Attachment of Two Antibiotics to a Titanium Alloy Surface to Inhibit Colonization by Gram-positive and Gram-negative Bacteria

Date of Award

8-2012

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Christopher Adams, PhD

Second Advisor

Farzaneh Daghigh, PhD

Third Advisor

Dawn Shell, PhD

Fourth Advisor

Marcus Bell, PhD

Abstract

Despite immense improvements in anti-infective measures and implant technology, periprosthetic infection (PPI) remains a major complication for individuals with percutaneous prosthetic attachment. While various methods and materials have been developed to counter infection, these methods are often ineffectual. To combat PPI in orthopaedic applications, titanium (Ti) alloy implants tethered with antibiotics active against both gram negative and gram positive organisms have been developed separately. However, as a percutaneous implant will be exposed to a highly variable soup of gram-positive and gram-negative pathogens, a broad based surface must be created. Thus, it is the goal of this study to develop methods to bind two separate antibiotics (Vancomycin, active against gram-positive organisms, and Tetracycline, active against gram-negative organisms) simultaneously to a single Ti surface to target both of these pathogen groups. Vancomycin and Tetracycline were covalently bound to Ti separately (Vanco-Ti NHS and Tet-Ti) and together (50V/50T-Ti) usmg a modified synthesis scheme involving N-Hydroxysuccinimide (NHS). Distribution of the surface-bound antibiotics was detected by immunofluorescence and confocal microscopy using an Olympus FV1000 Confocal Microscope. Stability and activity were evaluated after incubation in Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) cultures for one day. The effects of Vanco-Ti NHS, 50V/50T-Ti and Tet-Ti on bacterial adhesion and colonization were measured using scanning electron microscopy (SEM), fluorescently labeled bacteria evaluated by confocal microscopy and by direct colony counting of adherent bacteria. Immunofluorescence showed attachment of Vancomycin and Tetracycline to Ti. SEM showed adherent bacterial colonies on control surfaces that were absent on Vanco-Ti NHS. Live/dead staining of adherent bacteria showed that S. aureus was inhibited from colonizing Vanco-Ti NHS and 50V/50T-Ti, while E. coli were inhibited from colonizing Tet-Ti and 50V/50T-Ti. These results were confirmed by colony counts. Both Vancomycin and Tetracycline can be covalently tethered to the surface of Ti separately and simultaneously. Vancomycin effectively prevents S. aureus colonization while Tetracycline effectively prevents E. coli colonization on the Ti surface; the combined surface with both antibiotics attached prevented colonization by both gram-positive and gram-negative species.

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