الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 305 |  |  |
| | | from | 305 | | |
Abstract
In this study, a total of 123 of Pseudomonas aeruginosa isolates were identified and collected from sputum, urine and wound- burn infections. The isolated bacteria showed an observable high degree of antimicrobial resistance to several antibiotics specially; ampicillin, amoxicillin, oxacillin and Amoxycillin/ Clavulanic acid while, Amikacin, Gentamicin and Tobramycin were found to be more effective against most of tested isolates. Generally, 65% of P.aeruginosa isolates were MDR. In addition, more than 50% of MDR P.aeruginosa isolates were multi-virulent producing several virulence factors such as pigments, protease, lipase, hemolysin, gelatinase and biofilm. The efficiency of synthesized silver nanoparticles (AgNPs), asparticsilica silver nanoparticles (AS-AgNPs) and curcumin silver nanoparticles (Cur-AgNPs) as antibacterial agents was determined in vitro using agar well diffusion method. The obtained results revealed that; these nanoparticles showed high antibacreial activity against MDR P.aeruginosa isolates. Interestingly, the capping agents greatly increased the antibacterial activity of silver nanoparticles by observable increasing in the diameters of obtained inhibition zones. However, Cur-AgNPs were found to be more effective than AS-AgNPs. Moreover, the current study proved that capping silver nanoparticles with curcumin not only enhanced their antibacterial activity but also greatly decreased their MICs. Also, time kill kinetics and post agent effect, were better in case of Cur-AgNPs than AgNPs. Regarding stability and cytotoxicity of Cur-AgNPs; it was found that curcumin enhanced significantly the stability of silver nanoparticles over one month and showed less toxicity of these Cur-Ag particles to mammalian cells at concentrations higher than to bacterial cells. Besides, the in vitro wound healing activity of Cur-AgNPs was measured; and it was found that Cur- AgNPs completely closed the gap created by the scratch after 48 h only. The anitibiofilm activity of Cur-AgNPs was measured against both biofilm formation and pre-formed biofilm. It was found that Cur-AgNPs at sub-minimum inhibitory concentrations (sub-MICs) greatly inhibited the biofilm formation of P. aeruginosa. Also, Cur-AgNPs significantly reduced the amount of preformed biofilm and biofilm viability of the tested isolate at concentrations higher than MIC values. In order to prove the antibiofilm activity of Cur-AgNPs; quantitative PCR analysis was carried out to assess the changes in transcriptional levels of two genes necessary for biofilm formation (pelF and pslA) as well as one regulatory gene (gacA); the obtained results indicated that the transcriptions levels of these genes decreases after treatments with Cur-AgNPS which led to phenotypic changes. As observed by the TEM investigations for studying the possible effects of Cur-AgNPs on bacterial cell morphology and ultra- structure; it was clearly found that Cur-AgNPs caused great damage in bacterial cell wall and cell membrane affecting their permeability. Furthermore, the bacterial cell wall lysis resulted in increasing the amount of proteins and reducing sugar leakage from the cells; where tha maximum leakage was recorded after 24 h. Consequently, in order to prove the potential observed activity and to study the mode of action of Cur-AgNPs, the molecular docking study were performed on alginate compound, the major component of P.aeruginosa biofilm, and 6 other proteins involved in virulence of tested bacteria (as DNA gyrase, RNA polymerase, aminotransferase, pqsA, pqsE, pqsR). The results revealed that adding cucumin as capping agent greatly enhanced the binding affinity of silver nanoparticles towards the tested target proteins as well as alginate when compared to AgNPs alone which exihibited lower binding affinity. In conclusion, Cur–AgNPs and AS-AgNPS were synthesized where aspartic- silica and curcumin acted as reducing and capping agent. However, Cur–AgNPs were far better than chemically synthesized silver nanoparticles in terms of their stability. Antibacterial property and antibiofilm activity against P. aeruginosa isolates of these nanoparticles were also better than silver nanoparticles as evident from killing curve and TEM images. In addition, selective toxicity of these Cur–Ag particles to bacterial cells over mammalian cells suggest their potential application as wound dressing agent due to their high potential for combating MDR pathogens found in burn wounds. Additional in vivo studies are necessary to confirm the efficacy of the tested AgNPs composites as a novel antimicrobial agent in wound healing as well as novel biomolecules in various applications.