Date of Award

5-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Bioengineering

Advisor

VERTEGEL, ALEXEY

Committee Member

VYAVAHARE , NAREN

Committee Member

GUISEPPI-ELIE , ANTONY

Committee Member

WEBB , KEN

Abstract

The frequency of S. aureus infection and subsequent biofilm formation associated with vascular catheterization has been increasing in recent years and often begins as a local colonization at the site of the catheter insertion. Antimicrobial enzymes and peptides, which are effective against a broad range of pathogens and low rates of resistance, have attracted attention as promising alternative candidates in treatment of infections caused by antibiotic resistant bacteria. The use of nanoparticles as carriers for enzymes, in addition to their size, charge, high surface area per volume etc. offers targeted delivery of enzymes to pathogenic bacteria. We proposed to use nanoparticles as surfaces for targeted delivery of antibacterial enzymes and as `surrogate' surface coatings on indwelling central venous catheters (CVCs) to inhibit bacterial colonization and subsequent biofilm formation.
It was shown that nanoparticle charge can be used to enhance delivery and increase bactericidal activity of an antimicrobial enzyme, lysozyme. In the case of bacterial lysis assay with a Gram-positive bacterium Micrococcus lysodeikticus, activity of lysozyme conjugated to positively charged nanoparticles was approximately twice as high as that of free lysozyme. This was believed to occur through charge-directed targeting of enzyme-nanoparticle conjugates to negatively charged bacterial cell walls through enhanced electrostatic interactions. In a clinically more relevant model, we studied antimicrobial activity of lysostaphin against S. aureus for both lysostaphin-coated and lysostaphin-antibody coated nanoparticle conjugates at different enzyme: antibody: nanoparticle ratios. At the highest antibody loading, bacterial lysis rates for antibody-lysostaphin-coated samples were significantly higher than for plain lysostaphin-coated samples and free enzyme due to multiple-ligand directed targeting of antibody molecules to bacterial cell walls (p<0.05).
We also performed in vitro experiments to evaluate the inhibition of bacterial colonies adhering to a surface. Bacterial infections by S. aureus strains are among the most common postoperative complications in surgical hernia repair with synthetic polymer meshes. Colony counting data from the broth count (model for bacteria in wound fluid) and wash count (model for colonized bacteria) for the enzyme-coated samples showed significantly decreased number of colony forming units (CFU) when compared to uncoated samples (p< 0.05). A pilot in vivo study showed a dose-dependent anti-S. aureus efficacy of lysostaphin-coated meshes in a rat model.
Finally, we observed that that coating of nanoparticles overall did not significantly improve binding yield, leaching, durability and antibiofilm activity of enzymes adsorbed on catheter segments (p>0.05). Alternatives to coating catheter surfaces using covalent chemistry through functional groups on nanoparticles either directly or through appropriate crosslinkers could result in significantly higher enzyme loadings, better stability and long term durability for future applications. The approach developed here is universal and can potentially be used for treatment of other medical device-associated infections. Moreover, use of antibacterial enzyme-NP conjugates can eventually be expanded for intravenous administration, which will further broaden its range of application.

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