Microbiology

Research Organisation Partners

• 312 - Univerzitetni klinični center Ljubljana / University Medical Centre Ljubljana
•  381 - Univerza v Ljubljani, Medicinska fakulteta / University of Ljubljana, Faculty of Medicine
•  481 - Univerza v Ljubljani, Biotehniška fakulteta / University of Ljubljana, Biotechnical Faculty

Abstract

Biofilms — complex, organised microbial colonies in a self-produced matrix of extracellular polymeric substances (EPS) — cause periodontitis, especially biofilms developed at the gingival margin and in subgingival areas of periodontal pockets. The EPS matrix contains proteins, polysaccharides and bacterial eDNA in varying proportions. The biofilm microbiota supports symbiosis in the health but dysbiosis during disease development. It is unclear how EPS properties change during the transition from symbiosis to dysbiosis and how these changes affect treatment that could depend on the viscoelastic mechanical structure and chemical composition of the EPS. Our initial results suggest that dysbiotic biofilms are stickier and more difficult to remove than healthy symbiotic biofilms. A systematic investigation of the viscoelastic qualities of biofilm in conjunction with clinically important periodontitis variables could improve disease management, especially if its rheological properties could be modified.

The project aims to (1) identify key microbial species and extracellular polymeric substance (EPS) composition associated with the transition from symbiotic to dysbiotic biofilm rheology, (2) determine clinically relevant mechanical properties of dysbiotic biofilms essential for biofilm removal and recolonization, and (3) alter biofilm stickiness with selected surfactants and emulsifiers.

Using ex vivo biofilms from infected and healthy areas, the viscoelastic properties of symbiotic and dysbiotic subgingival biofilms will be investigated and the matrix components and bacterial species responsible for the phenotype of the dysbiotic biofilm will be determined. The viscoelasticity of the biofilm will be defined by material functions. The linear viscoelastic mechanical properties of symbiotic and dysbiotic biofilms will be determined by means of viscosity curves demonstrating amplitude and frequency sweeps, relaxation and creep tests. NGS of 16S rRNA will profile the microbial community, and dPCR will determine the total microbial load. We will investigate how rheological conditions relate to microbiome parameters such as species richness, alpha and beta diversity and subgingival microbiome dysbiotic index (SMDI).

We will mimic symbiotic or dysbiotic subgingival biofilms using in vitro biofilm models of 14 species, including primary colonisers, core species and pathobiont species in a separate work package. We will also determine their linear viscoelastic mechanical properties by determining the above-mentioned rheological parameters. We will separate the biofilm matrix and biochemically analyse the primary EPS constituents, including eDNA, proteins, polysaccharides and lipids. We will investigate the relationship between the viscoelastic properties and microbial composition, specifically the amount and ratio of 14 bacterial species in symbiotic and dysbiotic biofilms to identify important microbial species that influence biofilm rheology.

We will attempt to disperse the isolated biofilm matrix with Dispersin B or Surfactin. Both emulsifiers will be tested in a biofilm model with 14 species in vitro. We will investigate how the emulsifiers influence the mechanical properties during de novo biofilm formation and survival of the mature biofilm. The reduction in biofilm volume, structural organisation and surface coverage will be evaluated using confocal fluorescence microscopy and BiofilmQ software. The gel-based product for application to gingival pockets will be developed.

In the last part, stickiness-reducing agent will be selected and tested in a split-mouth clinical study with 20 patients with stage III/IV, grade B/C periodontitis and more than 20 natural teeth with symmetrically distributed residual pockets limited to root furcations and intrabony pockets.

Reducing the stickiness of the dysbiotic biofilm should help to facilitate the removal of the biofilm, prevent its re-formation and improve the treatment of periodontitis patients.

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The phases of the project and their realization

WP1: Rheological properties of symbiotic and dysbiotic biofilms obtained from periodontitis

patients
 WP 2: Key microbial species and matrix components responsible for dysbiotic biofilm stickiness

WP3: Modification of biofilm mechanical properties to reduce stickiness

WP4: Clinical study of biofilm stickiness modifiers for better control of therapy-resistant

periodontal defects