Abstract
BackgroundBiofilms are communal structures of microorganisms that have long been associated with a variety of persistent infections poorly responding to conventional antibiotic or antifungal therapy. Aspergillus fumigatus fungus and Stenotrophomonas maltophilia bacteria are examples of the microorganisms that can coexist to form a biofilm especially in the respiratory tract of immunocompromised patients or cystic fibrosis patients. The aim of the present study was to develop and assess an in vitro model of a mixed biofilm associating S. maltophilia and A. fumigatus by using analytical and quantitative approaches.Materials and MethodsAn A. fumigatus strain (ATCC 13073) expressing a Green Fluorescent Protein (GFP) and an S. maltophilia strain (ATCC 13637) were used. Fungal and bacterial inocula (105 conidia/mL and 106 cells/mL, respectively) were simultaneously deposited to initiate the development of an in vitro mixed biofilm on polystyrene supports at 37°C for 24 h. The structure of the biofilm was analysed via qualitative microscopic techniques like scanning electron and transmission electron microscopy, and fluorescence microscopy, and by quantitative techniques including qPCR and crystal violet staining.ResultsAnalytic methods revealed typical structures of biofilm with production of an extracellular matrix (ECM) enclosing fungal hyphae and bacteria. Quantitative methods showed a decrease of A. fumigatus growth and ECM production in the mixed biofilm with antibiosis effect of the bacteria on the fungi seen as abortive hyphae, limited hyphal growth, fewer conidia, and thicker fungal cell walls.ConclusionFor the first time, a mixed A. fumigatus—S. maltophilia biofilm was validated by various analytical and quantitative approaches and the bacterial antibiosis effect on the fungus was demonstrated. The mixed biofilm model is an interesting experimentation field to evaluate efficiency of antimicrobial agents and to analyse the interactions between the biofilm and the airways epithelium.
Highlights
Biofilm is composed of densely packed colonies of microorganisms enclosed in a matrix of self-produced extracellular polymeric substance, and adhered to an abiotic or biotic surface
Decrease of A. fumigatus growth and extracellular matrix (ECM) production in the mixed biofilm with antibiosis effect of the bacteria on the fungi seen as abortive hyphae, limited hyphal growth, fewer conidia, and thicker fungal cell walls
Among emerging microorganisms concomitantly isolated with A. fumigatus from the respiratory tract of immunocompromised patients or those suffering from chronic respiratory diseases, Pseudomonas aeruginosa, a non-fermentative Gram-negative bacillus, is the most studied [4]
Summary
Biofilm is composed of densely packed colonies of microorganisms enclosed in a matrix of self-produced extracellular polymeric substance, and adhered to an abiotic or biotic surface. Biofilms represent a protective environment enabling microorganisms to thrive in a hostile surrounding, and show inherent resistance to antimicrobial agents Both factors give rise to many persistent and chronic infections including chronic middle ear infection, chronic sinusitis or otitis, and chronic lung disease [1]. Among emerging microorganisms concomitantly isolated with A. fumigatus from the respiratory tract of immunocompromised patients or those suffering from chronic respiratory diseases, Pseudomonas aeruginosa, a non-fermentative Gram-negative bacillus, is the most studied [4]. Other bacteria, such as Stenotrophomonas maltophilia, have recently been identified as an important hospital-associated pathogen colonising the same population of patients. The aim of the present study was to develop and assess an in vitro model of a mixed biofilm associating S. maltophilia and A. fumigatus by using analytical and quantitative approaches
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