World Eye and Vision Congress
University of Nottingham, UK
Title: Early events of Pseudomonas aeruginosa microbial keratitis: An electron microscopic study
Biography: Elsahn A
Purpose: To study the behavior of Pseudomonas aeruginosa bacteria inside the corneal stroma after corneal infection in human microbial keratitis.
Methods: Human whole donor corneas obtained from the eye bank were used in this project. Bacterial suspensions of wild type and mCherry expressing P. aeruginosa PAO-1L were prepared to a concentration of 107 cfu/ml. Fifty microliters of bacterial suspension was injected into the middle of the corneal stroma from the endothelial using a 25G hypodermic needle. The corneas were then incubated in medium for 24 hours, after which they were either examined live under a Laser Confocal Fluorescence Microscopy (CFLM) or fixed and processed for Transmission Electron Microscopy (TEM).
Results: After 24 hours of infection, P. aeruginosa bacteria were found to internalize into Corneal Stromal Keratocytes (CSK). Using TEM, PAO-1L bacteria were found clustered inside CSK with much fewer bacteria scattered in the stroma outside cells. With CFLM live imaging, mCherry-expressing PAO-1L bacteria were seen inside CSK, also with much fewer extracellular bacteria.
Conclusion: We have established ex vivo models of bacterial keratitis and demonstrated that Pseudomonas aeruginosa internalize early into stromal keratocytes, possibly to avoid contact with resident immune cells.