الفهرس 
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Abstract
ABSTRACT Oral microbial flora constitutes an important part of the total human microbiome and any imbalance of this community is directly reflected upon the oral and whole-body health and disease status of diabetes and healthy people. Diabetes mellitus affects the flow of saliva and hence nutrients availability for mouth microbes. Studying and comparing the mouth microbial flora of healthy and diabetes are challenging process due to continuous changes which needed to be discovered. Molecular techniques such as protein banding patterns, DNA fingerprinting, the PCR analysis of the 16SrRNA gene DNA sequences and Matrix-Assisted Laser Desorption Ionization for identification bacteria were helpful in discovering the similarities and differences of microbial flora inhabiting this part of the human body. The molecular methods were not only superior to phenotypic methods in bacterial identification but they were faster and conclusive. This was realized even at the species and strain levels either in their protein contents or DNA. Moreover, it is important to note that some molecular methods worked well with cultivable bacteria, while, DNA sequencing of the gene encodes for the 16S rRNA would be important for the non-cultivable ones. The work and results can be summarized as follow: 1. Forty samples (swabs) were collected from the healthy and diabetic individuals. 2. Thirty nine microbial isolates were obtained, 11 colonies were yeast and 29 were bacteria, 14 bacteria from diabetes and 15 from healthy people. 3. Nine bacterial isolates (4h, 10h, 14h, 23h, 25h, D4, D8, D9 and D15). showed multidrug resistant (MDR). These were Trimethoprim/sulphamethoxazole 25mg, pencillins G 10mg, amoxicillin 30mg, ampicillin 10mg, oxacillin 1mg, vancomycin 30mg, clindamycin 2mg, erythromycin 30mg, Gentimicin 10µg and Kanamycin 30mg. 4. Molecular techniques such as SDS-PAGE, plasmid profile, RAPD-PCR, and toxin-antitoxin genes were used to identify the MDR bacteria. 5. MALDI-TOF-MS (VITEK-MS instrument at Zagazig University), a proteomic instrument, was also used for identification of the MDR bacteria. 6. The nucleotide sequences of the 16S rRNA genes of the nine MDR isolates were used to construct phylogenetic trees and identify them to the closest similar bacterium. 7. The protein patterns of isolates 10h and D4 were only differ in a single protein with electrophoretic motility close to 42kDa. 8. The 16SrRNA genes sequencing for isolates 4h, 25h, and D8 have identified to be species of the genus Staphylococcus. However, their RAPD-PCR fingerprints differ markedly from each other. Only one or two bands have electrophoretic motilities close to each other. These three isolates (4h, 25h, and D8) lack a complementary sequence for the OPK-02 primer and no PCR products; additionally, isolate D8 lacks a complementary site for primer OPU-16. 9. Isolate 10h identified by the sequencing of the 16SrRNA genes to be Acinetobacter schindleri, their RAPD-PCR fingerprints lack a complementary sequence for the OPK-02 primer and no PCR products. Isolate D4 have been identified by the sequencing of the 16SrRNA genes to be Enterobacter spp, their RAPD-PCR fingerprints lack a complementary sequence for the OPT-16. The two local isolates have been identified to be Bacillus species, their RAPD-PCR fingerprints differ markedly from each other. The two isolates D9 and D15 lack any complementary sequence for the OPK-02 primer and no PCR products; while, isolate D15 lacks any complementary site for primer OPU-16. 10. Toxin–antitoxin systems (TAs) are ubiquitous among bacteria and play a crucial role in the dissemination and evolution of antibiotic resistance, such as maintaining multi-resistant plasmids. Generally, activities of the toxins are neutralized by their conjugate antitoxins. 11. The presence/absence of four toxin–antitoxin systems (HipA toxin, HipB antitoxin, HicA toxin, HicB antitoxin, CcdA toxin, CcdB antitoxin, HigBA TA) were examined by using PCR technique in some bacterial isolates which isolated from oral cavity from healthy and diabetic individuals 12. The presence of toxin–antitoxin genes in Staphylococcus haemolyticus, Acinetobacter schindleri, Staphylococcus epidermidis (isolates 4h, 10h, 25h). In conclusion, MALDI-TOF-MS technology is simple and conclusive in identifying the local bacterial isolates. According to the built in databases the local isolates were identified as in Staphylococcus haemolyticus for isolates (4h, 14h, 23h), Acinetobacter schindleri for isolates (10h), Staphylococcus epidermidis isolates (25h), Enterobacter spp. Isolates (D4), staphylococcus aureus isolates (D8), Bacillus pumilus Isolates (D9) and Bacillus licheniformis isolates (D15). with 99.9% of confidence. It is also faster and cheaper than all other techniques used. Key words: Oral, microbiomes, diseases, molecular and phenotypic.