Purpose: To examine bacterial virulence factors in Pseudomonas aeruginosa isolates from contact lens (CL) wearers and non–CL wearers with P. aeruginosa keratitis, and to investigate relationships between virulence factors and clinical features of keratitis. Methods: The study involved 25 subjects including 18 CL and 7 non–CL-related P. aeruginosa keratitis patients. Slit-lamp photographs of all subjects were captured, and the focus occupancy ratio (FOR) was defined as the total focus area/entire cornea area, using image processing software. Twenty-five clinical P. aeruginosa isolates from keratitis were assessed for protease production, elastase production, biofilm formation, bacterial swimming and swarming motility, cell surface hydrophobicity, and genes encoding the type III secretion system (TTSS) effectors (ExoU and ExoS). Results: Ring abscess was found in 9 of 18 CL-related P. aeruginosa keratitis cases (CL[+] ring[+] group) but not in another 9 cases (CL[+] ring[−] group). Expression or prevalence of virulence factors in P. aeruginosa isolates from the CL(+) ring(+) group, CL(+) ring(−) group, and CL(−) group were compared. The FOR for CL(+) ring(+) or CL(−) was higher than for CL(+) ring(−) (P < 0.05 and P < 0.01, respectively). The rate of positive swimming motility for CL(+) ring(+) or CL(−) was higher than for CL(+) ring(−) (P < 0.05), whereas the rate of positive swarming motility for CL(+) ring(+) was higher than for CL(+) ring(−) or CL(−) (P < 0.05). Prevalence of an exoS+/exoU-genotype for CL(+) ring(+) or CL(−) was higher than for CL(+) ring(−) (P < 0.05). In the CL-related group, expression of elastase and swarming motility significantly correlated with FOR. Conclusions: Swimming motility, swarming motility, and TTSS ExoS could play a major role in the determination of clinical features of P. aeruginosa keratitis.

Purpose.: Pseudomonas aeruginosa is a leading pathogen of blinding keratitis worldwide. In this study, the role of the serine protease in the pathogenesis of P. aeruginosa keratitis in the mouse cornea was investigated by comparing the parent and rescue strains. Methods.: Cornea of C57BL/6 mice were infected with P. aeruginosa strain PAO1, serine protease (MucD protease or PA3535) mutants (ΔmucD or ΔPA3535), or a complemented strain. Corneal virulence was evaluated by determining clinical scores and bacterial enumeration. A myeloperoxidase assay was performed to determine the number of polymorphonuclear (PMN) cells infiltrating the cornea. An ELISA was used to quantify inflammatory cytokines and chemokines in the cornea. Results.: The clinical score and bacterial numbers in eyes infected with ΔmucD were significantly lower than in those infected with PAO1, ΔPA3535, or the MucD rescue strain after 48 hours (P < 0.001). A larger number of infiltrating PMN cells was observed in eyes infected with ΔmucD at 12 and 24 hours, compared with eyes infected with PAO1. IL-1β, KC, and MIP2 levels were higher in eyes infected with ΔmucD than in those infected with PAO1 after 12 hours. Conclusions.: The MucD protease suppressed IL-1β, KC, and MIP2 during the early stages of the infection and inhibited neutrophil recruitment in the cornea. Therefore, the MucD protease contributes significantly to the pathogenesis of keratitis. MucD protease plays a critical role in the establishment of Pseudomonas aeruginosa keratitis by facilitating evasion of the immune response.

Purpose.: To evaluate the disinfectant effect of methylene blue (MB)–mediated photodynamic therapy (PDT) on a pathogenic strain of Acanthamoeba. Methods.: Acanthamoeba castellanii (ATCC 50370) used in this study were treated under one of four experimental conditions: light irradiation and incubation in MB (L+M+), light irradiation and incubation in physiologic solution (L+M−), incubation in MB only (L−M+), and incubation in physiologic solution (L−M−). M+ trophozoites were incubated in either 0.25 or 0.5 mM MB for 10 minutes. L+ organisms were irradiated for 30 minutes following incubation in solution. A halogen lamp (660 ± 10 nm) with a maximum output of 6 mW/cm² was used as the PDT light source. After treatment, antiacanthamoeba activity was evaluated by checking the respiratory activity of the amoeba with 5-cyano-2,3-tetrazolium chloride (CTC) staining. We also determined whether the effect of PDT with MB had been retained or augmented when it was performed in combination with conventional antiamoebic agents. Results.: MB-PDT suppressed the respiratory activity of trophozoites in an MB-concentration–dependent manner at total light doses of 10.8 J/cm². The respiratory activity of each group as a percentage of that of L−M− is as follows: L+M+ 11.6% (0.5 mM), 60.9% (0.25 mM); L−M+ 116.5% (0.5 mM), 105.5% (0.25 mM); L+M− 107.6%; and L−M− 106.3%. (L+M+ versus L−M− P < 0.05). MB-PDT had a synergistic effect when used in combination with polyhexamethylene biguanide (PHMB) or amphotericin B, but not with voriconazole. Conclusions.: MB-PDT is effective against Acanthamoeba in vitro and has synergistic effects with PHMB and amphotericin B.

Phlyctenular keratitis is a hypersensitivity reaction of the cornea, and a complication of eyelid margin disease in children and young adults. In this study, we compared the morphology of the meibomian glands in eyelids between phlyctenular keratitis patients and healthy young adults, using noncontact meibography. The study included 16 eyes of 13 patients diagnosed with phlyctenular keratitis and 17 eyes of 17 healthy volunteers. Slit-lamp observations of the cornea and eyelid were performed on all subjects. The morphology of the meibomian glands was scored using non-contact meibography (meiboscore). The meiboscore in worse eye was used in bilateral phlyctenular keratitis. All eyes with phlyctenular keratitis, but not normal controls, showed corneal nodules, neovascularization, and superficial punctate keratopathy. The mean meiboscore in phlyctenular keratitis patients (upper lid: 2.9 ± 0.3, lower lid: 2.7 ± 0.5) was significantly higher than in controls (upper lid: 0.4 ± 0.6, lower lid: 0.1 ± 0.3). Noncontact meibography enabled visualization of meibomian gland loss in phlyctenular keratitis patients, suggesting a relationship between abnormalities of the meibomian glands in young individuals and the pathogenesis of phlyctenular keratitis.

The prostaglandin and leukotriene families of lipid mediators are formed via two distinct biosynthetic pathways that are initiated by the oxygenation of arachidonic acid by either cyclooxygenase-2 (COX-2) or 5-lipoxygenase (5-LOX), respectively. The 5-LOX product 5S-hydroxyeicosatetraenoic acid, however, can also serve as an efficient substrate for COX-2, forming a bicyclic diendoperoxide with structural similarities to the arachidonic acid-derived prostaglandin endoperoxide PGH₂[Schneider C, et al. (2006)J Am Chem Soc128:720–721]. Here we identify two cyclic hemiketal (HK) eicosanoids, HKD₂and HKE₂, as the major nonenzymatic rearrangement products of the diendoperoxide using liquid chromatography–mass spectrometry analyses as well as UV and NMR spectroscopy. HKD₂and HKE₂are furoketals formed by spontaneous cyclization of their respective 8,9-dioxo-5S,11R,12S,15S-tetrahydroxy- or 11,12-dioxo-5S,8S,9S,15S-tetrahydroxy-eicosadi-6E,13E-enoic acid precursors, resulting from opening of the 9S,11R- and 8S,12S-peroxide rings of the diendoperoxide. Furthermore, the diendoperoxide is an efficient substrate for the hematopoietic type of prostaglandin D synthase resulting in formation of HKD₂, equivalent to the enzymatic transformation of PGH₂to PGD₂. HKD₂and HKE₂were formed in human blood leukocytes activated with bacterial lipopolysaccharide and calcium ionophore A23187, and biosynthesis was blocked by inhibitors of 5-LOX or COX-2. HKD₂and HKE₂stimulated migration and tubulogenesis of microvascular endothelial cells, implicating a proangiogenic role of the hemiketals in inflammatory sites that involve expression of 5-LOX and COX-2. Identification of the highly oxygenated hemiketal eicosanoids provides evidence for a previously unrecognized biosynthetic cross-over of the 5-LOX and COX-2 pathways.