Role of pvdE Pyoverdine Synthesis in Pseudomonas aeruginosa Keratitis.

PURPOSE: Pseudomonas aeruginosa produces pyoverdine, encoded by the pvdE gene, for high-affinity iron uptake from transferrin and lactoferrin. This study investigated the contribution of pyoverdine to P. aeruginosa keratitis pathogenesis using in vitro and in vivo models. METHODS: The P. aeruginosa strains examined were parental strain PAO1 and isogenic mutant strain pvdE (ΔpvdE) defective in pyoverdine. Bacterial growth in vitro was determined by PAO1 and ΔpvdE optical densities in Luria-Bertani (LB) broth. PAO1 or ΔpvdE (10 colony-forming units/mL) was inoculated onto cultured human corneal epithelial cells (HCECs) for 1 hour. The monolayers were examined for bacterial adhesion and invasion. In addition, the corneas of C57BL/6 mice were infected with PAO1 or ΔpvdE. Corneal virulence was evaluated by determining clinical scores and bacterial counts during infection. RESULTS: The growth of PAO1 and ΔpvdE in LB broth was similar. Although adhesion of ΔpvdE onto HCECs was significantly increased compared with PAO1, the invasive capacity of ΔpvdE was significantly decreased. Clinical scores and bacterial numbers were significantly lower in ΔpvdE-infected eyes compared with PAO1-infected eyes at 6, 24, and 48 hours (P < 0.001). ΔpvdE was not detected in mouse corneas and did not induce corneal opacity at 6, 24, or 48 hours. CONCLUSIONS: ΔpvdE lost invasive ability toward HCECs. Moreover, ΔpvdE did not cause keratitis in vivo. Thus, pvdE pyoverdine synthesis has critical roles in proliferation and invasion on ocular surfaces and could be a target for prevention of P. aeruginosa keratitis.

Anterior Segment Optical Coherence Tomography of Patients With Late-Onset Tunnel Fungal Infections With Endophthalmitis After Cataract Surgery.

PURPOSE: To report the use of anterior segment optical coherence tomography for characterization of late-onset tunnel fungal infections with endophthalmitis after cataract surgery. METHODS: Case reports. RESULTS: A 77-year-old female (case 1) and a 76-year-old male (case 2) who received cataract surgery 15 and 1 year before their initial visits, respectively, were treated with topical steroids based on a diagnosis of uveitis, because they showed growing white lesions on the upper iris and beneath the cataract scleral wound. Irrigation of the anterior chambers and removal of the white lesions were performed in each case, and microbiological tests were positive for fungi (case 1, a positive culture of Fusarium sp.; case 2, a filamentous fungus present in a direct smear) in the white lesions. Both cases were diagnosed as late-onset fungal endophthalmitis after cataract surgery and were treated with topical and systemic antifungal agents. However, the white lesions reappeared, and the inflammation in the anterior chambers worsened. Anterior segment optical coherence tomography showed the spread of the white lesions into the scleral incisions from cataract surgery. Deroofing of the tunnel and sclerocorneal patch grafts were performed in both cases to treat the fungal tunnel infections. After these treatments, inflammation of both corneas and anterior chambers subsided. CONCLUSIONS: Anterior segment optical coherence tomography can be used to identify late-onset fungal tunnel infections with endophthalmitis after cataract surgery.

Relationship of Virulence Factors and Clinical Features in Keratitis Caused by Pseudomonas aeruginosa.

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.

Polymorphisms in cytomegalovirus genotype in immunocompetent patients with corneal endotheliitis or iridocyclitis.

Cytomegalovirus (CMV) that caused corneal endotheliitis and iridocyclitis in immunocompetent patients was genotyped. The gB type1 was detected in seven endotheliitis samples (77.8%) and five iridocyclitis samples (100%), and the gB type 3 was detected in two endotheliitis samples (22.2%). The UL144 type 1 was found in five endotheliitis samples (45.5%) and five iridocyclitis samples (83.3%). The UL144 type 2 was found in two endotheliitis samples (18.2%) and one iridocyclitis sample (16.7%). The gB type 1 was predominant in endotheliitis and iridocyclitis, and the CMV genotypes in eyes with endotheliitis and iridocyclitis were similar.

Genotypic analysis of Pseudomonas aeruginosa isolated from ocular infection.

Pseudomonas aeruginosa is the causative pathogen of keratitis, conjunctivitis, and dacryocystitis. However little is known about their clinical epidemiology in Japan. In this study we investigated the genotypic characterization and serotype of P. aeruginosa isolates from ocular infections. Thirty-four clinical P. aeruginosa isolates were characterized according to infection type, the type III secretion system (TTSS), serotype, and multilocus sequence typing (MLST). We divided the isolates into four clinical infection types as follows: Contact lens (CL)-related keratitis (CL-keratitis; 15 isolates), non CL-related keratitis (non CL-keratitis; 8 isolates), conjunctivitis (7 isolates), and dacryocystitis (4 isolates). Regarding the TTSS classification and serotyping classification, no significant differences were found among the infection types. Two clusters (I, II) and three subclusters (A, B, C) were classified according to MLST. CL-keratitis isolates with exoU positivity were clustered in II-B, and conjunctivitis was clustered in cluster I. Some linkage was found between the genetic background and CL-keratitis or conjunctivitis.

Pseudomonas aeruginosa MucD protease mediates keratitis by inhibiting neutrophil recruitment and promoting bacterial survival.

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-1ß, 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-1ß, 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.

In vivo and in vitro laser confocal microscopy to diagnose acanthamoeba keratitis.

PURPOSE: To determine the effectiveness of laser confocal microscopy in identifying Acanthamoeba cysts and trophozoites in the cornea of patients with Acanthamoeba keratitis (AK) and to evaluate its effectiveness in following AK after treatment. METHODS: The corneas of 9 patients clinically diagnosed with AK were monitored periodically with the Heidelberg Retina Tomograph II-Rostock Cornea Module (HRT II-RCM) to examine for Acanthamoeba cysts and trophozoites during the clinical course. RESULTS: Seven of 9 patients had positive corneal smears, and 5 of 9 patients had positive laboratory cultures. HRT II-RCM demonstrated the presence of highly reflective polygonal shadows with lower reflective borders in the cornea of all patients. In 1 patient, a highly reflective pleomorphic shadow with small less-reflective areas was detected inside the cell. The former finding resembled the image of Acanthamoeba cysts in culture as observed by HRT II-RCM, and the latter observation with that of Acanthamoeba trophozoites in culture. After treatment, the number of highly reflective inflammatory cells decreased and the number and morphology of the corneal epithelial cells with highly reflective nuclei recovered to normal levels. CONCLUSION: These results indicate that in vivo laser confocal microscopy can be a useful method to make a diagnosis and to follow patients with AK.

Demonstration of "owl's eye" morphology by confocal microscopy in a patient with presumed cytomegalovirus corneal endotheliitis.

PURPOSE: To report confocal microscopic observations of characteristic corneal endothelial lesions in a patient with presumed cytomegalovirus (CMV) corneal endotheliitis. DESIGN: Case report. METHODS: A 77-year-old, immunocompetent man was admitted with corneal edema, keratic precipitates, and coin-shaped lesions in the right eye. Confocal microscopy was performed to examine the corneal endothelium. Polymerase chain reaction (PCR) was used to identify viral DNA in an aqueous humor sample. RESULTS: CMV DNA was detected by PCR. Confocal microscopy showed large corneal endothelial cells with an area of high reflection in the nucleus surrounded by a halo of low reflection. This "owl's eye" morphology is characteristic of CMV infection. Topical and intravenous ganciclovir treatment resulted in rapid resolution of the corneal precipitates and edema, followed by disappearance of the owl's eye morphology. CONCLUSIONS: Confocal microscopy can detect the owl's eye morphology in the corneal endothelium of patients with presumed CMV corneal endotheliitis.