Proteomic analysis of the interaction Fusarium solani- Staphylococcus aureus isolated from human keratitis in presence of antimicrobial agents.

Abstract

Purpose: To study the proteomic profile of the coculture of Fusarium solani-Staphylococcus aureus in presence of antimicrobials agents and analyze biofilm formation.

Methods: Staphylococcus aureus and Fusarium solani were isolated from human keratitis. The microbial were characterized by microbiological and molecular means. The cocultures were established in Müller-Hinton Agar with or without Gatifloxacin and/or Itraconazole during 48h. The cocultures containing 48h biomass were processed to obtain the cellular extract; the proteins were quantified, precipitated and 2D electrophoresis performed. The proteomic profiles were analyzed by Dymension 2.0 software to determine the differential protein expression in the cocultures compared to cocultures in presence of antimicrobials. The differential expressed spots were identified by MALDI-TOF. The biofilms were established in 96 well with RPMI medium, the concentrations used for S. aureus was 1X105 and F. solani 1X105 for 24h, the biofilm formation was determined by crystal violet assay in ELISA microplate reader at 595 nm. Biofilms were pre-formed in coverslips and stain with DAPI for fluorescence microscopy.

Results: The coculture fungi grown was modified with antimicrobial treatment, combinated therapy may not showed the same effect as a single therapy with Itraconazole. Twenty-two proteins were identified from proteomics profiles: Enolase, Glyceraldehyde-3-phosphate dehydrogenase, Fructosa-bisphosphate aldolase, Protein disulfide-isomerase, Aconitate Hydratase cytoplasmic of F. solani. The biolfims microscopy fluorescence showed interactions between S. aureus and hypha of F. solani.

Conclusions: In the coculture F. solani-S. aureus-Itraconazole, F. solani showed low growth in presence of Itraconazole compared with coculture F. solani-S. aureus. Coculture F. solani-S. aureus showed differential protein expression compared to the cocultures with Itraconazole and/or Gatifloxacin. The biofilm of F. solani-S. aureus showed interactions of S. aureus with the hypha of F. solani.

Keywords: 594 microbial pathogenesis: experimental studies • 663 proteomics • 480 cornea: basic science