In vitro polymicrobial inter-kingdom three-species biofilm model: influence of hyphae on biofilm formation and bacterial physiology

Abstract

Biofilms are an important medical burden, notably for patients with orthopaedic device-related infections. When polymicrobial, these infections are more lethal and recalcitrant. Inter-kingdom biofilm infections are poorly understood and challenging to treat. Here, an in vitro three-species model including Staphylococcus aureus, Escherichia coli and Candida albicans was developed, to represent part of the diversity observed in orthopaedic infections or other clinical contexts. The importance of fungal hyphae for biofilm formation and virulence factor expression was explored. Two protocols were set up, allowing, or not, for hyphal formation. Culturable cells and biomass were characterised in both models, and biofilms were imaged in bright-field, confocal and electron microscopes. The expression of genes related to virulence, adhesion, exopolysaccharide synthesis and stress response was analysed in early-stage and mature biofilms. It was found that biofilms enriched in hyphae had larger biomass and showed higher expression levels of genes related to bacterial virulence or exopolysaccharides synthesis.

Keywords:

• Polymicrobial biofilm
• hyphae
• confocal microscopy
• Candida albicans
• Staphylococcus aureus
• Escherichia coli

Acknowledgements

The authors thank Prof. Patrick Van Dijck (KULeuven, Leuven, Belgium) for his critical insight in fungal biofilms, Alix Mangin and Virginie Mohymont (LDRI, UCLouvain, Brussels, Belgium) for their technical assistance, Dr Caroline Bouzin (IREC, UCLouvain, Brussels, Belgium) for advice and support in imaging studies and Dr Tiep Nguyen (LDRI, UCLouvain, Brussels, Belgium) for advice and support in RT-qPCR.

Disclosure statement

The authors declare no conflict of interest.

Additional information

Funding

A.R.S. was funded by the Orthenzy project, Win2Wal program from ‘Region Wallonne’, Belgium. H.P. was funded by ‘Fonds pour la Recherche dans l’Industrie et l’agriculture’ (FRIA) from FRS-FNRS. F.V.B. is a research director from the Belgian Fonds de la Recherche Scientifique (FRS-FNRS). This work was funded by the Orthenzy project, by the Belgian FRS-FNRS (grant J.0162.19) and by KU Leuven Internal Funds (STG/17/024 and C32/18/059).