Category: Manufacturing and Bioprocessing
Purpose: This study aims to develop an ex vivo rabbit cornea infection model suitable for evaluating the effectiveness of antibiotic eluting contact lenses in the treatment of corneal infections.
Methods: Lamellar keratectomy was performed on cadaver rabbit eyes and the corneoscleral buttons excised. These were subjected to different chemical sterilization techniques using PBS, sodium hypochlorite (NaOCl) and povidone iodine (PI) to determine a suitable sterilization method. The explants were separately inoculated with 1μl of 108 CFU/ml Pseudomonas aeruginosa (PAO1) and Staphylococcus aureus (SA 35556) and incubated for one-, two- and three-day period to investigate bacteria growth on the corneas. Some corneoscleral buttons were processed for histology to confirm the penetration of bacteria into the stroma. After incubation, the corneas were aseptically collected, vortexed multiple times in PBS-Tween 80 solution and the resultant suspension serially diluted and plated on tryptic soy agar (TSA) plates. Colonies were counted after incubation to determine viable bacteria cell count.
Results: Histological studies (Haematoxylin and Eosin staining) showed penetration of both micro-organisms into the stroma (Figure 1). Povidone iodine (PI) at 10% concentration was mild on the cornea compared to NaOCl (Figure 2) and effective in eliminating other organisms except the organism of interest; 16S RNA sequencing showed that 99% of the organism found on the cornea after decontamination was the inoculated organism. After 2 hours of incubation, there were ~105 CFU/ml of viable P.aeruginosa on the corneas. Incubation for 1 day increased the bioburden to ~108 CFU/ml where it appears to plateau for the next 2 days of incubation. The S. aureus inoculated corneas showed ~106 CFU/ml of viable bacteria after 2 hours which also peaked and plateaued at ~108 CFU/ml after 1 day of incubation . The growth of the inoculated organisms was sustained on the ex vivo corneas (Figure 3) while the growth of other microbes were prevented.
Conclusion: Infected ex vivo rabbit corneoscleral buttons could be used as a suitable alternative to in vivo studies for evaluation of antibiotic drug delivery with contact lenses, other solid devices or semi-solid ocular medications.
Anuj Chauhan– Professor, Dinesh O. Shah Fellow, University of Florida, Gainesville, Florida
Gregory Schultz– Director, Institute for Wound Research, University of Florida, University of Florida, Gainesville, Florida
Daniel Gibson– Gainesville, Florida