Phenotype of Pseudomonas aeruginosa isolates causing corneal infection between 1997 and 2000.

PURPOSE: To investigate the relationship between functional phenotype of and the associated human corneal infection. METHODS: This was an experimental pilot study of patients presenting with corneal infections at the Jules Stein Eye Institute with presumed infection during the period from 12/30/97 to 9/1/00. Thirteen patients were admitted to the study based on positive identification of the causative pathogen as and patient consent. Data were collected (including bacterial cultures, lens wear schedule and care, gender and age, completed history questionnaire, clinical photographs). Statistical analysis of possible correlations was performed. Phenotypes of were determined, and clinical factors associated with infection were explored. RESULTS: Both invasive and cytotoxic phenotypes of were isolated in equal proportion. Cytotoxic strains and invasive strains were found to be associated with patients younger than 50 years of age and older than 50 years of age, respectively. CONCLUSIONS: remains a significant pathogen in corneal infection, especially during contact lens wear. The age of the patient may influence the phenotype of causing infection. Since invasive and cytotoxic strains have different effects on corneal cells, treatment of the infection might require different approaches depending on this phenotype of the causative bacteria.

Pseudomonas aeruginosa strains with lipopolysaccharide defects exhibit reduced intracellular viability after invasion of corneal epithelial cells.

Pseudomonas aeruginosa is a leading cause of infectious keratitis. Many ocular isolates of this bacterium invade corneal epithelial cells in vitro and in vivo. Antibiotic survival assays have shown that a complete core lipopolysaccharide is required for full epithelial invasion by P. aeruginosa. In this study, we show that P. aeruginosa mutants with defects in their lipopolysaccharide core and O antigen exhibited reduced viability after internalization by corneal epithelial cells. Restoration of lipopolysaccharide core and O antigen expression by complementation with the plasmid pLPS1 restored intracellular survival. P. aeruginosa strains with a complete lipopolysaccharide survived and replicated within the cells. The data suggest that lipopolysaccharide is involved in the intracellular survival and/or replication of P. aeruginosa, indicating an additional mechanism by which this important virulence factor may contribute to the pathogenesis of corneal infection.

Mutation of csk, encoding the C-terminal Src kinase, reduces Pseudomonas aeruginosa internalization by mammalian cells and enhances bacterial cytotoxicity.

Clinical isolates of Pseudomonas aeruginosa are either invasive or cytotoxic towards mammalian epithelial cells, endothelial cells, and macrophages. Invasion requires host cell actin cytoskeleton function, and ExsA-regulated proteins of P. aeruginosa that inhibit invasion (ExoS and ExoT) can disrupt the cytoskeleton. Another ExsA regulated protein, ExoU, is involved in the cytotoxic activity of cytotoxic strains. Src-family kinases are thought to participate in the regulation of cytoskeleton function. Recent studies have suggested that Src-family tyrosine kinases, p60-Src and p59-Fyn, are activated during P. aeruginosa invasion. Using fibroblasts homozygous for mutation of csk (-/-), we tested the hypothesis that mutation of csk, encoding a negative regulator of Src-family tyrosine kinases, would be important in P. aeruginosa invasion and cytotoxicity. Mutation of csk was found to reduce invasion by approximately 8-fold, without reducing bacterial adherence to cells (P=0.0001). Conversely, csk (-/-) cells were approximately 5-fold more susceptible to ExoU-dependent cytotoxicity (P=0.024), which was accompanied by a small increase in ExsA-regulated adherence. ExoT-dependent invasion inhibitory activity of cytotoxic P. aeruginosa was attenuated in csk (-/-) cells as compared to normal fibroblasts. These data show that fibroblasts, like epithelial cells, are susceptible to P. aeruginosa invasion and cytotoxicity. They also show a role for Csk in P. aeruginosa invasion, while providing further evidence that actin cytoskeleton disruption contributes to ExsA-regulated P. aeruginosa cytotoxicity and invasion inhibition.