الفهرس 
IntroductionOnly 14 pages are availabe for public view
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Abstract
Summary and Conclusion
Emergence of multidrug resistant bacteria is a global concern and worrisome medical problem. Despite of the medical need for new antimicrobials, the number of antibacterial compounds available in clinical development is limited and most of the existing antibiotic classes are derivatives or modifications of antibiotics developed in the past decade. The persistence of antibiotic resistance urges the need of finding new therapies, or new natural alternatives having antimicrobial activity such as plant extracts and essential oils. One of the approaches currently investigated to overcome resistant bacteria is the use of combinations between antibiotics and bioactive medicinal plant extracts and essential oils. This association with antibiotics against resistant bacteria may expand the antimicrobial spectrum, reduce the emergence of resistant variants and minimize the use of antibiotics. With this in mind this work was carried out to investigate the antibacterial and resistance modulation activity of Egyptian and Chinese anise, also to study the effect of γ-irradiation (10 and 30 kGy) on the antibacterial activity of post-distillation extracts and essential oils of both Egyptian and Chinese anises.
In this study 100 clinical bacterial isolates belonging to four Gram-negative (Klebsiella spp., Escherichia coli, Pseudomonas spp. and Acinetobacter spp.) and two Gram-positive genera (Streptococcus spp. and Staphylococcus spp.) were collected from different samples. The isolates were investigated for their resistance to 18 antibiotics that differ in their mode of action. All isolates were found to be multidrug resistant (i.e resistant to 3 or more than 3 different classes of antibiotics).
Essential oils obtained by hydro-distillation from non-irradiated and irradiated Egyptian aniseeds (EEO) and fruits of Chinese anise (CEO) were subjected to GC-analysis. The spectrum obtained showed that both essential oils contain one major component identified as trans-anethole, other components could be
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observed in traces. Anethole concentration in EEO was 83.48 %, while a higher content of anethole (90.07 %) was detected in CEO. Results indicated that γ-irradiation at doses of 10 and 30 kGy resulted in 0.67, 3.4 and 1.45 , 2.74 % loss in the total concentration of the major component (trans-anethole) of EEO and CEO, respectively.
Preliminary investigation of EEO and CEO antibacterial potential against 100 collected MDR clinical bacterial isolates was carried out using agar disc diffusion method. The results showed that both Egyptian and Chinese anise essential oils have a noticeable antibacterial activity against all tested clinical isolates. In almost all cases the sensitivity of Gram-positive clinical isolates did not significantly differ from Gram-negative ones. CEO antibacterial activity (with inhibition zones varying from 21.6 to 38.3 mm) was found to be slightly higher than that of EEO (with inhibition zones varying from 20.0 to 35.3 mm). Effect of gamma irradiation on the antibacterial potential of both oils was also investigated; the results revealed that doses of 10 and 30 kGy did not significantly affect the antibacterial activity of both oils.
Preliminary evaluation of resistance modifying ability of both oils against the most potent six MDR clinical isolates (Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli and Pseudomonas aeruginosa) was carried out using disc diffusion assay. The results showed a noticeable variation in inhibition zone diameters of EEO, CEO, antibiotics and their combinations. In all combinations, Egyptian and Chinese essential oils were found to enhance the activity of tested antibiotics. The resistance modifying potencies of anise and Chinese anise oils were also evaluated through determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of conventional antibiotics (cephradine, amoxicillin, chloramphenicol and tetracycline) against the selected MDR clinical isolates in the presence and absence of essential oils. from MIC results, it was found that both oils were more effective in inhibiting MDR isolates
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than tested antibiotics. The results also revealed that all tested combinations resulted in synergistic interaction, which is detected by lowering the MIC of essential oils and antibiotics.
The detected synergistic patterns were also confirmed by calculating Fractional Inhibitory Concentration Index (FICI). FIC indices were found to range from 0.07 to 0.62 for Egyptian anise oil, and from 0.13 to 0.62 for Chinese anise oil. Among all tested combinations of oils/antibiotics antagonistic and additive interactions were not observed.
HPLC analysis of phenolics extracted from Egyptian and Chinese anise waste residual extract (EAWRE and CAWRE) showed qualitative and quantitative differences between EAWRE and CAWRE, the amount of phenolic compounds in CAWRE was higher than EAWRE with the exception of vanillic and reversetrol, which were higher in EAWRE. Catechein was not detected in CAWRE. Moreover, coumarine which could not be detected in EAWRE, were identified in CAWRE chromatograms.
The irradiated and non-irradiated extracts of Egyptian and Chinese anise waste residues (waste residues after essential oils extraction) were screened for their antibacterial activity against collected MDR clinical isolates using agar disc diffusion method. The results showed that both Egyptian (EAWRE) and Chinese anise waste residual extracts (CAWRE) have significant antibacterial activity against all tested clinical isolates with inhibition zones varying from 11.0 to 26.6 mm and from 14.3 to 30.6 mm, respectively; indicating a higher activity of CAWRE when compared to EAWRE. Data revealed that there were no significant differences between inhibition zones obtained for Gram positive bacteria and Gram negative ones. The results also interestingly reported that irradiation at 10 and 30 kGy did not significantly affect the antibacterial activity of both post-distillation extracts.
The resistance modifying ability of Egyptian and Chinese post-distillation extracts against six multidrug resistant clinical
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isolates (Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli and Pseudomonas aeruginosa) were screened using agar disc diffusion method. In the majority of combinations, both EAWRE and CAWRE were found to improve the activity of tested antibiotics against tested isolates. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of selected antibiotics against the MDR clinical isolates were determined in the presence and absence of post-distillation extracts. from the results it was found that the most active extract was CAWRE with MIC values between 16 to 64 μl/ml. However, the MIC values of EAWRE were in the range from 64 to 128 μl/ml, which were in most cases better than the antibiotics. The combination of anise waste extracts and the tested antibiotics mostly showed synergistic effect. Synergistic interaction was most expressed against Streptococcus pneumoniae and Staphylococcus aureus by tetracycline and chloramphenicol; Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii and Escherichia coli by cephradine, amoxicillin and Tetracycline.
The combination between both extracts and commercial antibiotics was further assessed on the basis of the fractional inhibitory concentration indices (FICI). The anise post-distillation extracts/antibiotics combinations indicated synergism (62.5 % for EAWRE and 54.1 % for CAWRE), additive/indifferent (16.6 % for EAWRE and 37.5 % for CAWRE) and antagonism (20.8 % for EAWRE and 8.3 % for CAWRE). The number of synergistic interactions between the EAWRE and the antibiotics against different bacteria in each treatment is in the following order: cephradine = amoxicillin = chloramphenicol > tetracycline (with FIC indices ranging from 0.50 to 0.75), While the number of synergistic interaction between the CAWRE and the antibiotics against different bacteria in each treatment is in the following order: tetracycline > cephradine, amoxicillin and chloramphenicol (with FIC indices ranging from 0.25 to 0.75).
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The effect of Egyptian and Chinese anise oils and residual extracts alone and in combination with tetracycline on the surface morphology of Pseudomonas aeruginosa were assessed using Scanning electron microscope (SEM). The bacterial cells surface were morphologically intact, regular, smooth and showed typical characters of rod shape in the control sample. Cell membrane injury was observed when exposed to tetracycline. P. aeruginosa cells treated with Egyptian and Chinese anise oils and post-distillation extracts showed deformations in shape and size. These deformations were more frequently occurred after treatment with synergistic mixtures of extracts/antibiotics. In contrast to control cells, cells exposed to both oils were found to be shriveled and aggregated; while micrograph of cells exposed to EEO combined with tetracycline showed irregular furrowed malformed cells. Also, treatment of Pseudomonas aeruginosa with CEO combined with tetracycline displayed wrinkled and aggregated cells and leakage of cellular content was observed.