الفهرس 
ABSTRACTOnly 14 pages are availabe for public view
 |  | from | 202 |  |  |
| | | from | 202 | | |
Abstract
Microbial Drug Resistance Is A Medical Crisis That Pose A Real Threat To The Public Health. In Particular, Acinetobacter Baumannii Has Attracted A Special Attention Due To Its Incredible Ability To Develop Resistance To Different Classes Of Antibiotics. A. Baumannii Infections Have Poor Prognosis With High Morbidity And Mortality Due To Limited Number Of Available Effective Solutions. The Spread Of This Challenging Pathogen Has Spurred Researchers To Mine For Suitable And Effective Alternatives Before Entering Post-Antibiotic Era. Bacteriophages (Phages) And Their Lytic Enzymes Are Presented As Promising Approaches To Control This Problem. They Developed Suitable And Effective Approaches To Overcome Bacterial Resistance For Their Infections. Polysaccharide Degrading Enzymes, Anchored To Tail Fiber Or Spikes, Are Important Weapons That Strip Bacteria from Their Capsules, Important Virulence Factors That Contribute In Bacterial Resistance And Persistence. Other Powerful Weapons Are Lysins Which Are Used By Phages To Breach Peptidoglycan Meshwork At The Beginning And End Of Phage Lytic Cycles. The Exploitation Of Phage And Its Recombinantly Produced Lytic Enzymes To Combat A. Baumannii Infections Is The Main Target Of This Project.
Firstly, A Set Of Ten A. Baumannii Strains Were Isolated from Different Health Care Settings In Fayoum And Beni-Suef Cities. The Isolated Strains Showed A Wild Resistance Profile With Resistance To At Least 4 Out Of 12 Used Antibiotics. A Lytic Phage (PMK34) Was Isolated Against Extensively Drug Resistant Strain (A. Baumannii MK34). PMK3 Showed A Favorable Kinetic, Stability And Genetic characters (Lack Of Virulence Genes). Nevertheless, Its Limited Host Range (Based On Capsular Recognition) In Addition To Emergence Of Resistant Mutants During Co-Incubation With Its Host Bacteria Encouraged Us To Investigate Its Genome In Seek Of Finding Antimicrobial Proteins. PMK34 Derived Lysin Encoding Gene (Lysmk34) Was Amplified from Phage Genome, Cloned, Expressed And Purified Then We Performed A Biochemical characterization (Activity Calculations, Ph And Temperature Optima And Stability) And An Analysis Of Its Antibacterial Activity. Lysmk34 Showed A Prominent Muralytic Activity (1499.9 ± 131 U/µm), A Thermal Stability Up To 55 °C And A Broad Ph Activity Range (4 To 10). Furthermore, It Showed A Turgor Pressure Dependent Intrinsic Antibacterial Activity Against A Problematic A. Baumannii And Pseudomonas Aeruginosa Strains. Surprisingly, Lysmk34 Reduced Colistin MIC Up To 32-Fold And Reverted Colistin Resistance In Colistin Resistant Strains.
Secondly, Dpomk34 (Depolymerase) Was Evaluated As Potential Anti-Virulent Protein Found Within PMK34 Genome. We Amplified, Cloned And Expressed A Putative Tail Fiber (Orf45) Within PMK34 Genome. In Silico Analysis Suggested Dpomk34 As A Polysaccharide Degrading Enzyme By Predicting Central Pectate Lyase Domain. Furthermore, Its Role In Determining PMK34 Host Spectrum And Adsorption Was Confirmed. Although Lacking Antibacterial Activity Alone And In Combination Of Different Antibiotics, Dpomk34 Showed Promising Novel Strategy To Fight A. Baumannii By Sensitizing It To Serum Killing.
Thirdly, Lysmk34 characters Were Improved, Using Versatile Platform To Produce Enhanced Version Of Lysmk34, Artlyd34 By Fusing Outer Membrane Permeabilizer (Cecropin A). The Engineered Version Displayed Faster Antibacterial Activity Than Wild One. Furthermore, It Was Able To Kill Stationary Grown A.Baumannii Strains Even In Low Intracellular Turgor Pressure In Contrary To Lysmk34. Moreover, Artlyd34 Showed Antibacterial Activity In Human Serum Model, Underlying The Possibility Of Using This Version In Wounds And Systemic Infections.
To Sum Up. This Study Presented An Experimental Comparison Between Using PMK34 And Its Derived Products (Lysmk34 And Dpomk34) To Combat A Challenging Pathogen Like A. Baumannii. Furthermore, We Presented How The Novel Shuffling Technique (Versatile Platform) Could Be Used To Improve characters Of Lysmk34.