Novel strategies for wound treatment

Conventional wound care involves topical applications of antibiotics, oral antibiotics and silver containing bandages. Traditional wound care merely treats wound infections while allowing body's immune system to take charge of the natural healing process. It is common during the treatment of wounds to encounter persistence of drug resistant infections caused by presence of biofilms and delay of wound healing caused by antimicrobial agents applied to treat wounds. As wound healing is a complex process, wound care cannot be a single dimensional entity. Modern approach proposed in this study of wound care entails 3 main factors into consideration: possible invasion of multidrug resistant bacteria, biofilms formation associated with the wounds and physiological stages of wound repair. Despite the rise of multidrug resistant bacteria, production of new antibiotics has been declined in the recent years. The urgent need to combat the multidrug resistant organisms caused a resurgence of interest in phytochemicals because of their ubiquitous and antimicrobial nature. Phytochemicals that act synergistically with antibiotics to kill bacteria are especially the center of interest as they can reduce toxicity and delay resistance development. In this study, the antimicrobial effects of phytochemicals in combination with the conventional antibiotics were investigated by employing checkerboard assay and time kill assay using 6 strains of Staphylococcus aureus. Ethyl gallate was found to be synergistic with mupirocin, fusidic acid, tetracycline, cefoxitin and indifferent with vancomycin. Mechanism of action of phytochemicals can be postulated depending on their behavior of interaction with antibiotics. Ethyl gallate's synergistic interaction with protein synthesis inhibitors and its indifference with a cell wall synthesis inhibitor raised an interest in its possible mechanism of action on cell wall. Scanning electron microscopy and atomic force microscopy observation of bacteria cells after treatment with ethyl gallate further confirmed its action on cell wall. Computational docking studies were performed with lipoteichoic acid synthase enzyme where the results revealed that ethyl gallate was inhibiting the cell wall synthesis by inhibition of polymerization of peptidoglycan. Furthermore, the cytotoxicity assays also ensured that the phytochemicals under study were not toxic to human peripheral blood mononuclear cells.Infected wounds plagued with biofilms of bacteria are recalcitrant to treatment with antimicrobials. Moreover, dissemination of biofilm associated bacteria can result in fatal complications. Eradication of bacteria in biofilms needs thousand fold concentrations of antibiotics which narrow the therapeutic index of antimicrobials. The biofilm inhibition potential of phytochemicals and antibiotics were studied and compared as single agents and in combinations. Synergistic phytochemical-antibiotic combinations were found to have anti-biofilm activities investigated by microtiter plate assay and scanning electron microscopy. Staphylococcus aureus which is a common casual organism of wound infection is infamous for its drug resistance properties. When antibiotics are used in therapeutic regimes, it is important to take mutant selection window into consideration. Mutant selection window and mutant prevention concentrations for each of the phytochemicals and antibiotics in focus were studied and compared. Mutant strains of Staphylococcus aureus were studied and the duration taken for each antimicrobial was noted along with the mutation frequency of bacteria under treatment. The presence of ethyl gallate was found to close the mutant selection window, decrease mutant prevention concentration and reduce mutation frequency while prolonging the duration of resistance development. Chitosan and alginate are well known biocompatible natural polymers and have potential to form polyelectrolyte membrane in solutions. Employing this concept a novel natural polymeric polyelectrolyte membrane was synthesized and synergistic combination of fusidic acid and ethyl gallate were incorporated as a model drug combination. Effects of polyelectrolyte membrane on various aspects of wound healing process were determined. Drug eluting polyelectrolyte membrane developed in this study was able to help the wound healing activity by allowing gaseous exchange, retaining moisture in the wound, enabling slow release of drug combination, and assisting the proliferation as well as the metabolic activity of fibroblastsDOCTOR OF PHILOSOPHY (MAE