Abstract
Purpose :
To study the behaviour 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 microlitres 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 24h, 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 24h of infection, P. aeruginosa bacteria were found to internalise 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
Conclusions :
We have established an ex-vivo models of bacterial keratitis and demonstrated that Pseudomonas aeruginosa internalise early into stromal keratocytes, possibly to avoid contact with resident immune cells.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.