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Inhibition Of Bacterial Biofilm Formation By Topical Antibiotic Treatment In Collagen Hydrogel Carrier
Evan Harris Jarman, BS, Ayushi Sharma, BS, James Chang, MD, Paige Fox, MD PhD.
Stanford University, Stanford, CA, USA.

PURPOSE: Chronic wounds are a growing medical concern due to their susceptibility to bacterial colonization, with approximately 60% of chronic wounds containing bacterial biofilms1. Biofilms are the coordinated growth of bacteria in which clusters of cells encase themselves in a dense polymer to protect against external disruption. Bacterial biofilms hinder tissue repair, decrease the efficacy of systemic antibiotics, and enhance antibiotic resistance development. Clostridium perfringens is of particular interest because of its ability to lead to Clostridial myonecrosis, a life-threatening condition. Our collagen hydrogel (cHG) has been shown to effectively elute antibiotic over time, inhibiting Pseudomonas aeruginosa biofilms both in vitro and in vivo. We hypothesize that cHG can be used as a carrier for controlled release of antibiotic to successfully inhibit Clostridium perfringens biofilms while maintaining mammalian cell viability critical for wound healing.
METHODS: Collagen hydrogel preparation: 2.5% cHG was manufactured from a previously established protocol. Once prepared, cHG was mixed with clindamycin to achieve a concentration of 100 μg/ml (10x the minimum inhibitory concentration for C. perfringens treatment). The hydrogel-antibiotic (cHG-abx) solution was incubated to induce gelation. Modified Kirby-Bauer: C. perfringens was plated on agar plates and incubated. cHG-abx was gelled onto polycarbonate filters and allowed to elute in PBS for various timepoints before being placed onto the inoculated agar. After 24 hours of treatment, the zone of inhibition (ZOI) was quantified. Crystal violet assay: cHG-abx eluted for various time points was added to bacterial suspensions with biofilm formations, incubated, then stained with crystal violet solution. Absorbance was measured at 595 nm and compared to a non-treated well. Mammalian cell cytotoxicity: Wells seeded with human and mouse fibroblasts and ADSCs from human were treated with cHG-abx for 24, 48, and 72 hours before staining and quantifying.
RESULTS: The modified Kirby-Bauer assay demonstrated that as hydrogel elution time increased, there was a significant decrease in the ZOI compared to non-eluted gel, as expected. Inhibition of cell growth indicating sustained antibiotic release was seen to 48 hours (Figure A/B). Biofilm disruption demonstrated that biofilm disruption was significantly different than control to 24 hours (Figure C). cHG-abx treatment resulted in no significant cell death of mammalian cell cultures at any time point in any cell lineage.
CONCLUSION: This study confirms the capacity of the cHG to provide sustained antibiotic release resulting in Clostridium perfringens biofilm inhibition while maintaining mammalian cell viability. These findings broaden the applications of our hydrogel and support its potential use as a future treatment option for chronic wounds, eliminating the need for systemic antibiotics. REFERENCE: 1) James, G.A., Swogger, E., Wolcott, R., Pulcini, E.d., Secor, P., Sestrich, J., Costerton, J.W. and Stewart, P.S. (2008), Biofilms in chronic wounds. Wound Repair and Regeneration, 16: 37-44.


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