الفهرس 
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Abstract
The antagonistic activity of isolated gut bacteria from the American cockroach, Periplaneta americana L. (collected from three different environmental sites) were tested against some multi drug resistant (MDR) human pathogens. The selected MDR human pathogens were (Streptococcus mutans, MRSA); as Gram-positive bacteria, (Enterobacter cloacae, Salmonella enterica); as Gram-negative bacteria, human pathogenic yeast and fungi manipulated during this study were (Candida albicans, Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus and Penicillium italicum).
The isolated gut bacteria from the American cockroach, Periplaneta americana L. were identified by using Vitek (MALDI-TOF MS), and their antagonistic activities against the selected MDR human pathogens were tested by using agar well diffusion method, The mode of action of antagonistic activity was studied on the most affected bacteria and fungi by transmission electron microscopy.
The whole-body extracts of the American cockroach, Periplaneta americana L. were prepared using two different solvents;(ethanol 95% and cyclohexane). Identification and characterization of the biologically active compounds present in these extracts were performed using liquid chromatography mass spectrometry; (LC-MS), the antimicrobial activities of the two different extracts were tested against the selected MDR human pathogens.
The most active antimicrobial extract;(cyclohexane extract) was fractionated using vacuum liquid chromatography, (VLC) for characterization and identification of the active fractions.
The antimicrobial activities of the obtained ten fractions were tested against MRSA and Candida albicans, the most active fraction was(F8), its antitumor activity and cytotoxicity were tested.
The most active fraction (F8) was identified using nuclear magnetic resonance, (NMR). Finally, the mode of action of the identified compound against the target tumor cells was studied using molecular modelling;(docking).
The results of the study can be summarized as follows:
1. Identification of isolated gut bacteria by using Vitek (MALDI-TOF MS):
The identified bacteria isolated from the paper factory were mostly belonging to families Enterobacteriaceae with percentage 25%, Brucellaceae with percentage 8% and Xanthomonadaceae with percentage 4%,while bacteria isolated from food store were mostly belonging to families Enterobacteriaceae with percentage 15%, Comamonadaceae with percentage 2% and Micrococcaceae with percentage 3%.Finally the isolated bacteria from sewage water were mostly belonging to family Bacillaceae with percentage 29%, Enterobacteriaceae with percentage 27% and Staphylococcaceae with percentage 2%. Most of isolated gut bacteria belonging to families Bacillaceae and Enterobacteriaceae.
2. Evaluating antagonistic activity of isolated gut bacteria:
In testing the antagonistic activity of the isolated gut bacteria, it was found that Stenotrophomonas maltophilia has the highest antagonistic growth effect against Streptococcus mutans with inhibition zone measuring (37±0.3mm) while Serratia marcescens proved its high multiple antagonistic effect against the growth of Streptococcus mutans with inhibition zone (35±0.1mm), MRSA (30±0.1mm) and Enterobacter cloacae (20±0.2mm). Anyhow Gram-positive bacteria (Streptococcus mutans and MRSA) were more sensitive than Gram-negative bacteria (Enterobacter cloacae and Salmonella enterica). In measuring the antagonistic activity of the isolated gut bacteria against the tested fungi; Bacillus cereus has the highest antagonistic effect against Candida albicans with inhibition zone measuring (25±0.4mm),Serratia marcescens showed multiple antagonistic effect against Aspergillus niger and Candida albicans with inhibition zones (13±0.4 mm) and (11±0.5 mm) respectively, while Delftia acidovorans induced good inhibition against Penicillium italicum and Aspergillus fumigatus recording (23±0.5 mm) and (20±0.4 mm) inhibition zones, respectively.
3. Ultra-structural changes showed by tested pathogens due to antagonistic effects of bacterial symbionts:
In studying the ultra-structures of the most affected cells with the antagonistic effect of the isolated insect gut bacterium, Streptococcus mutans ultra-thin sections, when treated with Stenotrophomonas maltophilia, and Serratia marcescens showed great alterations in cell structure resulted in: membrane damage which appeared to have ruptured potential with leaks of intracellular materials and cellular damage led finally to complete cell deformation. Also, Enterobacter cloacae treated with Serratia marcescens showed that all cells were lysed and void of cytoplasmic fluid with completely shrinkage of cytoplasmic membrane, while MRSA showed completely cellular damages, breaking in cell wall, and leaking in cytoplasmic materials after treatment with Serratia marcescens. Treatment of Candida albicans with Bacillus cereus showed irregular cell wall with changed in cytoplasmic membrane, increasing in thickness and the cytoplasmic materials constricting in the center of the cell. While, treated Penicillium italicum with Delftia acidovorans, represented deformations of both cell wall and cell membrane with disappearance of the identified cytoplasmic materials which concerted in the center of the cell and appearance of numerous vacuoles.
4. Identification of extracted compounds:
Forty five fractions were extracted from Periplaneta americana lysate using LC-MS spectrometry; (15 compound in ethanol extract and 30 compounds in cyclohexane extract).
5. The antimicrobial activity of the whole-body extract:
In measuring the antimicrobial activities, results showed that the cyclohexane extract was more active against the tested pathogens and the inhibition zones measuring:(15±0.4 mm for Streptococcus mutans,28±0.3mm for MRSA), (10±0.2mm for Enterobacter cloacae), (27±0.2mm for Candida albicans),
(18±0.2mm for Aspergillus fumigatus), (13±0.4mm for Aspergillus flavus) and (15±0.3 mm for Penicillium italicum).
6. Extraction and purification of biologically active components and measuring their biological activities:
Vacuum liquid chromatography, (VLC) was used to extract and purify the biologically active components in the cyclohexane extract, the total number of obtained fractions was 10 fractions. The antimicrobial activities of the ten obtained fractions were tested against MRSA and Candida albicans, and the most active fraction was F8 with inhibition zones measuring (27±0.5mm,27±0.6 mm) respectively.
7. Cytotoxic Effects of the most active fraction(F8) against human cancer cells:
The inhibitory activity against Lung carcinoma cells was detected using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- 2H-tetrazolium bromide) assay under experimental conditions with IC50 = 28.9 ± 1.5 ^g/ml, while inhibitory activity against Hepatocellular carcinoma cells was detected with 50% inhibitory concentration; Co)= 51 ± 2.6 ^g/ml.
8. Morphological changes of treated cancer cells:
The affected lung carcinoma cells represented profound morphological changes characteristic with shrinkage, granulation and irregular shape, also, the affected hepatocellular carcinoma cells showed shrinkage, aggregation of cells and irregular shape; in both cell lines there were release of their contents which finally lead to a reduction of cell number, compared with untreated control cells did not show any adverse effect.
9. Cytotoxic Effects of the most active fraction F8 against human normal cells:
The inhibitory cytotoxic activity against normal human lung fibroblast cells was detected using, (MTT) assay under these experimental conditions with50% cytotoxic concentration, (CC50)= 118 ± 3.4 ^g/ml.
10.Identification of f8 fraction:
The most active fraction was identified (configuraized) by nuclear magnetic resonance as (E)-3-Benzylidene-5- methyldihydrofuran-2(3H)-one. With chemical formula
(C12H12O2).
11.Molecular docking studies:
To understand the reaction between our compound and active cell receptors, molecular docking studies was used.