Abstract
# Purpose To examine over time, the electron microscopic changes associated with Pseudomonas aeruginosa (PA) and human corneal tissue interactions in the context of microbial keratitis. # Methods Corneal stromal fibroblast monolayer and whole tissue models were made from human eye bank eyes and from residual tissue after corneal transplantation. In the whole tissue model (WTM), donor buttons were infected with PAO1 by scoring and intrastromal injection. Tissue was examined after 3, 9 and 24 h post challenge by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). In the cell culture model (CCM), corneal fibroblasts (CF) were infected in vitro with PAO1. Bacterial adherence and internalization were assayed at 3, 6 and 9 h by SEM and TEM. Adherent and internalized bacteria were measured by the gentamicin protection invasion assay. A subset of infected fibroblasts was treated with gentamicin to study intracellular bacterial survival and cell viability using a lactose dehydrogenase assay (LDH). # Results In the WTM, bacteria were seen to penetrate the epithelium at the scored sites only. At 3 h bacteria were seen in the stroma and by 9 h distinct intrastromal bacterial colonies were observed. Clusters of intracellular bacteria were observed in keratocytes in the intrastromal injection model. In CCM, SEM demonstrated bacteria adherent to the surface of CF and the saponin lysis assay demonstrated adherence and internalization in a dose- and time-dependent manner. Bacterial internalization was detected as early as 3 h. Intracellular bacteria survived and replicated without affecting cell viability. # Conclusion PAO1 bacterial can infect stromal keratocytes only when the epithelium and basement membrane are breached, or bypassed by direct injection. PA interaction with CF occurs very early leading to internalization of bacteria where they are protected and can multiply intracellularly without affecting CF viability for 24 h. This may have relevance to ideal timing of medical intervention.
Highlights
Microbial keratitis remains a significant public health problem as a major cause of visual disability around the world, and yet it is alarmingly under-diagnosed and under-treated [1]
In the transmission electron microscopy (TEM) of the non-scarified human corneal buttons (hCB), the bacteria were seen in all layers of epithelium but not beyond the basement membrane (BM)
Studies of Pseudomonas aeruginosa (PA) microbial keratitis show progressive destruction and liquefaction of the cornea that rapidly leads to perforation unless intense antibiotic treatment is immediately initiated [25]
Summary
Microbial keratitis remains a significant public health problem as a major cause of visual disability around the world, and yet it is alarmingly under-diagnosed and under-treated [1]. Contact lens wear accounts for up to 50.3% of cases of microbial keratitis [5] in the western world, and Pseudomonas aeruginosa is the most commonly isolated causative organism for contact lens associated microbial keratitis, accounting for up to 68.8% of isolates [6,7,8,9,10,11]. P. aeruginosa microbial keratitis is one of the most devastating, rapidly progressing corneal infections, which can cause corneal perforation, endophthalmitis and, blindness. Numerous live animal models of microbial keratitis have been developed to study the effects of bacterial colonization and virulence factors, the host responses to the pathogen and the interplay between
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