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Background: Coinfections with fungi and bacteria in ocular pathologies are increasing at an alarming rate. Two of the main etiologic agents of infections on the corneal surface, such as Aspergillus fumigatus and Staphylococcus aureus, can form a biofilm. However, mixed fungal-bacterial biofilms are rarely reported in ocular infections. The implementation of cell cultures as a study model related to biofilm microbial keratitis will allow understanding the pathogenesis in the cornea. The cornea maintains a pathogen-free ocular surface in which human limbo-corneal fibroblast cells are part of its cell regeneration process. There are no reports of biofilm formation assays on limbo-corneal fibroblasts, as well as their behavior with a polymicrobial infection. Objective: To determine the capacity of biofilm formation during this fungal-bacterial interaction on primary limbo-corneal fibroblast monolayers. Results: The biofilm on the limbo-corneal fibroblast culture was analyzed by assessing biomass production and determining metabolic activity. Furthermore, the mixed biofilm effect on this cell culture was observed with several microscopy techniques. The single and mixed biofilm was higher on the limbo-corneal fibroblast monolayer than on abiotic surfaces. The A. fumigatus biofilm on the human limbo-corneal fibroblast culture showed a considerable decrease compared to the S. aureus biofilm on the limbo-corneal fibroblast monolayer. Moreover, the mixed biofilm had a lower density than that of the single biofilm. Antibiosis between A. fumigatus and S. aureus persisted during the challenge to limbo-corneal fibroblasts, but it seems that the fungus was more effectively inhibited. Conclusion: This is the first report of mixed fungal-bacterial biofilm production and morphological characterization on the limbo-corneal fibroblast monolayer. Three antibiosis behaviors were observed between fungi, bacteria, and limbo-corneal fibroblasts. The mycophagy effect over A. fumigatus by S. aureus was exacerbated on the limbo-corneal fibroblast monolayer. During fungal-bacterial interactions, it appears that limbo-corneal fibroblasts showed some phagocytic activity, demonstrating tripartite relationships during coinfection.

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Purpose: Viral infections such as herpetic keratitis (HSK) activate the innate immune response in the cornea triggering opacity and loss of vision. This condition is performed mainly by myofibroblasts that exacerbate secretion of inflammatory cytokines. Amniotic membrane transplantation (AMT) reduces ocular opacity and scarring inhibiting secretion of inflammatory cytokines and proliferation of myofibroblasts. We previously reported that the amniotic membrane (AM) favors an anti-inflammatory microenvironment inhibiting the secretion of inflammatory cytokines, expression of innate immune receptors, and translocation of nuclear NF-κB on human limbal myofibroblasts (HLMs). The aim of the present study was to determine whether the soluble factors of the AM decrease the immune response of HLMs stimulated with polyinosinic-polycytidylic acid sodium salt (poly I:C). Methods: The AM was incubated in Dulbecco's modified eagle medium (DMEM)/F12, and the supernatant was collected to obtain amniotic membrane conditioned medium (AMCM). HLMs were isolated from cadaveric sclera-corneal rims. HLMs were cultured in DMEM/F12 or AMCM and stimulated or not with poly I:C (10 µg/ml) for 12 h to analyze synthesis of CCL2, CCL5, CXCL10, MDA5, RIG-1, and TLR3 or for 2 h to analyze translocation of nuclear NF-kB, IRF3, and IRF7. The proteins contained on AMCM were analyzed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and the acquired peptide ions were analyzed with the Mascot program using both National Center for Biotechnology Information (NCBI) and expressed sequence tag (EST) databases. Results: AMCM downregulated the mRNA levels of CCL2, CCL5, CXCL10, MDA5, RIG-1, and TLR3. In addition, AMCM decreased secretion of CCL2, CCL5, and CXCL10 and translocation of nuclear NF-κB. Interestingly, AMCM increased translocation of nuclear IRF3 and synthesis and secretion of type I IFN-ß. We also identified small leucine-rich proteoglycan lumican in the AMCM. The administration of rh-lumican to poly I:C-stimulated HLMs reduced the mRNA levels of CCL2, CCL5, and CXCL10. Conclusions: These results suggest that the AM can trigger an anti-inflammatory response on HLMs through soluble factors, and that lumican could play an important role in these effects.

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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.

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The interactions between prokaryotes and eukaryotes are abundant in nature. These microorganisms also interact in the human body. Fungal-bacteria interactions are present in many diseases. In this study, we evaluated the microbial interaction of Fusarium falciforme and Staphylococcus aureus developing mixed biofilm in vitro. When both microorganisms grew up together the mixed biofilm biomass decreased than F. falciforme monobiofilm biomass. S. aureus was able to interact and form aggregates over the mycelium and conidia surface of F. falciforme. Our results suggest that S. aureus could bind to colloidal chitin. On another hand, the supernatants from S. aureus biofilm and S. aureus-F. falciforme presented an antifungal effect over F. falciforme biofilm formation. Finally we found that the pH had an inhibitory effect over fungal biofilm formation. We concluded that S. aureus can affect the F. falciforme growth negatively in mixed biofilm involving factors like pH, supernatants compounds, anchor to chitin, and bacterial viability.

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Biofilms are structures that confer adaptive ability to and facilitate the virulence of fungal pathogens. Certain multi-functional proteins have been shown to be involved in fungal pathogenesis and these proteins may also be implicated in biofilm formation. The aim of this study was to identify a fungal agent isolated from the human cornea, to analyze the ability of this organism to form biofilms in vitro and to investigate protein expression in this condition. The fungus was identified by phylogenetic inference analysis. Biofilm formation and structure were evaluated by colorimetric methods and by optical and electron microscopy. We also resolved proteins obtained from biofilms and planktonic cultures by two-dimensional gel electrophoresis and identified those proteins by mass spectrometry. The fungus was identified as Fusarium falciforme. Colorimetric analysis and microscopy revealed that the highest level of biofilm formation was obtained at a concentration of 1 × 106 conidia/mL with 96 h of incubation at 28 °C. The biofilm architecture consisted of an extracellular matrix that embedded fungal filaments. We found nineteen proteins that were over-expressed in biofilms, as compared with planktonic cultures, and six proteins with unique expression in biofilms. Among the more abundant proteins identified were: transketolase, a putative antigen 1, enolase, phosphoglycerate kinase and ATP-citrate synthase. Some of these proteins are involved in basal metabolism, function as multi-functional proteins or have been described as potential virulence factors. We focused on the expression in biofilm of the enzyme, enolase, which was determined by real-time PCR. Our findings provide a perspective on the proteins associated with the formation of biofilms in vitro by an F. falciforme keratitis isolate.

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Fibroblasts are present in all tissues but predominantly in connective tissues. Some of their functions include contractility, locomotion, collagen and elastin fiber production, and the regulation and degradation of the extracellular matrix. Also, fibroblasts act as sentinels to produce inflammatory mediators in response to several microorganisms. There is evidence that fibroblasts can synthesize toll-like receptors (TLRs), antimicrobial peptides, proinflammatory cytokines, chemokines, and growth factors, which are important molecules involved in innate immune response against microorganisms. Fibroblasts can express TLRs (TLR-1 to TLR-10) to sense microbial components or microorganisms. They can synthesize antimicrobial peptides, such as LL-37, defensins hBD-1, and hBD-2, molecules that perform antimicrobial activity. Also, they can produce proinflammatory cytokines, such as TNFα, INFγ, IL-6, IL-12p70, and IL-10; other chemokines, such as CCL1, CCL2, CCL5, CXCL1, CXCL8, CXCL10, and CX3CL1; and the growth factors granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) to induce and recruit inflammatory cells. According to their immunological attributes, we can conclude that fibroblasts are sentinel cells that recognize pathogens, induce the recruitment of inflammatory cells via cytokines and growth factors, and release antimicrobial peptides, complying with the characteristics of real sentinels.

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BACKGROUND: Infectious keratitis is a sight-threatening condition for children. The purpose of this study was to describe the clinical profile, risk factors and microbiological profile of infectious keratitis in children. METHODS: Retrospective review of clinical records of patients under 16 years of age with history of microbial keratitis seen at a tertiary referral center. Clinical characteristics, risk factors, visual and surgical outcomes as well as the microbiological profile are analyzed. RESULTS: Forty-one eyes of 41 patients. Mean age was 8.7 years. Time between the onset of symptoms and ophthalmological examination was 12.7 days. Predisposing factors were found in 78%; ocular trauma was the most common (25%). Visual acuity equal or worse than 20/200 at admission correlated positively with a poorer visual outcome, p=0.002. Positivity of cultures was 34%. Gram-positive bacteria were isolated in 78.5%; Staphylococcus epidermidis (28.6%) was the most common microorganism. CONCLUSIONS: Our study emphasizes the importance of a prompt diagnosis and treatment of infectious corneal ulcers in children. Trauma and contact lenses were the main predisposing factors. Gram-positive organisms were isolated in the vast majority of cases and visual outcomes are usually poor.

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