermentation of the most potent endophytic isolate under the optimum growth conditions in large scale and extraction of antibacterial compound(s) by different organic solvents and screening the antibacterial activity against two isolates of MDRGNB.
Results of this study can be summarized as follows:
A) Multidrug resistant Gram negative bacteria
• The six clinical isolates were identified biochemically by both Oxoid™ Microbact™ Identification GNB 12A Kit and Oxoid™ Microbact™ software program as three different isolates of Acinetobacter baumannii (A. baumannii) and three different isolates of Klebsiella pneumoniae (K. pneumoniae). They were resistant to almost the commercially standard antibiotics proving that the isolates are multidrug resistant.
B) Medicinal plants
• The collected medicinal plants were identified as: Origanum majorana L. No.1 (El-Adleya farm, SEKEM), Origanum majorana L. No.2 (El-Maghara station, North Sinai, DRC), Origanum syriacum L. (El-Sheikh Zowied station, DRC), Thymus capitatus L. (Wadi Habbes, Marsa Matrouh), Thymus vulgaris L. (El-Adleya farm, SEKEM), Salvia fruticosa Mill. (El-Maghara station, North Sinai, DRC), Mentha viridis L. (El-Adleya farm, SEKEM) and Lavandula officinalis L. (El-Adleya farm, SEKEM).
1) Essential oils
• The antibacterial activities of eight essential oils were assayed against identified six isolates of MDRGNB by disc diffusion method. Essential oil of Mentha viridis L. showed the least effect and no effect was found for both Salvia fruticosa Mill. and Lavandula officinalis L., while essential oils of Origanum majorana L. No.1, Origanum majorana L. No.2, Origanum syriacum L., Thymus capitatus L. and Thymus vulgaris L. were effective. The MIC values for the effective five essential oils were determined. Essential oil of T. capitatus L. was the most effective essential oil that showed the highest antibacterial activity against all MDRGNB isolates.
• GC-MS analysis of the extracted essential oils from Origanum majorana L. No.1, Origanum majorana L. No.2, Origanum syriacum L., Thymus capitatus L., Thymus vulgaris L., Salvia fruticosa Mill., Mentha viridis L. and Lavandula officinalis L., revealed the presence of terpinen-4-ol (21.99%), terpinen-4-ol (23.56%), cymene (27.98%), carvacrol (41.55%), benzene,1,2,3,4-tetramethyl- (27.26%), eucalyptol (54.84%), carvone (47.79%) and eucalyptol (36.42%) as major components, respectively.
2) Endophytic microorganisms
• A total of eighty-four endophytic isolates were isolated from different fresh aerial parts of the studied eight medicinal plants.
• All endophytic isolates were tested against two isolates of MDRGNB (A. baumannii and K. pneumoniae) to assess the efficiency of antibacterial activity of biometabolites of the endophytes. The most potent endophytic isolate was from the stem of Thymus vulgaris L.
• The most potent endophytic isolate was identified by macroscopic and microscopic characteristics as Alternaria tenuissima.
• Optimization of physical factors by using one-variable-at-a-time approach for Alternaria tenuissima were as follows; for incubation temperature, the maximum growth and the highest efficiency of antibacterial activity of biometabolites was recorded at 30ºC. For incubation period, the highest efficiency was on the 7th day and the maximum growth was on the 15th day. For the initial pH, pH 7.5 was the optimum for the maximum growth while pH 5.5 was the optimum for the highest efficiency. Finally for speed of agitation, 100 rpm was the optimum for the maximum growth, while static condition was the optimum for the highest efficiency.
• Optimization of nutritional factors by using one-variable-at-a-time approach were as follows; for the carbon source, glucose was the optimum carbon source for the highest efficiency of antibacterial activity of biometabolites and sucrose was the optimum for the maximum growth. For the best concentration of glucose, 25 g/L was the best concentration for both the maximum growth and the highest efficiency. For nitrogen source, KNO3 was the optimum nitrogen source as it showed the highest efficiency of antibacterial activity of biometabolites, however, yeast extract was the optimum for the maximum growth. For the best concentration of KNO3, 2.5 g/L was the best concentration for both the maximum growth and the highest efficiency of antibacterial activity of biometabolites.
• Alternaria tenuissima had to undergo further optimization experiments by using all variables by statistical analysis by Design-Expert® sotware, by both PBD and CCD with two responses, for the first response the optimum medium for the highest efficiency of antibacterial activity of biometabolites contains glucose (25 g/L), KNO3 (3.38 g/L), KH2PO4 (1.47 g/L) and FeSO4.7H2O (0.049 g/L) while for the second response the optimum medium for the highest efficiency of antibacterial activity of biometabolites contains glucose (20 g/L), KNO3 (2.74 g/L), KH2PO4 (0.51 g/L) and FeSO4.7H2O (0.037 g/L) and ZnSO4 (0.15 g/L).
• After fermentation of Alternaria tenuissima under the optimum growth conditions in large scale, the antibacterial activity of biometabolites was screened against two isolates of MDRGNB, ethyl acetate crude extract was the most effective.
• Finally, both essential oils and biometabolites of endophytic microorganisms of some Egyptian medicinal plants can be used as an alternative natural drugs to treat infections caused by MDRGNB.