Diana Gabriela Ponce-Angulo1, Luis Antonio Bautista-Hernández2, Rosa Paulina Calvillo-Medina3, Franco Ivan Castro-Tecorral4, Gerardo Aparicio-Ozores5, Edgar Oliver López-Villegas6, Rosa María Ribas-Aparicio5, Victor Manuel Bautista-de Lucio7. 1. Departamento de Microbiología y Proteómica Ocular, Instituto de Oftalmología "Fundación de Asistencia Privada Conde de Valenciana", Mexico City, Mexico; Programa de Posgrado en Biomedicina y Biotecnología Molecular, Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City, Mexico. 2. Departamento de Microbiología y Proteómica Ocular, Instituto de Oftalmología "Fundación de Asistencia Privada Conde de Valenciana", Mexico City, Mexico; Programa de Doctorado en Ciencias Biológicas y la Salud, Universidad Autónoma Metropolitana, Mexico City, Mexico. 3. Departamento de Microbiología y Proteómica Ocular, Instituto de Oftalmología "Fundación de Asistencia Privada Conde de Valenciana", Mexico City, Mexico. 4. Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Mexico City, Mexico. 5. Programa de Posgrado en Biomedicina y Biotecnología Molecular, Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City, Mexico. 6. Central de Microscopía, Departamento de Investigación, Sección de Estudios de Posgrado e Investigación, Escuela Nacional de Ciencia Biológicas, Instituto Politécnico Nacional, Mexico City, Mexico. 7. Departamento de Microbiología y Proteómica Ocular, Instituto de Oftalmología "Fundación de Asistencia Privada Conde de Valenciana", Mexico City, Mexico. Electronic address: vbautistal@institutodeoftalmologia.org.
Abstract
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.
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.
Authors: Gislaine G O S Silveira; Marcelo D T Torres; Camila F A Ribeiro; Beatriz T Meneguetti; Cristiano M E Carvalho; Cesar de la Fuente-Nunez; Octávio L Franco; Marlon H Cardoso Journal: ACS Pharmacol Transl Sci Date: 2021-01-27