AUTHOR=Muchova Maria , Balacco Dario L. , Grant Melissa M. , Chapple Iain L. C. , Kuehne Sarah A. , Hirschfeld Josefine 

TITLE=Fusobacterium nucleatum Subspecies Differ in Biofilm Forming Ability in vitro

JOURNAL=Frontiers in Oral Health

VOLUME=3

YEAR=2022

URL=https://www.frontiersin.org/journals/oral-health/articles/10.3389/froh.2022.853618

DOI=10.3389/froh.2022.853618

ISSN=2673-4842

ABSTRACT=<p>Development of dysbiosis in complex multispecies bacterial biofilms forming on teeth, known as dental plaque, is one of the factors causing periodontitis. <italic>Fusobacterium nucleatum</italic> (<italic>F. nucleatum</italic>) is recognised as a key microorganism in subgingival dental plaque, and is linked to periodontitis as well as colorectal cancer and systemic diseases. Five subspecies of <italic>F. nucleatum</italic> have been identified: <italic>animalis, fusiforme, nucleatum, polymorphum</italic>, and <italic>vincentii</italic>. Differential integration of subspecies into multispecies biofilm models has been reported, however, biofilm forming ability of individual <italic>F. nucleatum</italic> subspecies is largely unknown. The aim of this study was to determine the single-subspecies biofilm forming abilities of <italic>F. nucleatum</italic> ATCC type strains. Static single subspecies <italic>F. nucleatum</italic> biofilms were grown anaerobically for 3 days on untreated or surface-modified (sandblasting, artificial saliva, fibronectin, gelatin, or poly-L-lysine coating) plastic and glass coverslips. Biofilm mass was quantified using crystal violet (CV) staining. Biofilm architecture and thickness were analysed by scanning electron microscopy and confocal laser scanning microscopy. Bioinformatic analysis was performed to identify orthologues of known adhesion proteins in <italic>F. nucleatum</italic> subspecies. Surface type and treatment significantly influenced single-subspecies biofilm formation. Biofilm formation was overall highest on poly-L-lysine coated surfaces and sandblasted glass surfaces. Biofilm thickness and stability, as well as architecture, varied amongst the subspecies. Interestingly, <italic>F. nucleatum</italic> ssp. <italic>polymorphum</italic> did not form a detectable, continuous layer of biofilm on any of the tested substrates. Consistent with limited biofilm forming ability <italic>in vitro, F. nucleatum</italic> ssp. <italic>polymorphum</italic> showed the least conservation of the adhesion proteins CmpA and Fap2 <italic>in silico</italic>. Here, we show that biofilm formation by <italic>F. nucleatum in vitro</italic> is subspecies- and substrate-specific. Additionally, <italic>F. nucleatum</italic> ssp. <italic>polymorphum</italic> does not appear to form stable single-subspecies continuous layers of biofilm <italic>in vitro</italic>. Understanding the differences in <italic>F. nucleatum</italic> single-subspecies biofilm formation may shed light on multi-species biofilm formation mechanisms and may reveal new virulence factors as novel therapeutic targets for prevention and treatment of <italic>F. nucleatum</italic>-mediated infections and diseases.</p>