الفهرس 
Review of LiteratureOnly 14 pages are availabe for public view
 |  | from | 265 |  |  |
| | | from | 265 | | |
Abstract
Salmonella enterica serovar typhi (S. typhi) is the main causative agent of enteric fever which is members of the family of Enterobacteriaceae and it is the most important Gram-negative motile bacterium cause typhoid fever. Antibiotics are a well-known tool in the fight against typhoid fever because they have significantly decreased the number of infectious illnesses death rates and morbidity. However, excessive antibiotic prescription and abuse in human medicine have accelerated the establishment and dissemination of multidrug-resistant (MDR) S. typhi strains. The present study was carried out to detect and to identify S. typhi from stool samples by serotyping especially that has the multi-drug resistant (MDR) S. typhi by antibiotic disc diffusion method. The study is concerned with the identification of specific genes responsible for multi-drug resistant using the PCR technique by genotypic analysis and the sequence of genes was determined by using sequencing method. The study also aimed to detect the effect of different Nano-particles on this multi-drug resistant strain and indicate the mechanism of action of these Nano-particles with comparing the cytotoxicity effect of the most effective Nano-particles to the effect of the drug of choice currently used. The current study showed that seventy-six isolates out of 113 (67.25%) were identified as S. typhi from various hospitals in Egypt: 84 samples from Abbassia Fever Hospital and 29 samples from Abo El-Reesh Al Mounira Hospital with research ethics approval (no. 30-2020/10) by the Egyptian National Center for Research and Health Development. Males were more
influenced by S. typhi than females with a rate of 62 and 38% respectively and the age rate was categorized as 18-25 (male 90% & female 10%), >25-35 (male 65% & female 35%), and >35-45 (male 54% & female 46%). It is obvious that males were infected by S. typhi more than females in each category. S. typhi strains were tested against 12 different antibiotics belonging to nine groups, Penicillins group (Ampicillin 10μg), Carbapenems group (Meropenem 10μg), Macrolide group (Azithromycin 15μg), Cephalosporins group (Cefaclor 30μg, Cefoperazone 75μg and Cefepime 30μg), Quinolones group (Ciprofloxacin 5μg and Nalidixic acid 30μg), Aminoglycosides group (Gentamycin 10μg), Sulfonamides group (Trimethoprim/Sulphamathoxazole 1.25/23.7μg), Phenicols group (Chloramphenicol 30μg) and Cyclines group (Doxycycline 5μg). Fifty- four percent (41 out of 76) of them were found to be multidrug- resistant strains. S. typhi strains were resistant to macrolides group (azithromycin 95.12%), sulfonamides group (trimethoprim/sulphamathoxazole 92.68%), penicillins group (ampicillin 90.24%) and cephalosporins group (cefaclor 95.12%, cefoperazone 95.12% and cefepime 95.12%). Elderly patients are most susceptible to MDR S. typhi in clinical samples (61.0%). The use of PCR analysis to find the blaCMY-2 gene is in charge of Cephalosporin resistance, DHFR gene is in charge of Sulfonamide resistance and acrB & rplD genes answerable for Macrolide resistance. The PCR product revealed that 39 strains out of 41 (95.12%) harbored the blaCMY-2 gene, 38 isolates (92.68%) harbored the DHFR gene, 20 isolates (48.78%) harbored the acrB gene, and 35 isolates (85.36%)
harbored the rplD gene, whereas thirteen isolates (31.70%) harbored the four resistance genes. Series alignment was performed for each detected gene and compared to the World Wide Web’s gene bank database, revealing the presence of several types of mutation.. In the case of the blaCMY-2 gene, the deletion mutations were recorded at nine query positions, while the inversion mutations were detected at thirty-one query positions. At the same time, transversion mutations were recorded at sixty query positions. The insertion mutations were recorded at eleven query positions. Nevertheless, in the case of the DHFR gene, one type of mutation was substitution (inversion and transversion mutations). There were three types of mutations in the acrB gene were deleted at three query positions, substitution (inversion recorded at twelve query positions and transversion were recorded at thirteen query positions) and insertion mutations were recorded at one query position. One type of deletion mutation was recorded at three query positions in the rplD gene. It’s possible that Egypt’s excessive or incorrect usage of antibiotics is to blame for the abnormalities in both genes.
The effect of the medicinal plants on multi-drug resistant S. typhi was carried out using Achillea millefolium L., Ecballium elaterium, Costus speciosus, Cymbopogon citratus, Peganum Harmala L. and Vitex agnus-castus extract plants. A. millefolium L. and E. elaterium plant extracts had a higher effect against MDR S. typhi than using another plant extract. In the current study, A. millefolium L. and E. elaterium essential oil, extract, and nanoparticles were tested for their
antibacterial activity on MDR S. typhi strains containing four resistance genes (blaCMY-2, DHFR, acrB and rplD) by the filter paper disc method. The mean diameter of the growth inhibition zone of A. millefolium L. nanoparticles (15mm) on MDR S. typhi was larger than that of A. millefolium L. extract (8mm) and A. millefolium L. essential oil (11mm), while The mean diameter of the growth inhibition zone of
E. elaterium nanoparticles (18mm) on MDR S. typhi was larger than that of E. elaterium extract (13mm) and E. elaterium essential oil (14mm). The MBC of nano A. millefolium L. was 30µg/ml and the MIC was 20µg/ml for MDR S. typhi that harboured the blaCMY-2, DHFR, acrB and rplD genes, while the MBC of nano E. elaterium was 20µg/ml and the MIC was 10µg/ml for MDR S. typhi that harboured the blaCMY-2, DHFR, acrB and rplD genes. GC-MS was used to chemically characterize the essential oil of A. millefolium L. nanoparticles. Twenty one chemical structure and quantities of each component were discovered. In this study, Cyclohexane,1-methylene- 4-(1-methylethyl) compound (44.5%), chamazulene (10.64%) and eucalyptol (8.2%), were the most prevalent compound, while Biochanin B compound (19.31%) was the most prevalent compound in essential oil of E. elaterium nanoparticles. The effect of A. millefolium
L. nanoparticles on MDR S. typhi strains harboring four genes was determined by TEM. Cell wall was damaged and cytoplasmic structures were not seen. Protein bands were detected by protein profile (SDS-PAGE) of bacteria alone (16 protein band were appeared) and after addition of A. millefolium L. nanoparticles as treatment, 6 protein bands were lysed and disappeared. The polymerase chain
reaction was reanalyzed, and the four MDR S. typhi genes that were disappeared for A. millefolium L. nanoparticles at a 20% dilution on a 1.5% agarose gel, while the effect of E. elaterium nanoparticles on MDR S. typhi strains, cell wall was damaged, nuclear materials and cytoplasmic structures were not seen. 12 protein bands were lysed and disappeared; the four resistance genes MDR S. typhi were not presented on 1.5 g % agarose gel tested for E. elaterium nanoparticles 10% dilution.
The IC50 is the concentration of an inhibitor where the response is reduced by half E. elaterium nanoparticles is 1230.05ug/ml, A. millefolium L. nanoparticles is 552.023ug/ml, gentamicin drug is 151.386ug/ml and chloramphenicol drug is 73.6924ug/ml. These results show that gentamicin and chloramphenicol drugs more cytotoxic on GES1 normal cells than E. elaterium nanoparticle and A. millefolium L. nanoparticles.
.
Conclusion
Using natural compounds is safer for humans than using chemical compounds as antibacterial, E. elaterium nanoparticles and A. millefolium L. nanoparticles have a higher bactericidal effect against MDR S. typhi that contain blaCMY-2, DHFR, rplD, and acrB resistance genes and have lower cytotoxicity on GES1 normal cells than chloramphenicol and gentamicin drugs (the most effective drugs of choice to treat MDR S. typhi), so we recommend using natural products in the treatment of multi-drug-resistant bacteria.
Biochanin B compound was the most prevalent chemical present in E. elaterium nanoparticles oil while Cyclohexane,1-methylene-4-(1- methylethyl) compound was the most prevalent chemical present in A. millefolium L. oil nanoparticles and many studies proved its antibacterial effect.