ABSTRACT
BACKGROUND: Infectious keratitis is the main cause of preventable blindness worldwide, with about 1.5-2.0 million new cases occurring per year. This inflammatory response may be due to infections caused by bacteria, fungi, viruses or parasites. Fungal keratitis is a poorly studied health problem. OBJECTIVES: This study aimed to identify a new fungal species by molecular methods and to explore the possible efficacy of the three most common antifungals used in human keratitis in Mexico by performing in vitro analysis. The capacity of this pathogen to cause corneal infection in a murine model was also evaluated. METHODS: The fungal strain was isolated from a patient with a corneal ulcer. To identify the fungus, taxonomic and phylogenetic analyses (nrDNA ITS and LSU data set) were performed. An antifungal susceptibility assay for amphotericin B, itraconazole and voriconazole was carried out. The fungal isolate was used to develop a keratitis model in BALB/c mice; entire eyes and ocular tissues were preserved and processed for histopathologic examination. RESULTS AND CONCLUSION: This fungal genus has hitherto not been reported with human keratitis in Mexico. We described a new species Purpurecillium roseum isolated from corneal infection. P roseum showed resistance to amphotericin B and itraconazole and was sensitive to voriconazole. In vivo study demonstrated that P roseum had capacity to developed corneal infection and to penetrate deeper corneal tissue. The global change in fungal infections has emphasised the need to develop better diagnostic mycology laboratories and to recognise the group of potential fungal pathogens.
Subject(s)
Antifungal Agents/therapeutic use , Hypocreales/classification , Hypocreales/drug effects , Hypocreales/isolation & purification , Keratitis/microbiology , Aged , Amphotericin B/therapeutic use , Animals , Cornea , DNA, Fungal , Drug Resistance, Fungal/drug effects , Female , Humans , Hypocreales/pathogenicity , Itraconazole/therapeutic use , Keratitis/drug therapy , Keratitis/pathology , Mexico , Mice , Mice, Inbred BALB C , Microbial Sensitivity Tests , Mycological Typing Techniques , Mycoses/drug therapy , Mycoses/microbiology , Phylogeny , Voriconazole/therapeutic useABSTRACT
PURPOSE: To report the characterization and analysis of the biofilm formation in mixed keratitis induced by the coinfection of Staphylococcus aureus and Fusarium falciforme in a novel murine model. METHODS: Clinical ocular microbial isolates and female BALB/c mice were used to develop the murine model. Immunosuppression was achieved with cyclophosphamide and methylprednisolone. A corneoscleral lesion was performed with a micro-pocket technique. Mice received an inoculum with a concentration of 1 × 105 conidia of F. falciforme and S. aureus with 1 × 105 UFC/ml. Mice were sacrificed at 72 h after induction of infection, the right eye was enucleated and preserved in 10% formaldehyde to perform the PAS staining. In addition, cuts were obtained for the labeling with the fluorophores propidium iodide and Calcofluor White, and other eye cuts were processed to transmission microscopy. RESULTS: F. falciforme and S. aureus were able to developed mono and mixed biofilm in vitro. Keratitis of F. falciforme, S. aureus and mixed, were established at immunosuppressed mice. Clinical symptoms were observed at murine cornea. Histological analysis by special stains identified bacterial, fungal and mixed biofilm structures at epithelial and stromal level. Extracellular matrix was observed surrounded clusters of bacterial, fungi and mixed by fluorescence and transmission electronic microscopy. CONCLUSION: This study provides direct evidence of the establishment and formation of mixed biofilm in vitro, as well as in vivo on the corneal surface of mice in an experimentally induced S. aureus and F. falciforme mixed keratitis infection.
