Biofilm formation by zygomycetes: quantification, structure and matrix composition

Most studies on fungal biofilms have focused on Candida in yeasts and Aspergillus in mycelial fungi. To the authors' knowledge, biofilm formation by zygomycetes has not been reported previously. In this study, the biofilm-forming capacity of Rhizopus oryzae, Lichtheimia corymbifera, Rhizomucor pusillus and Apophysomyces elegans was evaluated. At appropriate seeding spore densities, Rhp. oryzae (10⁵ c.f.u. ml⁻¹, L. corymbifera (10⁴ c.f.u. ml⁻¹) and Rhm. pusillus (10⁴ c.f.u. ml⁻¹) produced highly intertwined, adherent structures on flat-bottomed polystyrene microtitre plates after 24 h at 37 °C. The adhered fungal hyphae were encased in an extracellular matrix, as confirmed by phase-contrast and confocal microscopy. The thickness of Rhp. oryzae, L. corymbifera and Rhm. pusillus biofilms was 109.67±10.02, 242±23.07 and 197±9.0 µm (mean±sd), respectively. Biochemical characterization of the biofilm matrix indicated the presence of glucosamine, constituting 74.54-82.22 % of its dry weight, N-acetylglucosamine, glucose and proteins. Adherence and biofilm formation were not observed in A. elegans. Although A. elegans spores germinated at all three seeding densities tested (1×10⁷, 1×10⁶ and 1×10⁵ c.f.u. ml⁻¹), no significant difference was observed (P>0.05) between the A₄₉₀ of wells inoculated with A. elegans and the cut-off A₄₉₀ for biofilm detection. This study highlights the potential for biofilm formation by at least three medically important species of zygomycetes.