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P. aeruginosa  and  S. pneumoniae  are major bacterial causes of corneal ulcers in industrialized and in developing countries. The current study examined host innate immune responses at the site of infection, and also expression of bacterial virulence factors in clinical isolates from patients in south India. Corneal ulcer material was obtained from 49 patients with confirmed  P. aeruginosa  and 27 patients with  S. pneumoniae , and gene expression of Toll Like Receptors (TLR), cytokines and inflammasome proteins was measured by quantitative PCR. Expression of  P. aeruginosa  type III secretion exotoxins and  S. pneumoniae  pneumolysin was detected by western blot analysis. We found that neutrophils comprised >90% cells in corneal ulcers, and that there was elevated expression of TLR2, TLR4, TLR5 and TLR9, the NLRP3 and NLRC4 inflammasomes and the ASC adaptor molecule. IL-1α IL-1β and IFN-γ expression was also elevated; however, there was no significant difference in expression of any of these genes between corneal ulcers from  P. aeruginosa  and  S. pneumoniae  infected patients. We also show that 41/49 (84%) of  P. aeruginosa  clinical isolates expressed ExoS and ExoT, whereas 5/49 (10%) of isolates expressed ExoS, ExoT and ExoU with only 2/49 isolates expressing ExoT and ExoU. In contrast, all 27  S. pneumoniae  clinical isolates produced pneumolysin. Taken together, these findings demonstrate that ExoS/T expressing  P. aeruginosa  and pneumolysin expressing  S. pneumoniae  predominate in bacterial keratitis. While  P. aeruginosa  strains expressing both ExoU and ExoS are usually rare, these strains actually outnumbered strains expressing only ExoU in the current study. Further, as neutrophils are the predominant cell type in these corneal ulcers, they are the likely source of cytokines and of the increased TLR and inflammasome expression.

Host Response and Bacterial Virulence Factor Expression in Pseudomonas aeruginosa and Streptococcus pneumoniae Corneal Ulcers