Microbiological and electron microscopic assessment of Pseudomonas aeruginosa infection of primary human corneal fibroblasts and epithelial cells in-vitro

Elsahn, Ahmad, Hossain, Parwez and Christodoulides, Myron (2013) Microbiological and electron microscopic assessment of Pseudomonas aeruginosa infection of primary human corneal fibroblasts and epithelial cells in-vitro. Investigative Ophthalmology & Visual Science, 54 (15).

Abstract

Purpose
To test the hypothesis that Pseudomonas aeruginosa spp. invasion of human corneal epithelial cells (HCEC) and fibroblasts (CF) contributes to the pathology of microbial keratitis.


Methods
Primary HCEC and CF were extracted from clinical samples from several patients, cultured separately to confluence in vitro and infected with various doses of Pseudomonas aeruginosa strain PAO-1. Total bacterial association was quantified at 15 and 30 min and at 1, 3, 6 and 9 h using the saponin-lysis and viable counting method. Invasion was quantified using the standard gentamicin protection assay. Scanning and transmission electron microscopy (TEM/SEM) were used to confirm bacterial attachment and invasion. Pathogen-induced host cell death and innate responses were analysed by LDH release and cytokine measurement.


Results
PAO-1 adherence to human corneal cells in vitro and subsequent invasion was observed in a dose and time dependant manner. Bacterial interactions also induced cytotoxicity, as demonstrated by increased LDH release, and influenced cellular innate responses.


Conclusions
We have established in-vitro cell culture models of microbial keratitis and demonstrated the direct adherence of Pseudomonas aeruginosa. Invading bacteria appear to induce significant deleterious changes to corneal cells and the mechanism of PAO-1 invasion is currently being investigated.

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Contributors

Author: Parwez Hossain

Author: Myron Christodoulides

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