Biofilm

Dr. Priyam Mishra

P.G first year
S.B.D.C.H
 Introduction

• Biofilms are collection of microorganisms

surrounded by the slime they secrete, attached to
either living or inert surface.

• E.g. : the plaque on your teeth, slippery slime on river

stones, fish tanks, contact lenses etc.

• Reason for existence is that it allows micro organisms

to stick to & multiply on surfaces.
 Definition
• Biofilms are defined as ‘ matrix-enclosed
bacterial populations adherent to each other
&/or to surfaces or interfaces’.
(Costerton J W, 1994)
• However, the concept of biofilm was first
described in 1978. (Costerton JW, scientific
american 1978;238(1):86-95)
 Properties of biofilm
• Open architecture eg presence of channels & pores.

• Protection from host defences eg. Production of extracellular

polymers & physical protection from phagocytosis.

• Enhanced tolerance to antimicrobials-

-retarded or incomplete penetration
-microbes are physiologically altered due to differential
gene expression. (Davies 2003)

• Neutralization of inhibitors eg beta lactamase production.

• Novel gene expression eg. Synthesis of novel proteins upon

attachment of plaque.
• Co-ordinated gene responses.

• Cell- cell signaling by Competence Stimulating Peptide, AI-2

• Spatial & environmental heterogeneity.

• Broader habitat range comprising of obligate anaerobes in an

aerobic environment.

• Enhanced virulence eg pathologic synergism observed in

abscesses & periodontal diseases.

• More efficient metabolism eg in plaque, brought by complete

catabolism of complex host macromolecules like mucin.
 Nature of biofilm

• Biofilms are fascinating structures.

• Preferred method of growth for many and perhaps

most species of bacteria. (Cardiff Bioline 1999).

• Major advantage: protection to colonizing species

from competing micro-organisms from environmental
factors such as host-defence mechanism.

 
• Biofilms can facilitate :

- processing and uptake of nutrients

- cross feeding: one specie providing nutrients for

other.

- removal of potentially harmful metabolic products

• - the development of an appropriate physiochemical

environment (such as a properly reduced oxidation
reduction potential). (Periodontology 2000, vol 28, 2002)
•  
Functions of biofilm
 Structure of biofilm

• Biofilms are composed of micro colonies of bacterial

cells(15-20% by volume) that are non-randomly
distributed in a shaped matrix or glycocalyx(75-80%
by volume).

• The presence of micro colonies between water

channels permit the passage of nutrients and other
agents throughout the biofilm acting as a primitive
“circulatory” system.
• Presence of water channels :

-provides nutrients to colonizing organism

-removal of waste products
-transport of cells to new colonizing sites
-increased rate of genetic transfer among
bacteria.
• Exopolysaccharides : backbone of biofilm, composed
predominantly of water & aqueous solutes.

• Dry material is a mixture of exopolysaccharides,

proteins, salts & cell material. (Cardiff, Bioline 1999)

• Play a major role in maintaining the integrity of biofilm.

• Some are neutral, eg mutans from St. Mutans whereas

others are highly charged polyanionic macromolecules.

• Can be degraded & utilized by bacteria within biofilm.

 Exopolysaccharide
• Maintains the structural integrity

• Effective adhesive

• Protects the microbial cells by preventing

• dessication & attack by harmful agents.

• Creates a local nutritionally enriched environment

favouring specific micro-organisms.

• Acts as a buffer & retains extracellular enzymes

enhancing substrate utilization by bacterial cells.
 Growth of biofilm
• Biofilm develops in stacked layers of different bacterial
species.

• Early colonizers are Actinomyces & Streptococcus

species & they release small amounts of signaling
molecules, in the form of peptides.

• These indicate the other free floating bacteria that

conditions are now favourable.

• Late colonizers are periodontal pathogens.

• As the biofilm grows, fluid channels develop.

• Bacteria aggregated on biofilm produce

hundreds of proteins & some of them trigger
genetic activity such as bacterial aggregation,
matrix formation & adhesion to other biofilm
bacteria.
 Plaque as a biofilm
(caries research, P.D Marsh 2004)

• CFM has confirmed plaque as a open structure similar

to other biofilms with channels & voids.

• Gradients develop in areas of dense biomass over short

distances in key parameters that influence microbial
growth & development.

• Bacteria exhibit an altered pattern of gene expression

either as a direct result of being on surface or indirectly
in response to local heterogenicity within biofilms.
• Bacteria communicate via small diffusible
signaling molecules increasing rates of gene
transfer & AMA resistance.

• Oral bacteria in plaque does not exist as an

independent entity but function as a
coordinated & fully metabolically integrated
community.

• Hence plaque is an example of both a biofilm

& microbial community. (Marsh & Bradshaw
1999, Marsh & Bowden 2001)
 Biofilm formation
• It’s a step wise process.

1. Adhesion of planktonic micro-organisms to a surface.

2. Colonization & co-adhesion (binding of planktonic organisms

to sessile, already adhering cells)….Ciardi et al 1987

3. Growth & maturation

4. Detachment of some organisms.

• Detachment is the least understood biofilm phenomenon

(Querynan et al)
WHAT KEEPS BIOFILM TOGETHER ?
 Quorum sensing

• Quorum sensing in bacteria “involves the regulation

of expression of specific genes through the
accumulation of signaling compounds that mediate
intercellular communication”. (Prosser JI,1999)

• The possible role of quorum sensing in influencing

the properties of biofilms was first suggested by
Cooper et al in 1995.
 The stimuli for quorum sensing systems are signaling
molecules, called autoinducers.

 Gram positive – oligopeptide signals.

(Dunny & Leonard,1997)

 Gram negative – acyl homoserine

lactones(AHLs).(Whitehead et al.2001)

• Quorum sensing allows intra species communication

purpose.

.
• Gives biofilm distinct properties, eg,
expression of genes for antibiotic resistance at
high cell densities may provide protection.

• Can influence community structure by

encouraging growth of beneficial species &
discouraging the growth of competitors.

• Can alter physiological properties.

• Autoinduction leads to increased
concentration as cell density increases.

• Once the signaling compounds reach a

threshold level, gene expression is activated.
(Davies et al,1998;Stoodley el al,2002)

• Conjugation, transformation, Plasmid transfer

have all shown to occur in naturally occurring
or mixed species biofilms prepared in vitro.
• A second communication system
:Autoinducer system 2(AI-2)allows for
interspecies communication. (Bassler,1999)

• AI-2 signal molecule is a furanosyl borate di-

ester, its synthesis depends on the luxS gene
product . (Winans & Bassler, 2002)

• AI-2 is produced by P. gingivalis, P. intermedia, &

F. nucleatum. ( Frias et al, 2001)
 Horizontal gene transfer
• Horizontal gene transfer is more feasible in multiple
species biofilm & transfer of resistance genes from
commensol to pathogenic sp is well documented.
(Molin & Tolker,Neilson 2003)

• The presence of pathogenicity islands in P.gingivalis is

also indirect evidence of gene transfer, which explains
the evolution of more virulent strains.(Chen et al,2004)

• These findings suggest that plaque can function as a

genotypic reservoir by harboring transferable mobile
elements & genes. (Loo,2003)
 Attachment of bacteria
• Adhesion is the first step in development of biofilm.

• The current view of the adhesion process is that it

takes place in two distinct phases.

• In Phase 1 the microbe is held, for a brief period, by

a weakly attractive force some 10nm from the
surface .

• Shear force or Brownian Motion may disrupt this

initial attraction.
• A number of specific adhesion mechanisms may hold
the cell close to the surface for a significant time
period. These specific interactions may be a
combination of lectin-like , electrostatic and
hydrophobic interactions with fimbriae or fibrils.

• Fimbriae – are proteinaceous hair like appendages, 2-8

nm in diameter, composed of protein subunits called
fimbrillins. eg Porphyromonas gingivalis ,
Actinomycetemcomitans.

• Fibrils – found in mitis group. Morphologically shorter

than fimbriae. May be dense or sparsely distributed on
the cell surface .
• In phase 2- The biofilm is held together and
protected by a matrix of excreted polymeric
compounds called EPS.

• This matrix protects the cells within it and

facilitates communication among them
through biochemical signals.
 Coaggregation of bacteria

• Cell- cell adherence is known as co-aggregation

that is ability of two genetically distinct
bacteria to recognize & adhere to one another.

• Based on specific interaction of a adhesin

produced by one bacteria & a carbohydrate or
protein receptor found on the surface of
another bacterium.
• Streptococcus species unlike other oral
species show a high degree of co-aggregation
hence dominant as early colonizers.

• Secondary colonizers include co-aggregation

of F. nucleatum with St. sanguis.
• Fusobacterium nucleatum, however, is unusual and is
intentionally placed at the border between early and late
colonizers for the following reasons.

- the most numerous gram-negative species in healthy

sites, and its numbers increase markedly in periodontally
diseased sites.

- co aggregates with all of the early colonizers and the

late colonizers.

- F. nucleatum acts as a bridge between early and late

colonizers, which may partially explain why fusobacteria are
so numerous in samples from both healthy and diseased
sites.
• Bridging is also a property of co-aggregating
cells.

• Refers to 2 non co-aggregating strains, may

participate together in a multigeneric
aggregate if they recognize a common partner
by distinct mechanisms. Eg- P. loescheii with A.
israeli & S. oralis. (Weiss et al 1988)
 Microbial complexes
• Association of bacteria in the biofilm is not
random.

• Presence of specific microbial groups within

dental plaque (Socransky et al 1999)
 E
Blue complex Various actinomyces species A
R
L
Y
Purple complex Actinomyces odontolyticus C
O
L
Eikenella corrodens,Capnocytophaga O
Green complex gingivalis N
Actinobacillus actinomycetemcomitans I
serotype(A) Z
Streptococcus mitis,streptococcus E
Yellow complex R
oralis,sp.sanquis,sp intermedius
LATE COLONIZER

Orange Camphylobacter,Fusibacterium
nucleatum,Provetella intermedia
complex

Porphyromonas gingivalis.Treponema
Red complex denticola
Tanerella Forsythia
• Red complex is of particular interest because
its associated with bleeding on probing which
is the important clinical parameter of
destructive periodontal diseases.

• The existence of complexes of species in

plaque shows the bacterial independency in
the biofilm environment.
 Physical heterogeneity within
biofilms
• Cells of the same species can exhibit extremely
different physiological states in a biofilm even though
separated by as little as 10µm.

• pH can vary over short distances in a biofilm.

• The number of metal ions differ, so that a difference in

ion concentration can produce measurable potential
differences.
• Indicates sessile cells growing in mixed biofilms
can exist in an almost infinite range of chemical
& physical micro habitats within microbial
communities. ( Costerton JW,1994)

• Environmental heterogeneity that develops in

biofilms can accelerate phenotypic & genotypic
diversity in bacterial populations & might be a
mechanism whereby cells are better prepared
to cope with adverse conditions; form of
BIOLOGICAL INSURANCE. (Boles et al,2004)
 Factors affecting biofilm formation
• 1.Substratum – rough is better than smooth surface
- as increase surface area so increased colonization
- also provides protection from shear forces &
- increases difficulty of cleaning

• 2.Hydrophobic is better than hydrophilic- detachment of cells

from biofilm & establishment in new sites .

• 3.Conditioning film - provides several receptors for the

binding of oral bacteria such as acidic proline-rich proteins
that bind Streptococcus gordonii and A. Naeslundii.
( Busscher HJ et al,1999)
• 4.Flow velocity versus shear forces- High flow will not
prevent bacteria attachment nor completely remove
existing biofilm, but it will limit biofilm thickness.

- Biofilms grown under laminar flow conditions

(low shear) developed as patchy microcolonies and
consisted of roughly circular cell clusters separated by
interstitial voids.

- turbulent flow conditions : patchy but

consisted of patches of ripples and elongated
streamers that oscillated in the flow
• 5. Chemical composition - also impacts on bacterial
colonization since it may contain beneficial or
detrimental components.

• For example, metals such as brass (an alloy of copper

and zinc) have antimicrobial properties due to
dezincification and the antimicrobial properties of
copper.

• Polyvinyl chloride contains carbon, hydrogen &

chloride which may encourage bacterial growth.
 Factors affecting structure
• Hydrodynamics- affects the shear stress & the rate at
which nutrients are transported to the surface of biofilm
(Vieira MJ, 1999)

• Adding nutrients- increases mass & structure

( Stoodley et al)
• Variation with dentition- faster formation in lower arch, in
molar areas, on buccal surfaces & in interdental regions.
( JP 44:396, 1973)

• Gingival inflammation- increased GCF enhances plaque

formation. ( JCP 2002:29;524-530)
 Methods to study biofilm
• Confocal Scanning Laser Microscopy(CSLM) - Biofilm structure
is studied without chemical fixation or embedding techniques.
(Lawrence JR et al.,1991)
• Combined with reporter gene technology identifies genes that are
expressed only within a biofilm.

• Miniature micro-electrodes that permit measurement of local

environment.

• Programmed microscope permit accurate images of biofilms in 3-D

• These approaches have shown that biofilms are
usually highly structured with channels traversing the
depth of the biofilm, from outside to enamel surface.

( Wood, Auschill 2001)

• Novel microscopic techniques combined with the use

of a pH sensitive fluoroscent dye demonstrated
considerable pH heterogeniety over very short
distances. (Vroom et al)
 Mechanism of increased antibiotic
resistance
• Almost without exception, organisms in a biofilm are
1000-1500 times more resistant to antibiotics than
their planktonic cells. This is called Biofilm inhibitory
conc/ biofilm eradicating conc/ biofilm killing conc.
(Anwar & Costerton1990, Johnson .et .al 2002 &
Shoni et al 2000)

• The mechanism of this increased resistance differs

from species to species, from antibiotic to antibiotic
& in biofilms growing in different habitats.
• Resistance to penetration of AMA:

• Strongly charged or highly reactive agents bind to

opposite charged polymers in matrix & get neutralised
• Extracellular enzymes inactivate some antibiotics,
exception being macrolides

• Slower rate of growth of bacterial species

• Drug target modified or unexposed

• Phenotypic variation in bacterial cells

• Novel gene exprssion
• Indirect pathogenicity (Walker & winkelhoff)
• Super resistant bacteria

• Presence of persister organisims (Keren et

al,2004)

• Homeostatic function of biofilm matrix

 Antiplaque biocides & resistance
(Srinivasan P, Gaffar A-JCP-2002)

• Antiplaque biocides such as triclosan, essential oils,

& CHX contain surfactants, ant nucleating agents &
polymers that are effective in penetrating biofilms.

• The results of various studies on real life use of oral

formulations with antiplaque biocides shows no
emergence of resistant microflora nor alterations in
the oral microbiota, while such formulations have
been found to provide the benefits of reducing
plaque & gingivitis.
 COMMONLY USED BIOCIDES
BIOCIDE MODE OF ACTION USE
Chlorhexidine Loss of structural Mouth rinse. Oral
organisation. coagulation spray
of cytoplasm Dentrifce
disinfectant

Triclosan Membrane damage. Dentrifice

release of cellular Liquid soap
components

Cetyl pyridium Membrane damage Mouth rinse

chloride
Phenolic flavours Protein denaturation Mouth rinse

Detergents Initiation of autolysis Foaming agent

Povidone iodine Binding of thiol comp Oral irrigant

 Biofilm & therapy

• For therapeutic purposes,it is necessary to

attack the established biofilm.

• For prophylactic purposes,it seems reasonable

to target processes involved in biofilm
formation. (Honcock 1999, Payne et al 2001)
• Therapies directed towards control of biofilm are grouped into :

o Those that physically remove biofilm & its subsequent

microorganisms
- Mechanical Debridement

o Those that attempt to kill or affect the metabolism of

organisms
- AMA & Antiplaque Biocide

o Those that affect the environment of organisms

- Surface modifying agents, replacement therapy & methods
interfering with signaling pathways

o Immunization
• Mechanisms contribute to colonization resistance
includes more effective competition for nutrients &
attachment sites, the production of inhibitory factors, &
creation of unfavourable growth conditions for invading
specie by normal microflora.

• It is important that resident microflora are maintained as

the reduction of colonization resistance will result in
overgrowth by previously minor components of
microflora, establishment of exogenous sp, which can
lead to physiological changes or enrichment of resistant
organisms. (Lacey,1983 Woodman 1985)

• Hence the purpose should be plaque control & not

elimination
 Surface modification

• Functional groups like phosphate may be used to

anchor water-soluble, protein-repelling
substances to the mineral surface.

• It has been shown in vitro that the combination

of alkyl phosphate & a non ionic surfactant alters
the surface characteristics of tooth, making it less
attractive for microorganisms. (Olsson,1998)
 Replacement therapy
• Replace pathogenic microorganisms with
genetically modified organisms that are less
virulent.

• However modified replacement strain might

later undergo transformation in oral biofilms
& then become a oppurtunistic pathogenic
strain
 Immunization

• The main aim is to inhibit adhesion or

virulence of putative microbial etiological
agents.

• There is data to show that passive

immunisation in humans impedes
recolonisation of selective target micro-
organisms in both caries & periodontal
diseases. (Booth et al 1996)
 Other methods to control biofilm
• CONTROL OF NUTRIENTS
-Addition of base generating nutrients
-Reduction in GCF flow through anti-inflammatory agent
-Inhibition of key microbial enzymes.
• CONTROL OF BIOFILM pH
-Sugar substitutes
-Antimicrobial agents
-Fluroide
-Stimulate base production
• CONTROL OF REDOX POTENTIAL
-Redox agents( reduction and oxidation potential)
-Oxygenating agents
 Conclusion

• The oral cavity harbors a diverse, abundant &

complex microbial community. Bacteria
accumulate on both hard & soft tissues in a sessile
biofilm. Under certain circumstances, however,
the oral microbiota can be directly or indirectly
responsible for diseases. An understanding of the
basis of microbial colonization thus provides
insight into both oral ecology & the underlying
pathogenic mechanisms of oral bacteria
 Refrences
• Dental plmaque:biological significance of a biofilm &
community life style.
JCP 2005 ; 32 :7-15.
• Dental biofilms: difficult therapeutic targets
Periodontology 2000;vol 28:12-55.
• Antiplaque biocides & bacteial resistance:A review.
JCP 2002; 29:965-974
• Microbial complexes in subgingival plaque.
JCP 1998;25 : 134-144
• Biofilm & plaque control- H I Busscher.
• Jan Lindhe
• Biofilm concept: consequences for future prophylaxis
of oral diseases.
Crit Rev Oral Biol Med 15(1); 4-12
• Introduction to microbial aspects of biofilm,
communities, development & treatment.
Periodontology 2000, vol 42, 2006
• Effect of 2 antimicrobial agents on early in situ biofilm
formation. JCP 2005;32: 147-152
• Antimicrobial therapy in periodontitis: the use of
systemic antimicrobials against subgingival biofilm.
JCP 2008 (suppl 8)
• Bacterial interactions & successions during plaque
development. Periodontology 2000 vol 42, 2006, 47-
49
Thank you