Abstract
Development of dysbiosis in complex multispecies bacterial biofilms forming on teeth, known as dental plaque, is one of the factors causing periodontitis. Fusobacterium nucleatum (F. nucleatum) 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 F. nucleatum have been identified: animalis, fusiforme, nucleatum, polymorphum, and vincentii. Differential integration of subspecies into multispecies biofilm models has been reported, however, biofilm forming ability of individual F. nucleatum subspecies is largely unknown. The aim of this study was to determine the single-subspecies biofilm forming abilities of F. nucleatum ATCC type strains. Static single subspecies F. nucleatum 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 F. nucleatum 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, F. nucleatum ssp. polymorphum did not form a detectable, continuous layer of biofilm on any of the tested substrates. Consistent with limited biofilm forming ability in vitro, F. nucleatum ssp. polymorphum showed the least conservation of the adhesion proteins CmpA and Fap2 in silico. Here, we show that biofilm formation by F. nucleatum in vitro is subspecies- and substrate-specific. Additionally, F. nucleatum ssp. polymorphum does not appear to form stable single-subspecies continuous layers of biofilm in vitro. Understanding the differences in F. nucleatum 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 F. nucleatum-mediated infections and diseases.
Highlights
The oral environment consists of a multitude of bacterial species living on both soft and hard tissues in complex multispecies communities known as biofilms [1]
Differences in biofilm mass were seen amongst the subspecies and on the different surfaces (Figures 1A,B): Generally, sandblasted glass surfaces supported biofilm formation best with significantly higher biofilm mass in most subspecies compared to untreated glass
FNF adhered significantly better to uncoated Thermanox coverslips when compared to uncoated glass (p = 0.002), the other subspecies showed no difference in biofilm formation between glass and plastic surfaces
Summary
The oral environment consists of a multitude of bacterial species living on both soft and hard tissues in complex multispecies communities known as biofilms [1]. A type of biofilm which forms on the surface of teeth, has been extensively studied for decades due to its association with periodontitis, a chronic inflammatory disease of the toothsupporting tissues [2]. One of the factors leading to periodontitis is the accumulation of dental plaque and emergence of dysbiosis therein [2, 3]. Studies analysing diseased periodontal sites exhibited a shift toward communities containing anaerobic, Gram-negative periodontal pathogens belonging to Socransky’s “red complex” and leading to dysbiosis with an associated destructive immune-inflammatory response resulting in tissue damage [8–12]
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