1. Biofilm, dental plaque MUDr. Černohorská Lenka, PhD. Department of Microbiology Masaryk University Medical School and St. Anna´s Faculty Hospital, Brno, Czechia

2. Microbial growth Planktonic form M icrobial cells float freely in a fluid Biofilm form M icrobial cells stick to one another and to a solid surface and form a community connected by an extracellular matter

3. Examples of biofilm Have you ever slipped on a wet stone in a creek? It was biofilm that you slipped on Have you an aquarium and do you clean its walls? If you do, what you wipe from them is the biofilm formed by algae Do you clean your teeth regularly? I hope so and by doing this you remove the biofilm called dental plaque

4. Definition of biofilm Microbial biofilm is a 3D strucuture which: forms at the boundary of phases (usually of the solid and fluid phase) is surrounded by an extracellular matter, in which a complex system of channels forms

5. Stages of biofilm development Direct contact of a planktonic bacteria with a surface + Attachment to this surface Adhesion, growth, and aggregation of cells into microcolonies Production of polymeric matrix Formation of three-dimensional structure known as biofilm

6. Development of biofilm 1. Attraction – in mobile bacteria due to flagella 2. Adhesion – via bacterial adhesins fimbriae (pilli): colonization factors of enteropathogenic E. coli proteins and lipopolysaccharides of outer membrane ( G-negative bacteria) slime ( coagulase-negative + S. aureus) curli ( E. coli)

7. Development of biofilm – 3. Aggregation Movement - by flagella (E. coli), by means of fimbriae (P.aeruginosa), convergent – aggregates of different species (coaggregation of Str. gordonii + F. nucleatum in dental plaque) Multiplication - aggregation + cell division in aggregates lead to the development of microcolonies Quorum sensing- during division individual cells emit chemical signals (homoserinlactones in P. aeruginosa). After reaching a particular number of cells (quorum) the elevated concentration of signals causes the change of cellular properties: switching off some genes, expression of other genes, production of new molecules

8. Development of biofilm 4. Accumulation: production of exopolysaccharides leads to the development of typical biofilm architecture - colanic acid (E. coli), alginate (P. aeruginosa), polysaccharide intercellular adhesin (S. epidermidis) 5. Dispersal: after reaching the critical amount of biomass + after the reduction of nutrients in the environment the character of surface cells changes (for. ex. P. aeruginosa the superficial cells produce lyase and flagellin, superficial layer of biofilm starts to disintegrate, cells grow flagella and get loose of biofilm) colonize new surfaces. The cycle closes… The cells drift away as a planktonic population to look for more suitable environment and to colonize new surfaces. The cycle closes…

9. Architecture of biofilm Alcian blue has coloured extracellular polysaccharides. Photo: Veronika Holá  Candida albicans biofilm. Toluidin blue - mushroom-like structure of the biofilm

10. Architecture of biofilm Depends on the concentration of nutrients <10 mg/L (mountain streams, lakes, open sea) heterogeneous mosaic - a thin layer + columns of microcolonies 10-1000 mg/L (majority of our rivers and ponds) complex system with channels (created by mushroom-like, partially merging microcolonies)  1000 mg/L (in the environment of macroorganism) compact biofilm (almost without traces of channels)

11. Main importance of biofilm formation Bacteria harbored inside are protected against:  antibiotic action  host´s immune response  disinfection

12. Researching methods  In bacterial strains (S. aureus, E. coli, P. aeruginosa etc.) biofilm can be detected by the modified Christensen method  Biofilm susceptibility testing: MBIC (minumum biofilm inhibitory concentration) was determined  MBIC was compared with MIC  Synergy testing: FBIC (fractionate biofilm inhibitory concentration) was calculated as follows: FBICs (∑FBIC) = MBIC ATB A in combination + MBIC ATB B in combination MBIC ATB A alone MBIC ATB B alone Combinations of antimicrobial agents: synergistic (∑FBIC ≤ 0,5) partially synergistic (∑FBIC > 0.5 a ≤1) indifferent (∑FBIC > 1 a ≤ 4) antagonistic (∑FBIC > 4) + -

13. Antibiotic susceptibility of staphylococci isolates Antibiotic susceptibility of staphylococci isolates Abbreviations: ams - ampicillin/sulbactam, ch - chloramphenicol, ery - erytromycin, te - tetracyclin, cli - clindamycin, tei - teicoplanin, van - vancomycin, ofl - ofloxacin Planktonic bacteria (MIC)Biofilm-forming bacteria (MBIC)

14. The inefficiency of antibiotics may be due to: Polyanionic charge Polyanionic charge of sessile cells Decreased growth Decreased bacterial growth Diffusion barrier Diffusion barrier of glycocalyx Reaction Reaction with biofilm matrix phenotypes Formation of protected phenotypes intercellular signalling Mechanism of intercellular signalling immune response mechanisms… Host´s immune response mechanisms…

15. Dental plaque Sticky microbial layer on the teeth surface, composed of living and dead bacteria and their products + components of host origin originated from salivas Cannot be washed, is removable only mechanically Supragingival and subgingival Differs in morphology and microbial components Subgingival plaque is divided into adherent and non- adherent Composition of plaque depends on its age and localization, speed of plaque formation is individually Contains many bacterial species

16. Development of dental plaque First 24 hours In supragingival plaque Group of Streptococcus mutans, sanguinis and mitis Days Bad hygiene of oral cavity = plaque increase = G+ more G+ rods and filamentous microorganism- lactobacili and actinomycets 1 week old plaque Column microcolonies of microbes, on the plaque surface adhesion of rods/filaments 3 weeks old plaque Filamentous forms of microbes, ear of corn-like on the surface: central fibre G+ cocci (Eubacterium yurii) surrounded by G+ cocci

17. Plaque development 1. On the teeth surface forms thin layer of saliva glycoproteins = pelicula. 2. The salivas transport oral bacteria to this surface 3. First adhere G+ cocci and rods due to surface adhesins. To G+ bacteria adhere the other 4. Aggregated bacteria divides and form microcolonies 5. Production of exopolysaccharides

18. 6. Metabolism of bacteria changes their environment and enable plaque formation also to another microbial species 7. Presence of saccharosis accelerates mature of plaque 8. Bacteria free themselves from the outer layers, inner layers form dental stone 9. pH under 5,5 -demineralisation of enamel and formation of dental caries

19. Reaction of gingiva Exsudate formation Inflammation – gingivitis - damages function of joining epitelium and plaque penetrates to subgingival area Older and stronger plaque – more symptoms Kolenbrander et al., 2002

20. Subgingival plaque 2 types: adherent and non-adherent Adherent plaque - sessile on the stub = G+ rods and fibres (actinomycets) and G+ cocci Non-adherent plaque - between adherent plaque and surface of soft gingival tissue = G- mobile anaerobes

21. Non-adherent plaque Mobile G- anaerobes: Porphyromonades (P. gingivalis), Prevotela (P. nigrescens) Fusobacterium (F. nucleatum subsp. polymorphum) Treponema (T. denticola). More patogennous than G+ cocci and rods. Gingivitis became worse = development of parodontal snout.

22. Plaque on the teeth implantates Various bacteria Group of Streptococcus mutans and sanguinis, yeast (Candida) - on contact area with mucous Anaerobes: G+ rods + Actinomyces israelii+ veillonela Staphylococci (S. aureus)

23. Teeth stone 80 % minerals, mainly hydroxylapatit, less calcium carbonate and magnesium phosphate and organic substances (rest of microbial cells, epitelia and mucin) Stone above gingiva contains mainly G+ bacteria, subgingival stone G - bacteria Structure - porosity and rough surface of dental stone - filamentous bacteria in plaque are oriented vertical+palisade to surface of the teeth - storage of microbial components toxic for parodontic tissue