Reliable, Efficient Measurement of Antibiotic Elution from Hydrogel for Topical Wound Therapy

Introduction: Chronic wounds affect 6.5 million Americans and cost $50 billion per year. With high biocompatibility and bactericidal elution of antibiotics, topical collagen-rich hydrogels (cHG) may be a solution. Prolonging antibiotic elution from cHG requires real-time monitoring of antibiotic concentrations. Using liquid chromatography mass spectrometry (LCMS) for this is costly, slow, and impractical. Thus, we propose using a modified Kirby-Bauer (KB) assay to build a regression model, such that the zone of inhibition (ZOI), correlating with bacterial killing, will estimate the eluted antibiotic concentration. This will allow optimization of antibiotic elution from cHG to be monitored and modified in real-time without LCMS.

Methods: Stock Gentamicin solution was diluted to concentrations between 0.05-1.00mg/ml at 0.05mg/ml intervals. KB discs with 20μl of solution were placed onto 108 CFU/ml Pseudomonas aeruginosa agar lawns. Plates were incubated at 37°C for 16 hours and imaged by ChemiDoc. Experiments were in triplicate. ZOI areas were semiautomatically measured via ImageJ and their average was correlated with concentration.

Results: Quadratic correlation of average area with concentration shows R2 of 0.988 with standard error 6.073 (df=19, F=712.615, p<0.001). 

Conclusion: With a well-fit regression, ZOI area is a reliable, fast, and inexpensive way to estimate concentrations of Gentamicin. This is an important new tool in the endeavor to lengthen cHG elution of antibiotics and improve topical treatment of chronically infected wounds.