Transcript Bakterie tvořící biofilm - IS MU

Biofilm, dental plaque
MUDr. Černohorská Lenka, PhD.
Department of Microbiology Masaryk University Medical School and
St. Anna´s Faculty Hospital, Brno, Czechia
Microbial growth
Planktonic form
Microbial cells float freely in a fluid
Biofilm form
Microbial cells stick to one another and to a
solid surface and form a community
connected by an extracellular matter
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
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
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
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)
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
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)
The cells drift away as a planktonic population to look for more
suitable environment and to colonize new surfaces. The
cycle closes…
Architecture of biofilm
Candida albicans biofilm. Toluidin blue - mushroom-like structure of the biofilm
Alcian blue has coloured extracellular polysaccharides. Photo: Veronika Holá
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)
Main importance of biofilm
formation
Bacteria harbored inside are protected
against:
 antibiotic action
 host´s immune response
 disinfection
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)
+
-
Antibiotic susceptibility of
staphylococci isolates
Planktonic bacteria (MIC)
Biofilm-forming bacteria (MBIC)
log
100
10
1
ams
ch
ery
te
cli
tei
van
ofl
Antibiotics
Abbreviations: ams - ampicillin/sulbactam, ch - chloramphenicol, ery - erytromycin, te tetracyclin, cli - clindamycin, tei - teicoplanin, van - vancomycin, ofl - ofloxacin
The inefficiency of antibiotics
may be due to:
Polyanionic charge of sessile cells
Decreased bacterial growth
Diffusion barrier of glycocalyx
Reaction with biofilm matrix
Formation of protected phenotypes
Mechanism of intercellular signalling
Host´s immune response mechanisms…
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 nonadherent
Composition of plaque depends on its age and
localization, speed of plaque formation is individually
Contains many bacterial species
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 =
more G+ rods and filamentous microorganismlactobacili and actinomycets
1 week old Column microcolonies of microbes, on the plaque
plaque
3 weeks
surface adhesion of rods/filaments
Filamentous forms of microbes,
old plaque ear of corn-like on the surface: central fibre
(Eubacterium yurii) surrounded by G+ cocci
Plaque development
1.
2.
3.
4.
5.
On the teeth surface forms thin layer of saliva
glycoproteins = pelicula.
The salivas transport oral bacteria to this
surface
First adhere G+ cocci and rods due to surface
adhesins. To G+ bacteria adhere the other
Aggregated bacteria divides and form
microcolonies
Production of exopolysaccharides
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
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
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
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.
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)
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