Design And Evualuation Of Aminoglycoside Antibiotics-based Reagents Against Microbial Biofilm And Intracellular Bacteria

The chronic infections related to biofilm and intracellular bacteria are always hard to be cured because of their inherent resistance to both antimicrobial agents and host defenses, which always need a high does of antibiotics therapy for a long time. On the contrary, antibiotic overuse is also one of major drivers to generate antibiotic resistant "super bugs" that can potentially cause serious effects on health. And aslo high dose of antibiotics will increase their side effects, threatening health of patients. Up to date, the aminoglycoside antibiotics are one of the most important drugs to treat bacterial infections, however, these antibiotics are not so effective in trating infectious diseases caused by biofilm and intracellular bacteria. In this study we designed several aminoglyside drugs using chitosan or nanoparticle carriers and the activities against biofilm and intracellular bacteria were evaluated. 1. Chitosan Improves Anti-biofilm Efficacy of Gentamicin through Facilitating Antibiotic PenetrationMicroorganisms on living or inert surfaces usually form biofilms which makes microbes highly resistant to antibiotics and immune clearance. In this study we reported that the polycationic polysaccharide, chitosan could improve the efficacy of a given antibiotic(gentamicin) to combat bacterial biofilms, the universal lifestyle of microbes in the world. Short- or long-term treatment with the mixture of chitosan and gentamicin resulted in the dispersal of L. monocytogenes biofilms. In this combination, chitosan with a moderate molecular mass(~13 kDa) and high N-deacetylation degree(~88% DD) elicited an optimal anti-biofilm and bactericidal activity. Mechanistic insights indicated that chitosan facilitated the entry of gentamicin into the architecture of L. monocytogenes biofilms. Finally, we showed that this combination was also effective in the eradication of biofilms built by two other Listeria species, Listeria welshimeri and Listeria innocua. Thus, our findings pointed out that chitosan supplementation might overcome the resistance of Listeria biofilms to gentamicin, which might be helpful in prevention of gentamicin overuse in case of combating Listeria biofilms when this specific antibiotic was recommended. 2. Gold nanoparticles make chitosan-streptomycin conjugates effective to gram-negative bacterial biofilmThe emergence of biofilm-associated resistance of microbe to traditional antibiotics has resulted in an urgent need for novel antimicrobial agents. Herein we developed a facile approach to overcome the problem through chitosan-streptomycin gold nanoparticles(CA NPs). The synthesized CA NPs were characterized by ultraviolet-visible absorption spectra(UV-vis), scanning electron microscopy(SEM), transmission electron microscopy(TEM) and dynamic light scattering(DLS). The resulted CA NPs maintained their antibiofilm activities to Grampositive organisms. More importantly, CA NPs damaged established biofilms and inhibited biofilm formation of Gram-negative bacteria pathogens. Mechanistic insight demonstrated CA NPs rendered streptomycin more accessible into biofilms, thereby available to interact with biofilm bacteria. Additionally, CA NPs was observed to kill more biofilm-dispersed cells than C-S conjugate or streptomycin and inhibit the planktonic cell growth of Gram-positive and-negative bacteria. Thus, this work represent an innovative strategy that gold nanoparticles linked to carbohydrate-antibiotic conjugation can overcome antibiotic resistance of microbial biofilms, suggesting the potential of using the generated CA NPs as antimicrobial agents for bacterial infectious diseases. 3. Chitosan conjugation enables intracellular bacteria susceptible to aminoglycoside antibioticMost chronic infections are difficult to eradicate because bacteria capable of surviving in host infected cells may be protected from the killing actions of antibiotics, leading to therapy failures and disease relapses. Here we demonstrated that covalent coupling chitosan to streptomycin significantly improved intracellular bactericidal capacity towards multiple organisms within phagocytic or non-phagocytic cells. Structure-activity relationship investigations indicated that antibiotic contents, molecular size and positive charges of the conjugate were the key to retain this intracellular bactericidal activity. Mechanistic insight demonstrated the conjugate was capable to target and eliminate endocytic or endosomal escape bacteria through facilitating the direct contact between the antibiotic and intracellular organism. In vivo acute infection models indicated that compared to equal dose of the antibiotic, C-S conjugate and especially the HCS complex formed by human serum album and C-S conjugate greatly decreased the bacteria burden in the spleen and liver in both wild type and immunosuppressive mice. Furthermore, the HCS complex remarkably reduced mortality of infected TLR2 deficient mice, mimicking immune-compromised persons who were more susceptible to bacterial infections. These findings might open up a new avenue to combat intracellular bacterial infection by aminoglycosides antibiotics at a lower effective dose. 4. Potent Antibacterial Nanoparticles against Biofilm and Intracellular BacteriaHerein we develop a facile approach to overcome the above conundrum through phosphatidylcholine-decorated Au nanoparticles loaded with gentamicin(GPA NPs). The nanoparticles were characterized by scanning electron microscopy(SEM), dynamic light scattering(DLS) and ultraviolet-visible(UV-vis) absorption spectra which demonstrated that GPA NPs with a diameter of approximately 180 nm were uniform. The loading manner and release behaviors were also investigated. The generated GPA NPs maintained their antibiotic activities against planktonic bacteria, but more effective to damage established biofilms and inhibited biofilm formation of pathogens including Gram-positive and Gram-negative bacteria. In addition, GPA NPs were observed to be nontoxic to RAW 264.7 cells and readily engulfed by the macrophages, which facilitated the killing of intracellular bacteria in infected macrophages. These results suggested GPA NPs might be a promising antibacterial agent for effective treatment of chronic infections due to microbial biofilm and intracellular bacteria.For the first time, we used modified chitosan-steptomycin conjugation by gold nanoparticles to overcome antibiotic resistance of microbial biofilms effectively. We used chitosan as a covalent carrier for streptomycin, which was useful to treat intracellular bacteria infections with high efficacy. Moreover, we used phosphatidylcholine-decorated gold nanoparticles loaded with gentamicin to improve the performance of gentamicin against bifilm and intracellular bacteria. These strateties will enhance efficacy and reduce the side effects of antibiotics and prevent bacterial resistance. This proposal will extend the application of traditional aminoglycosides in the filed of infectious deseases related to biofilm and intracellular bateria.