Abstract
Chronic periodontitis has a polymicrobial biofilm aetiology and interactions between key bacterial species are strongly implicated as contributing to disease progression. Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia have all been implicated as playing roles in disease progression. P. gingivalis cell-surface-located protease/adhesins, the gingipains, have been suggested to be involved in its interactions with several other bacterial species. The aims of this study were to determine polymicrobial biofilm formation by P. gingivalis, T. denticola and T. forsythia, as well as the role of P. gingivalis gingipains in biofilm formation by using a gingipain null triple mutant. To determine homotypic and polymicrobial biofilm formation a flow cell system was employed and the biofilms imaged and quantified by fluorescent in situ hybridization using DNA species-specific probes and confocal scanning laser microscopy imaging. Of the three species, only P. gingivalis and T. denticola formed mature, homotypic biofilms, and a strong synergy was observed between P. gingivalis and T. denticola in polymicrobial biofilm formation. This synergy was demonstrated by significant increases in biovolume, average biofilm thickness and maximum biofilm thickness of both species. In addition there was a morphological change of T. denticola in polymicrobial biofilms when compared with homotypic biofilms, suggesting reduced motility in homotypic biofilms. P. gingivalis gingipains were shown to play an essential role in synergistic polymicrobial biofilm formation with T. denticola.
Highlights
Polymicrobial biofilms are dynamic structures that can alter form or composition based on environmental conditions including nutrient supply, shear forces and temperature and on the synergies and antagonisms between the species that comprise the biofilm and the emergent properties that result from these interactions [1,2,3]
P. gingivalis was grown in brain heart infusion (BHI), T. denticola was grown in oral bacteria growth medium (OBGM), and T. forsythia was grown in tryptic soy broth supplemented with 0.3% yeast extract (TSBYK), vitamin K (0.4 mg/mL) and N-acetylmuramic acid (NAM) (10 mg/ mL) (Sigma Aldrich, MO, USA) as described previously [26,28]
The structures of P. gingivalis and T. denticola homotypic biofilms were quantified from Confocal Laser Scanning Microscopy (CLSM) images taken at five randomly chosen positions each containing a single microcolony from two biological replicates (Table 1)
Summary
Polymicrobial biofilms are dynamic structures that can alter form or composition based on environmental conditions including nutrient supply, shear forces and temperature and on the synergies and antagonisms between the species that comprise the biofilm and the emergent properties that result from these interactions [1,2,3]. Proteolytic bacterial species including Porphyromonas gingivalis, Treponema denticola and/or Tannerella forsythia are consistently found in elevated numbers in subgingival plaque samples taken from periodontally diseased subjects [5,6,7]. Among the three species that are frequently found associated with the clinical measures of chronic periodontitis, P. gingivalis has been shown to be a major pathogen, with well-defined virulence factors including cell surface located proteolytic enzymes and adhesins (gingipains) [12,13].Recent research suggests that a synergistic microbial community is more relevant to disease progression than individual species and it has been suggested that the abilities of micro-organisms to interact with one another are crucial for disease progression [14,15]. P. gingivalis has been shown to exert a community-wide pathogenic influence on the microbiota in an animal model and it has been suggested that the communication of P. gingivalis with other inhabitants of subgingival biofilm is crucial for the elevation of pathogenicity and disruption of host immune surveillance [16]
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