Bio films / orthodontics courses

BIOFILMS
INDIAN DENTAL ACADEMY
Leader in continuing Dental Education
www.indiandentalacademy.com

• A Historical Basis
• Biofilm Definition
• Beneficial and detrimental attributes of Biofilms
• Attachment of the biofilm
• Biofilm Structure
• The Established Community: Biofilm Ecology
• How do Biofilms form?
• Oral Biofilms
• Communication in oral Biofilms
• Biofilm formation in root canal surfaces
• Biofilm in Dental unit lines
• Control of water line contamination
• Prospects for Prevention
• Concluding Remarks and Future Directions

• Biofilms may be found on essentially any
environmental surface in which sufficient moisture is
present.
• Biofilms are well-organized communities of
cooperating microorganisms that can include bacteria,
protozoa, diatoms, and fungi.
• These microorganisms are usually encased in an
extracellular polysaccharide that they themselves
synthesize.

• Van Leeuwenhoek, using his simple microscopes, first observed
microorganisms on tooth surfaces and can be credited with the
discovery of microbial biofilms (Emerg Infect Dis 8(9), 2002 )
• Zobell observed that the number of bacteria on surfaces were
higher than in the surrounding medium (Seawater).
• Jones et al used scanning and transmission electron microscopy
to examine biofilms on trickling filters in a wastewater treatment
plant and showed them to be composed of a variety of organisms
(based on cell morphology).
(Centers for Disease Control and Prevention (CDC) 2002)
Historical Basis

• By using a specific polysaccharide-stain called Ruthenium red
and coupling this with osmium tetroxide fixative, these
researchers were also able to show that the matrix material
surrounding and enclosing cells in these biofilms was
polysaccharide.
• In 1973, Characklis studied microbial slimes in industrial water
systems and showed that they were not only very tenacious but
also highly resistant to disinfectants such as chlorine
(Emerg Infect Dis 8(9), 2002)
• Costerton et al in 1978 put forth a theory of biofilms that
explained the mechanisms whereby microorganisms adhere to
living and nonliving materials and the benefits accrued by this
ecologic niche.
(Emerg Infect Dis 8(9), 2002)

Biofilm Definition
A biofilm is an assemblage of microbial
cells that is irreversibly associated (not
removed by gentle rinsing) with a
surface and enclosed in a matrix of
primarily polysaccharide material.
(can J Micro 1998;44:1019-28)

• Biofilms are heterogeneous, containing microcolonies
of bacterial cells encased in an EPS matrix and
separated from other microcolonies by interstitial
voids (water channels).
• Biofilm-associated organisms differ from their
planktonic (freely suspended) counterparts with
respect to the genes that are transcribed.

Biofilms may form
• On solid substrates in contact with moisture.
• On soft tissue surfaces in living organisms.
• At liquid air interfaces.
Typical locations for biofilm production include rock and
other substrate surfaces in marine or freshwater
environments.

• Biofilms are also commonly associated with
living organisms, both plant and animal
• Tissue surfaces such as teeth and intestinal
mucosa which are constantly bathed in a rich
aqueous medium rapidly develop a complex
aggregation of microorganisms enveloped in an
extracellular polysaccharide they themselves
produce.
(J Periodontol nov 2003)

Beneficial and detrimental attributes
of biofilms
• Water treatment plants,
• waste water treatment plants and
• septic systems associated with private homes
remove pathogens and reduce the amount of
organic matter in the water or waste water through
interaction with Biofilms.
• On the other hand Biofilms can be a serious threat to
health especially in patients in whom artificial
substrates have been introduced.

Biofilms may form on a wide variety of surfaces, including living
tissues, indwelling medical devices, industrial or potable water
system piping, or natural aquatic systems.
Scanning electron micrograph
of a native biofilm that developed
on a mild steel surface in an
8-week period in an industrial
water system
Scanning electron micrograph of
a staphylococcal biofilm on the
inner surface of an indwelling
medical device.

• patients with indwelling catheters for urine
excretion, for continuous ambulatory peritoneal
dialysis (CAPD) or for any other reason are subject to
frequent and persistent bouts of infection.
• These recurrent infections are due to the
accumulation of mixed Biofilms on the artificial
surfaces provided by the catheter or other implant.
• The Glycocalyx in which the bacteria live protects
them from the effects of antibiotics and accounts
for the persistence of the infection even in the face
of vigorous chemotherapy

In vitro experiments suggest that bacteria
encased in Biofilms may be 50 to 500 times
more resistant to chemotherapy than
planktonic bacteria of the same strain.
(Can J Microbiol 1998)

The solid-liquid interface between a surface and
an aqueous medium (e.g., water, blood)
provides an ideal environment for the
attachment and growth of microorganisms
A clear picture of attachment cannot be obtained
without considering the effects of the
• Substratum,
• Conditioning films forming on the substratum,
• Hydrodynamics of the aqueous medium,
• Characteristics of the medium, and
• Various properties of the cell surface.
Attachment (Braz Dent J vol14 june2003)

• Characklis et al noted that the extent of microbial
colonization appears to increase as the surface
roughness increases.
• The physicochemical properties of the surface may
also exert a strong influence on the rate and extent
of attachment.
• This is because shear forces are diminished, and
surface area is higher on rougher surfaces
Substratum Effects

• Most investigators have found that microorganisms
attach more rapidly to hydrophobic, non-polar
surfaces such as Teflon and other plastics than to
hydrophilic materials such as glass or metals.
• hydrophobic interaction apparently occurs between
the cell surface and the substratum that would
enable the cell to overcome the repulsive forces
active within a certain distance from the substratum
surface and irreversibly attach.

• A material surface exposed in an aqueous medium will
immediately become conditioned or coated by polymers
from that medium, and the resulting chemical modification
will affect the rate and extent of microbial attachment
• A prime example may be the proteinaceous conditioning
film called "acquired pellicle," which develops on tooth
enamel surfaces in the oral cavity.
• Pellicle comprises albumin, lysozyme, glycoproteins,
phosphoproteins, lipids, and gingival crevice fluid; bacteria
from the oral cavity colonize pellicle-conditioned surfaces
within hours of exposure to these surfaces.
Conditioning Films

• Mittelman noted that a number of host-produced
conditioning films such as blood, tears, urine, saliva,
intervascular fluid, and respiratory secretions
influence the attachment of bacteria to biomaterials
• Ofek and Doyle also noted that the surface energy of
the suspending medium may affect hydrodynamic
interactions of microbial cells with surfaces by
altering the substratum characteristics.

Hydrodynamics
• Fluid at the center of any lumen travels fastest; as it
moves outward from the center toward the tubing, its
rate of flow is incrementally slowed by friction.
• Water at the tubing walls is virtually stagnant,
allowing bacteria to adhere and colonize the internal
surfaces.

• Cells behave as particles in a liquid, and the rate of
settling and association with a submerged surface will
depend largely on the velocity characteristics of the
liquid.
• Higher linear velocities would therefore be expected
to equate to more rapid association with the surface,
at least until velocities become high enough to exert
substantial shear forces on the attaching cells,
resulting in detachment of these cells (Rijnaarts et al
and Zheng et al)

characteristics of the aqueous medium, such as
• pH,
• nutrient levels,
• ionic strength,
• temperature,
may play a role in the rate of microbial
attachment to a substratum.
• Cowan et al showed in a laboratory study that an
increase in nutrient concentration correlated with an
increase in the number of attached bacterial cells.
Characteristics of the Aqueous Medium

Properties of the Cell
• Cell surface hydrophobicity,
• presence of fimbriae and flagella, and
• production of EPS
all influence the rate and extent of attachment
of microbial cells
• The hydrophobicity of the cell surface is important in
adhesion.

• Fimbriae play a role in cell surface hydrophobicity and
attachment, probably by overcoming the initial
electrostatic repulsion barrier that exists between
the cell and substratum
• Rosenburg et al and Bullitt and Makowski provided
evidence for the role of fimbriae in bacterial
attachment to surfaces.

• An increase in flow velocity,
• water temperature, or
• nutrient concentration
may also equate to increased attachment.
• The attachment of microorganisms to surfaces is a
very complex process, with many variables affecting
the outcome.
• In general, attachment will occur most readily on
surfaces that are rougher, more hydrophobic, and
coated by surface "conditioning" films

• Biofilms are composed primarily of microbial cells and
EPS
• EPS may account for 50% to 90% of the total organic
carbon of biofilms and can be considered the primary
matrix material of the biofilm
• The exopolysaccharides (EPS) synthesized by
microbial cells vary greatly in their composition and
hence in their chemical and physical properties.
Biofilm Structure

Sutherland noted two important properties of EPS that
may have a marked effect on the biofilm…
First, the composition and structure of the polysaccharides
determine their primary conformation
Second, the EPS of biofilms is not generally uniform but
may vary spatially and temporally
(JOE 2005 0ct)

• These researchers' results showed that different
organisms produce differing amounts of EPS and that
the amount of EPS increases with age of the biofilm
• EPS production is known to be affected by nutrient
status of the growth medium; excess available carbon
and limitation of nitrogen, potassium, or phosphate
promote EPS synthesis
• EPS may associate with metal ions, divalent cations,
other macromolecules (such as proteins, DNA, lipids)

• Slow bacterial growth will also enhance EPS
production because EPS is highly hydrated, it
prevents desiccation in some natural biofilms
(J Microbiology 2001; 147,3-9)
• EPS may also contribute to the antimicrobial
resistance properties of biofilms by impeding the
mass transport of antibiotics through the biofilm,
probably by binding directly to these agents.

• It is clear from a number of studies that mutants
unable to synthesize the EPS are unable to form
biofilms, although they may still attach to surfaces
and form micro-colonies to a limited extent
(Allison & Sutherland, 1987; Watnick & Kolter, 1999),
• However, in a study of a natural biofilm isolate
attaching to glass, most of the EPS- mutant bacteria
were seen as well-separated cells; under calcium-
limiting conditions, where little EPS was synthesized,
the effect was very similar

structure and properties of the
biofilm polysaccharides?
• Many of these polysaccharides are relatively soluble,
and because of their large molecular mass, yield
highly viscous aqueous solutions
• A few will form weak gels, which dissolve in excess
solvent, thus sloughing off the exposed surface of
biofilms

• The EPS contribute directly to the properties of
biofilms in that they normally permit considerable
amounts of water to be bound.
(Applied & Environmental Microbiolo 2000 Aug)
• The EPS will also contribute to the mechanical
stability of the biofilms, enabling them to withstand
considerable shear forces. (Mayer et al., 1999),

• In some polymers, the interaction with ions may yield
relatively rigid gels which are less readily deformed
by shear, thus producing a much more stable biofilm
• Mayer et al. (1999) suggested that biofilms might
indeed represent gel-like structures, but these may
be very weak and consequently may be readily
destroyed by shear or dissolution of the
polysaccharides.

Do the biofilm polysaccharides offer any protection to
the cells within the biofilm?
• By maintaining a highly hydrated layer surrounding
the biofilm, the EPS will prevent lethal desiccation in
some natural biofilms and may thus protect against
diurnal variations in humidity
• Skillman et al. (1999) observed that biofilms
composed of mixed enteric species, hydrolysis of one
EPS caused greater destruction of the biofilm

• In oral biofilms, many of the component bacteria are
capable of synthesizing several different EPS,
including dextrans (D-glucans) and levans (ß-D-
fructans)
(Crit Rev Oral Biolo Med 2004)
• In addition, both dextranases and fructan hydrolases
may be secreted
• Little is known of the effects such polysaccharide
hydrolases have on oral biofilms, but recent studies
on regulation of expression of the fructan-degrading
enzyme in Streptococcus mutans may start to provide
an insight (Burne et al., 1999)

The Established Community: Biofilm
Ecology
• The basic structural unit of the biofilm is the
microcolony.
(JADA 2001)
• Proximity of cells within the microcolony (or between
microcolonies) provides an ideal environment for
creation of nutrient gradients, exchange of genes,
and quorum sensing

Since microcolonies may be composed of multiple species,
the cycling of various nutrients (e.g., nitrogen, sulfur, and
carbon) through redox reactions can readily occur in
aquatic and soil biofilms

Gene Transfer
• Biofilms also provide an ideal niche for the exchange
of extrachromosomal DNA (plasmids)
• Conjugation (the mechanism of plasmid transfer)
occurs at a greater rate between cells in biofilms
than between planktonic cells.
• Ghigo has suggested that medically relevant strains
of bacteria that contain conjugative plasmids more
readily develop biofilms

• The probable reason for enhanced conjugation is that
the biofilm environment provides minimal shear and
closer cell-to-cell contact
• Since plasmids may encode for resistance to multiple
antimicrobial agents, biofilm association also provides
a mechanism for selecting , and promoting the spread
of, bacterial resistance to antimicrobial agents.

Quorum Sensing
• Cell-to-cell signaling has recently been demonstrated
to play a role in cell attachment and detachment from
biofilms
• Xie et al showed that dental plaque bacteria can
modulate expression of the genes.

Dispersal
Because of flow effects…Biofilm cells may be dispersed
either by
• shedding of daughter cells from actively growing
cells,
• detachment as a result of nutrient levels or
• quorum sensing, or
• shearing of biofilm aggregates (continuous removal of
small portions of the biofilm)

• Gilbert et al showed that surface hydrophobicity
characteristics of newly divided daughter cells
spontaneously dispersed from either E. coli or P.
aeruginosa biofilms differ substantially from those of
either chemostat-intact biofilms or resuspended
biofilm cells
• The mechanisms underlying the process of shedding
by actively growing cells in a biofilm are not well
understood.

Brading et al have emphasized the importance of
physical forces in detachment, stating that the three
main processes for detachment are
(JADA 1996)
• erosion or shearing (continuous removal of small
portions of the biofilm),
• sloughing (rapid and massive removal), and
• abrasion (detachment due to collision of particles
from the bulk fluid with the biofilm)

• The mode of dispersal apparently affects the
phenotypic characteristics of the organisms
• Eroded or sloughed aggregates from the biofilm are
likely to retain certain biofilm characteristics, such
as antimicrobial resistance properties, whereas cells
that have been shed as a result of growth may revert
quickly to the planktonic phenotype.

Variables important in cell
attachment and biofilm formation
Properties of
the substratum
Properties of
the bulk fluid
Properties of
the cell
–Texture or
roughness
–Hydrophobicity
-Conditioning
film
–Flow velocity
–Ph
–Temperature
–Cations Presence
•Cell surface
hydrophobicity
•Fimbriae
•Flagella
•Extracellular
polymeric substances
of antimicrobial
agents

The formation of this biofilm is far from a random process.
To the contrary, the formation of a biofilm follows a course
the nature of which can be predicted and recorded.

• These initial attractions may be considered weak and
reversible.
• After their initial adherence on a conditioned surface, the
microorganisms enter a quiet phase, termed the surface-
associated lag time, during which they may be changing the
expression of their genes.
• Once they make the phenotypic shift and divide, the
microorganisms enter a rapid growth phase and begin to
secrete complex exopolysaccharides, a mucilaginous slime
that cements the organisms to the surface and resists
detachment by fluid shear forces.
• The exopolysaccharides form a coating on the bacteria and a
fibrous matrix.

• other microorganisms may be trapped in the tangled
matrix or adhere by the molecular interactions
• The growth of microcolonies within the matrix and the
coaggregation of other bacteria increase the depth of
the biofilm; however, it might not exceed 1,000 μm in
thickness in a turbulent flow setting.
• The adherence of bacteria increases their density
compared to their former free-floating planktonic state,
and the signals they express may become concentrated
enough to serve as autoinducer signal molecules.
• Thereby the concentration may exceed a threshold, and
the bacteria sense they have a “quorum.”

• The signals are thought to allow cross-talk between
species, causing them to increase their production of
exopolysaccharide and the factors that increase
their virulence.

• Microorganisms on the surface are not as strongly
embedded as those deep within the biofilm and are faster
growing.
• Surface bacteria are susceptible to detachment by
predator protozoans, abrasion or shear of fluids or
particles in the stream, and periodically slough individuals
or clumps.
• The detachment may serve the community of
microorganisms by seeding downstream surfaces with the
start of a new colony, much like the swarming of bees.

In the mouth, Biofilms naturally form on the surfaces of
(JADA 1997)
• Teeth
• Dental prostheses
• Implants
• Oral epithelium.
These Biofilms can be benign or pathogenic, releasing harmful
products and toxins from harboring pathogenic microbes.

ORAL BIOFILMS
• Oral diseases, such as dental caries and periodontal
disease, should be considered as consequences of
ecologically driven imbalances of oral microbial
biofilms.
(Crit Rev Oral Biolo Med 2004; 15(1):4-12)
• Dental plaque is an example of biofilm; it has a
diverse microbial composition
• Both diseases are caused by micro-organisms
belonging to the resident oral micro flora rather than
by classic microbial pathogens

• A change in a key environmental factor can alter the
competitiveness of individual species. This can result
in the enrichment of a previously minor component of
the community or a loss of a dominant organism. This
relationship may be fundamental to explaining how
plaque-related dental diseases arise
• Dental plaque, the presence of various specific
microorganisms in the plaque (including mutans
streptococci) and the sugar consumption will attack
the tooth and cause cavities.
• For example, mutans streptococci would be favoured
by the low pH conditions from eating sugary food.

• Teeth are normally negatively charged and plaque is
positively charged. Opposite charges attract and
bond to each other
• Plaque, therefore, is attached to the tooth surface
by ionic bonding. (J Dent Res 1961;40:739-740).
• An average daily brushing of approximately 2 minutes
duration will remove only half the plaque, leaving the
other half to promote rapid re-growth. (J Periodontol
1979;50:661-664).

• Thus, most individuals harbor the micro-organisms
involved in these diseases
• In the case of dental caries, a low pH environment
caused by microbial fermentation of carbohydrates
selects a population of acid-tolerant and acid-
producing strains like mutans streptococci and
lactobacilli.
• This in turn increases acid formation that may cause
demineralization. Mixed anaerobic micro-organisms
are involved in periodontal disease, which develops
when the plaque community equilibrium is altered and
inflammation is induced
• The environment is altered by an increased flow of
gingival crevicular fluid, increased nutrients, and pH
rise that favors growth of periodontal pathogens
which may contribute to periodontal destruction

• Control of oral biofilms is fundamental to the
maintenance of oral health and to the prevention of
dental caries, gingivitis, and periodontitis.
• One probable explanation for this low efficacy is the
fact that the micro-organisms involved organize into
complex biofilm communities with features that
differ from those of planktonic cells, whereas micro-
organisms have traditionally been studied in a
planktonic state
• Apart from chlorhexidine and fluorides, only a few of
the existing oral prophylactic agents have significant
effects (Petersen and Scheie, 1998; Wu and Savitt,
2002; Scheie, 2003)
• However, oral biofilms are not easily controlled by
mechanical means and represent difficult targets for
chemical control (Socransky, 2002)

COMMUNICATION IN ORAL BIOFILMS
• Biofilms are likely to represent a natural scenario for
bacterial communication (Davey and O’Toole, 2000;
Kolenbrander et al., 2002).
• The ability to communicate through quorum-sensing
has been shown in some oral streptococci and some
periodontal pathogens
• For most oral biofilm micro-organisms, however, the
presence and function of signal transduction
pathways and quorum-sensing communication remain
to be clarified. Evidence for the involvement of two-
component signal transduction systems in oral biofilm
formation was first found in Streptococcus gordonii
(Loo et al., 2000)

DENTAL PLAQUE AS A BIOFILM
• Dental plaque is a structurally-functionally organized
biofilm.
• Dental plaque is the community of organisms found on
the tooth surface as a biofilm, embedded in a matrix
of polymers of host and bacterial origin.

Development of dental plaque biofilm
• Dental plaque forms via an ordered sequence of
events, resulting in a structurally-functionally –
oraganized, species rich microbial community.
• Stages include……
• Acquried pellicle formation
• Reversible adhesion involving weak long range physio
chemical interactions between the cell surface and
pellicle
• Co-adhesion resulting in attachment of secondary
colonizers to already attached cells
• Multiplication and biofilm formation.
• detachment

BIOFILM FORMATION ON ROOT CANAL WALL
• When bacteria grow as biofilm, the altered genetic
and metabolic processes of the bacteria along with its
complex matrix, prevent the entry and action of the
antimicrobial agents.
• Subsequently the colonizing organism gains protection
against unfavorable, environmental and nutritional
conditions.
• there is constant detachment of cells from a fully
matured biofilm and the detached cells serve as a
steady source for chronic infection.

• The environmental niche of root canal after
chemomechanical is manifested by reduced oxygen
tension, limited nutrient availability and the presence
of antimicrobials that act as driving forces in the
selection of microbes in the root canal system
(Triple O 1998;85:86-93)
• The inherent antimicrobial resistance and the ability
to adapt to changing environment help E.faecalis to
persist in harsh environmental conditions existing in
the endodontically treated tooth.
(Triple O 2001;91:579-86)
• E. faecalis is the most common and occasionally
isolated bacteria from root canals of the teeth with
persistent periapical periodontitis
(JOE 2003)

• Moreover, the harsh environmental conditions
existing in the root canal may favour the growth of
bacteria as a biofilm
• This aspect is supported by the fact that clinically
isolated E.faecalis posses increased adhering
capacity, increased virulence factors and increased
resistance to antimicrobials that are all
characteristics of biofilm style of growth.
(J Bio Med Research 2006)

• Tronstad et al demonstrated that bacteria might live
and maintain endodontic infections within periapical
lesions .
(Endo Dent Traumatolo 1987; 3:86-
93)
• Clinical examination by Noiri was done to examine the
surface of extracted root tips & GP Points removed
during the surgical or endodontic procedure for the
presence of biofilm formation in periapical lesions.
(JOE 2002;oct 28(10): 679)

SEM image of extruded gutta-percha
Bacterial cells aggregate without
a covering of Glycocalyx like
structure
Others areas are covered with glycoclyx
Like structure.
Glycocalyx area
Filamentous or spirochete shaped
bacteria.

SEM image of an overfilled area from a gutta percha
Short rods located on the
glycocalyx structure
Small colony of cocci observed at the
cracking the biofilm structure

SEM image of the periapical
area of the extracted tooth
Filaments, rods and fusiform bacteria
form a bacterial biofilm at internal
wall of periapical foramen

Filaments and rods predominantly
colonize on the periapical root
surface area
Mature Glycocalyx like structure
and filaments form the biofilm

Filaments, rods and fusiform bacteria
form a bacterial biofilm at the internal
wall of the periapical foramen
Microcolony seen next to the
periodontal ligament

• Dahle et al reported that spirochetes isolated from
root canals were 140 microns long and 2 microns thick
(Oral Microbio & Immunol 1993;8:251)
• Nair et al demonstrated the predominance of
filaments and low frequency of cocci and rods in the
apical root canals affected with periapical
periodontitis. (JOE 1990;16:580-8)

• Dabelion et al investigated bacteremia in conjuction
with endodontic therapy. They reported that
bacterial species were recovered from the blood
stream and suggested that those bacteria invaded
the blood via the periapical foramen of the root
canals. (Endo Dent Trauma 1995;11:142-9)
• The monotypes of bacteria isolated from blood were
predominently rods and cocci……
• Propionibacterium acne
• Prevotella intermedius
• Streptococcus sanguis
• Streptococcus intermedius

• Filamentous and spirochetes – formed biofilms at the
periradicular area.
• Hence in case of periapical peridontitis, bacteria in
the infected root canal might invade extraradicular
sites and form a biofilm on the periapical root
surface within the lesion.

• When bacteria grow as biofilm, the altered genetic and
metabolic processes of bacteria along with its complex
matrix, prevent the entry and action of antimicrobial
agents.
(Anti Microbial Agents Chemotherp 1996;40:2571-22)
• Subsequently, colonizing organisms gain protection
against unfavorable environmental and nutritional
conditions
(J Antimicrob Chemotherap 2001;48:141-2)
There is constant detachment of the cells from a fully
matured biofilm, and the detached cells serve as a
steady source of infection.
(Trends Microbiolo 2001;9:50-2)

• Antibiotic therapy generally ameliorates the acute
clinical symptoms, which lead to planktonic cells
being released from the biofilm, but cannot kill the
bacteria completely within the biofilm.
• Disengagement of planktonic bacterial cells from
the biofilm is a natural pattern of genetic program
• Dental caries and marginal periodontitis are caused
by plaque biofilm and biofilms are involved in
osteomyeliytis, cystic fibrosis.

• Biofilms offer their member cells several benefits,
the foremost of which is protection from killing by
antimicrobial agents
Four mechanisms that confer antimicrobial tolerance to
cells living in a biofilm have been elicited (JOE 2002)
• The first is the barrier properties of the EPS matrix.
Extracellular enzymes such as ß lactamase may
become trapped and concentrated in the matrix,
thereby inactivating ß lactam antibiotics
• The second mechanism involves the physiological
state of biofilm microorganisms. Bacterial cells
residing within a biofilm grow more slowly than
planktonic cells, as a result, biofilm cells take up
antimicrobial agents more slowly. Further more, the
depletion of nutrients can force bacteria into
dormant or stationary growth phase in which they are

• The third suggested mechanism responsible for
antimicrobial tolerance is that microorganisms within
the biofilm experience metabolic heterogeneity.
• Studies have shown that oxygen can be completely
depleted by cells at the biofilm surface leaving
anaerobic niches deeper in the community.
• Some antibiotics like aminoglycosides are more
effective against bacteria growing in aerobic
conditions than the same organism growing in anerobic
conditions; therefore not all cells within the biofilm
will be effected in the same way.

Study by George was done to examine the
(JOE 2005;31:867)
• Ultrastructure of biofilm formed on root canal
• To examine the penetration of dentinal tubules by
E.faecalis, both under nutritional and environmental
conditions.

Bacterial biofilm when grown in nutrient rich medium
Bacterial biofilm when grown in nutirent deprived area

Interaction between E.faecalis biofilm and root canal
dentin substrate – was studied by Kishen
(J BioMed Research 2006;
77A;406)
• By examining the shift in chemical composition of
biofilm structure with time.
• By studying the topography and ultrastructure of the
biofilm and dentin substrate.
This study showed
• E.faecalis formed biofilm on root canal dentin
• Bacteria induced dissolution of the mineral fraction
from the dentin substrate
• A reprecipitated apatite layer was formed in the

• As E.faecalis present in the root canals, various root
canal irrigants have been tested against E.faecalis
biofilm (JOE 2006;32:527)
• It was found that 1% and 6% NaOCl were effective in
eliminating E.faecalis biofilm than 2% CHX,
REDTA,MTAD tested.
• As concentration of NaOCl increased, the time taken
to reduce the CFU to zero reduced.

Bacteria in biofilms also respond differently depending
on their
• growth phase
• The dose
• Frequency of exposure to antimicrobial agents
In a study by Gulabivala,
• NaOCl, iodine and CHX were effective against
P.micros and P. intermedia
• Iodine was efective against S.intermedius
• NaOCl was effective against E.faecalis
(IEJ 2001;34:300-7)

• E. faecalis seems to be highly resistant to the
medications used during treatment and is one of the
few organisms that has been shown to resist the
antibacterial effect of calcium hydroxide.
• E. faecalis is not indigenous to oral cavity, indicating
that it is an exogenous infection that can enter the
root canal, survive the intracanal medication
treatment and persist after obturating.
(Triple O 1998;85:86-93)

E.faecalis colonizing root canals medicated with calcium hydroxide

• If E.faecalis can form biofilms in root canals, this
might explain its ability to persist in that
environment.
• Compared with planktonic bacterial cells, biofilm
bacteria are up to 1000 fold more resistant to
phagocytosis, antibiotics and antibodies.
(J Bacteriol 1994; 176:2137-42)

Factors contributing to the resistance include:
• Impenetrable polysaccharide coating on the biofilm bacteria
• Ability of the biofilm bacteria to survive without dividing.
In addition, the physical conditions available to support the
growth of bacteria,
• pH
• Ionic concentration
• Nutrient availability
• Oxygen supply
(JOE2002;28:690)

• The proximity of the individual bacteria in biofilm also
increases the opportunity for gene transfer, making it
possible to convert a previously avirulent organism into a
highly virulent pathogen or a bacterium that is succeptible
to antimicrobics into a resistant one.
• This potential for gene transfer within biofilms is
particularly significant in case of E faecalis, because a
number of E faecalis virulence factors are encoded on
transmissible plasmids.
These include
• Collagenase
• Gelatinase
• Adhesins
All with the potential to contribute to survival in and
colonization of the root canal.

In recent years concerns, have been raised about
microbial growth in slow moving waterlines such as
those in dental offices.
• The quality of dental-unit water is of considerable
importance since patients and dental staff are
regularly exposed to water and aerosols generated
from the dental-units..

Their presence in dental-unit waterlines has been known
since 1963
The discovery that biofilms contribute to the
microbial contamination of dental unit waterlines has
made the need for cleansing systems apparent, to
minimize the potential danger of infection and cross
contamination

• In dental-unit waterlines, Biofilms have been
measured to be 30-to-50 micrometers thick.
• Layers upon layers of organisms form structures,
including nutrient channels, utilizing polysaccharide
adherence and matrix compounds,
• Various potential pathogens, both environmental and
human-derived, have consistently been cultured from
dental units worldwide .

• The American Dental Association’s goal is to reduce
microbial counts to below 200 colony-forming units
(CFU) per ml in the unfiltered output from dental-
water supply lines.
• Average living microbial counts in water from
handpieces and air-water syringes are in the range of
300,000 to 400,000 CFU per ml and can easily contain
millions of CFUs per ml of water
( J Periodontol 2003 nov)

Microbial populations of the biofilms found in dental-unit
waterlines include the most common opportunistic pathogens
linked to hospital related waterborne infections; e.g.,
• Pseudomonas,
• Legionella, and
• non-tuberculous Mycobacterium
Predominant early colonizers include
• Pseudomonas spp.,
• Pasteurella
• Moraxella,
• Ochrobactrum,
• Aeromonas spp.,
• Flavobacterium,
• Acinetobacter spp.

Oral flora, most likely deriving from “suck-back” events,
are also commonly reported; e.g.,
• Lactobacillus,
• Streptococcus,
• staphylococcus
• Bacteroides,
• Actinomyces
• Veillonella,
• Candida .

MICROBIAL SPECIES ISOLATED FROM DUWL (JOE 2001)

Detachment of surface microorganisms from the
biofilms in DUWL allows them to exit in the
• coolant of high-speed dental handpieces,
• in the flow of air-water syringes (AWS),
• from ancillary equipment such as ultrasonic scalers
attached to the dental units.
These bacteria can then be flushed into the mouths of
dental patients and become airborne as aerosols and
droplets of splatter.
(Scien Issu Impact Dent
1999)

Portal of entry of organisms:
• The inhalaion of contaminated aerosols
• direct exposure of open wounds.
• swallowing the water
• The use of instruments such as ultrasonic scaler,
which potentially could force the organisms into
gingiva, may raise the possibility of introducing the
organisms into the blood stream.

• An aerosol cloud of particulate matter and fluid often
is clearly visible during dental procedures.
• This cloud is evident during tooth preparation with a
rotary instrument or air abrasion, during the use of
an air-water syringe, during the use of an ultrasonic
scaler and during air polishing.
• This ubiquitous aerosolized cloud is a combination of
materials originating from the treatment site and
from the dental unit waterlines, or DUWLs.
• Many dental procedures produce aerosols and
droplets that are contaminated with bacteria and
blood

• Dental handpieces, ultrasonic scalers, air polishers
and air abrasion units produce the most visible
aerosols
• Each of these instruments removes material from the
operative site that becomes aerosolized by the action
of the rotary instrument, ultrasonic vibrations or the
combined action of water sprays and compressed air.
(JADA 2001)

Several features of dental-unit waterlines are
responsible for biofilm formation, (JADA 1997)
• surface area,
• surface chemistry,
• flow rates.
• Most plastic dental tubing has an inside diameter of
1/16-to-1/8 inch, and thus has a very large surface
area to volume ratio.
• At peak usage, the flow rate in a dental handpiece can
be between 2-to-10 ml per minute
• The hydrophobic surface of waterline plastics
promotes the attachment and colonization of biofilm
organisms.

• The exopolysaccharides are mostly insoluble in water.
• Deep inside the accumulated biofilm, nutrients are
transferred from one species to another, but the
inward diffusion of oxygen and absorbed nutrients
decreases.
• Studies with microelectrodes showed that oxygen
penetrated no deeper than 25 or 30 μm. As a result,
bacterial growth becomes very slow or almost static.

• The matrix resists the physical displacement of
biofilm bacteria, and it limits the inward diffusion of
adverse agents by consuming them through chemical
reactions.
• The overall result is that microorganisms in a biofilm
are many times more resistant to disinfection than in
their planktonic phase.

• The water in the dental lines is also completely
stagnant on weekends and evenings.
• The layered structure of biofilms (limiting diffusion)
combined with the low flow conditions renders these
microbial colonies intrinsically resistant to many
biocides and cleansing schemes.

• Active biofilms then become the primary reservoir for
continued contamination of the system.
• Improving the microbial quality of dental-unit water,
as means become available, is a natural part of
maintaining a high quality of patient care and staff
protection.
• It takes less than five days before initial microbial
counts reach a plateau of 200,000 CFU per ml in newly
installed waterlines.

• The biofilm may start as a surface patch, but as it
spreads and develops, it may take on the form of
pillars or mushroom-like shapes, forming channels
that improve the circulation of nutrients and the
discharge of wastes and toxins.
• In a severely starved biofilm, stacks of colonies may
extend up into the fluid bulk like dendrites; in a richly
fed biofilm such as dental plaque, the colonies form a
thick, dense film.

There is no doubt that exposure of the dental staff and
patients to high levels of microbial contamination leads
to a high risk of infection.
Infection control is of major importance in routine daily
procedures of a dental office.
According to Ito et al., a series of actions,
• anti-sepsis,
• laying of barriers,
• use of conservatives,
• disinfections,
• disposal and
• sterilization

• When the dental units are not in use–between
patients, at night, over weekends–the planktonic
bacteria entering from the city water distribution
system and those shed from the biofilm surfaces
accumulate in large numbers.
• Counts as high as 1,000,000 colony forming units
(CFU)/ml have been recorded.
(Braz Dent J 2003 June)
CONTROL OF WATERLINE
CONTAMINATION

• autoclaving of handpieces,
• handpiece replacement between patients,
• flushing of the unit prior to use,
• anti-contamination' devices to prevent retrograde
aspiration of oral secretions into the water supply
line,
• connection to a separate water supply (eg connection
to bottles of distilled water),
• chemical disinfection of waterlines,
• ultra-violet radiation disinfection and
• the use of in-line water filters.
These have been developed and implemented in many
dental practices

Schemes to reduce microbial counts in dental treatment
water fall into four broad categories
(i) use of water systems that are independent of public
systems, including those designed to deliver sterile
water (INDEPENDENT WATER SYSTEMS)
(ii) chemical treatments that are provided either
continuously or intermittently (CHEMICAL
TREATMENT PROTOCOLS)
(iii) filters placed inline just before the point of use
(i.e. handpiece, three-way syringe, ultrasonic scaler)
(POINT-OF-USE FILTERS)
(iv) devices to create turbulent and/or high energy flow
conditions to cleanse fine tubing (STERILE WATER

Independent water delivery system
• These systems bypass the municipal water supply,
instead drawing fluid from the reserviors holding a
selected treatment water or treatment solution.
• These systems provide a means of administering
chemical solution regimens for reduction of microbial
contamination.

• Such devices alone cannot improve the quality of
treatment water
• Regular chemical or mechanical treatment is required
to reduce adherent microbial biofilm.
• Proper maintenance of these systems, as outlined by
the manufacturer is required to reduce the adherent
biofilm.

Chemical treatment protocols
Solutions for control of dental unit contamination
• Sodium hypochlorite
• Chlorine,
• Chloramines,
• Chlorine dioxide
• Hydrogen peroxide
• Chlorhexidine gluconate
• Ethanol
• Povidine iodine

Use of solutions for disinfection
Intermittently used
(SHOCK TREATMENT)
Continuously used
•Deliver the agent for a specific
contact time and frequency
using an independent reservior
•Active agent is purged from the
system before patient treatment.
Disadv:
•Potential for surviving biofilm organisms
to rebound between treatments
•Potential staff exposure to chemicals
•Adverse effect on metals, rubber &
synthetic dental unit components
•Uses lower concentration of solutions
•This regimen also employ initial shock
treatment to inactivate biofilms.
•Offers less potential for
recolonization of waterlines
Disadv:
•May damage the equipment
•Chronic exposure to chemicals
through aerosols.
•Enamel & dentin bonding strength
of dental adhesive materials may
be affected.

• Sodium hypochlorite solutions (NaOCl), or diluted
bleach, have effectively reduced planktonic counts,
but biofilms are 150 to 3,000 times more resistant to
hypochlorite.
• Using a combination of periodic shock and continuous
treatment in dental units with independent
reservoirs, Karpay et al. reduced the heterotroph
plate counts (HPC) in 10 dental units to less than 10
CFU/ml 5 days after the shock treatment.
(JADA 1999)

• Fiehn and Henriksen reported that the use of 1 to 2
ppm NaOCl in intermittent doses reduced the HPC in
ultrasonic scalers to 100 to 6,500 CFU/ml, while
continuous treatment resulted in 270 to 610 CFU/ml.
• Removal of tubings from dental units and treatment
in the laboratory with 5.25% bleach for 15 hours
eliminated counts in the tubing effluent, but they
tended to recur by 15 days.
(JADA 1995)

• In a clinic water distribution system, use of a
chlorinator to provide a shock treatment with 50 ppm
overnight and then to dispense 1 ppm for 4 weeks
failed to eliminate counts or resident Legionella.
(JADA 1997, oct)
• The use of 0.5% to 1% bleach once a week for 10
minutes over a 4-year period kept microbial
contamination in check but caused a slow corrosion of
metal fittings in the dental units. (JADA 1999)

• Treating DUWL for 10 minutes with 1:6 bleach
solution and then flushing it out eliminated bacteria in
the effluent, but there were compliance problems in
private practice settings.
• In vitro tests on biofilms of waterline bacteria
showed that the matrix reduced the penetration of
chlorine over time
• problem with chlorine or its hypochlorite solution is
that it reacts with the organic matrix to create
chlorinated by-products. The chemical reactions in
the surface layers of the biofilm would prevent its
penetration.

• The manufacturer recommended that the waterlines
of the self-contained water system be purged Tightly
with air and disinfected weekly with 1:10 solution of
household bleach for at least 10 but never more than
30 minutes.
• Clinical reports indicated that after 4 or 5 weeks of
treatment with alkaline peroxide, all units had counts
under 200 CFU/ml, and the biofilm on tubing samples
examined by SEM was absent or spotty (JADA 1999)

•Alkaline peroxide treatments might be beneficial, as
hydrogen peroxide has been recommended, and
chelation of cations by EDTA is a possible treatment
plan

• When chlorine reacts with water it forms
hypochlorous acid.
• The hypochlorous acid can then undergo acid-base
reactions to form hypochlorite ion
• The distribution of chlorine into HOCl and OCl- is pH
dependent.
Chlorine

• HOCl is a stronger disinfectant than OCl-, and
therefore a lower pH is preferred for disinfection
with chlorine.
• The chlorine (HOCl or OCl-) attacks bacterial cells
and the protein coat of viruses, effectively killing
both bacteria and viruses.
• Chlorination, while highly effective at inactivating
pathogens, produces several potentially harmful by-
products.

Chloramine Disinfection
• Chloramines are an alternative disinfectant to
chlorine.
• Chloramination does not cause the taste and odor
problems often experienced when disinfecting with
chlorine.
• The main disadvantage to chloramination is that it
requires a very large CT (concentration * time) value
to provide effective disinfection.

• Chloramination involves the addition of chlorine and
ammonia to the water source.
• When chlorine reacts with ammonia, monochloramine
(NH2Cl), dichloramine (NHCl2) or trichloramine
(NCl3) are formed.
• Monochloramine is the best chemical for disinfecting
water because unpleasant taste and odors can arise
when dichloramines or trichloramines are formed.

Chlorine Dioxide Disinfection
• The main disadvantages of using chlorine dioxide as a
water disinfectant compared to chlorine are higher
operating costs, health risks caused by residual
oxidants and the creation of harmful by-products.
• disadvantage of chlorine dioxide is that it is a very
unstable chemical and it rapidly dissociates into
chlorite and chlorate.
• High concentrations of chlorite and chlorate can
cause an increase in methemoglobanemia

Commercially available disinfectants
• Bio 2000 – 12% ethanol & flavoring agent
• Glycerin based lubricant – 0.12% chlorhexidine
gluconate
• Sterilex ultra – hydrogen peroxide based
• Bioclear – citric acid based
• Dentacid- iodine based

Disinfection By-products
• Disinfection by-products (DBPs) are defined as the
class of chemicals that are formed when
disinfectants react with the organic compounds in
water. (JADA 1995 nov)
• Some of these compounds are carcinogens and some
are suspected of causing acute health effects.

• DBPs are chemical compounds produced as an
undesirable result of water disinfection and
oxidation.
• The chemical compounds of most serious concern
contain chlorine and bromine atoms.
• These compounds have been shown to be carcinogenic,
mutagenic or hepatotoxic, and have caused negative,
reproductive or developmental effects in animal
studies.

• Trihalomethanes are formed when chlorine has a
chemical reaction with the organic material that is
already present in the water supply. (JADA 2001)
Trihalomethanes (THMs) include
• chloroform (CHCl3),
• dibromochloromethane (CHBr2Cl),
• bromodichloromethane (CHBrCl2),
• bromoform (CHBr3).
• Chloroform is the THM most commonly found in drinking
water and is usually present in the highest
concentration (Vogt and Regli, 1981).

• Haloacetic acids (HAAs) are disinfection by-products
which were first detected in chlorinated drinking
waters by Christman et al. (1983)
• HAAs are the second most common group of DBPs and
are very soluble in water.
• When using a chlorine disinfectant, dichloroacetic and
trichloroacetic acids are the most common HAAs.

Filters
• To stop the effluent of waterline bacteria, filters
near the end of a waterline have been recommended,
but they often foul rapidly and may have to be
changed daily or more often.
• Downstream from the filter, there is almost certain
to be more biofilm.
• Filters trap the planktonic bacteria only; they do
nothing to remove the biofilm.

Point-of-use filters can be quite effective and simple
to install. However,
• filters do not address the central issue of a large
retained biomass within the dental unit
• many filters will not remove bacterial endotoxins,
• filter systems can be high-maintenance in terms of
both time and cost .

• The dentapure point of use filters employs an
iodinated resin in combination with a 0.22um point of
use filters.
• The release of small amounts (5ppm) of iodine
retards the growth of biofilm formation in the post
filtration tubing system

• Clear line one day filter (disposable) 0.22um
• Clear line plus disposable filter built in with
retraction valve
• Filters offer a built in anti retraction valve, iodine
eluting resin to inhibit down stream biofilm formation
and filter materials designed to remove bacterial
endotoxin.
• To be effective, filters must be placed on each water
bearing line as close as possible to the handpiece or
the air water syringe.

• Dental water filtration system, which consists of a
single reusable filter housing with two different
0.2um filter elements.
• The standard filter removes bacteria and has a one
day use life.
• The high performance filter element also removes
endotoxin and has an optimal performance duration of
one patient.

Drying
• Since biofilms are usually thin (200 or 300 μm) and
are mostly water (95%), drying the DUWL at night
and on weekends by purging the waterlines with
compressed air would seem to be rational.
• Furuhashi and Miyamae reported that air drying
resulted in no CFU/ml when used in combination with
flushing and 70% ethanol. (JADA 1995)
• In other tests, however, drying alone seemed to
offer no benefit.
• The exopolysaccharide matrix and the static growth
conditions probably protect the bacteria from
dessication.

• The mean endotoxin unit (EU) level in samples of
DUWL effluent has been measured at 80.7 EU/ml,
which is considered enough to cause a fever in a
normal, healthy patient
• In samples from 11 dental units with extensive biofilm
contamination and high viable counts, the endotoxin
level was 2,560 EU/ml before flushing (JADA 1999)
• Filters would not stop the flow of endotoxin
• Using sterile saline in a self-contained dental unit
water system would not prevent the flow of
heterotrophs and endotoxin from biofilms located
downstream from the reservoir bottle.

• Biomaterials pose a risk for the creation of biofilms
and subsequent chronic infections.
• Sutures and orthopedic implants may acquire the
seeds of a biofilm at the time of surgery,
• Direct examination of necrotic bone removed from
two patients with osteomyelitis after treatment for
leg fractures revealed biofilms consisting of Gram-
positive and Gram-negative bacteria within a
ruthenium red-positive glycocalyx on the bony
surface; Bacteroides melaninogenicus, Clostridium
clostridiforme, Corynebacterium, Enterococcus,
Fusobacterium, Proteus mirabilis, and Streptotoccus
morbillorium were isolated from cultures of the bone
samples. (Br.D J 2002)

• The bottom line for clinicians is that they cannot risk
letting DUWL contamination cause “refractory
periodontitis” in their patients, or possibly the
removal of a failing dental implant that has been
carefully and skillfully placed
• Clinicians need a sure remedy for control of biofilms
in dental unit waterlines.

• The chlorine dioxide products were very effective in
lowering the HPC
• both the shock treatment and daily care for the
freshly mixed chlorine dioxide took longer than
treatment with the buffer stabilized chlorine dioxide

• Commercial water-testing laboratories can enumerate
microbial counts and water-testing kits are available
for in office use
• Expert consensus appears to indicate that there is
little need to evaluate water quality prior to
implementing a treatment program or is there a need
to identify specific organisms.
• Information regarding specific organisms is highly
useful in only a few circumstances, such as where a
waterline is refractory towards treatment or a
waterborne illness is suspected

Flushing
• Flushing for 2 minutes in the morning and for 20 to
30 seconds between patients should be considered
the norm for dental office procedures, and longer
flushing is suggested after weekends.
• Flushing the waterlines remove the bulk of amassed
bacteria.

• both the American Dental Association and the
Centers for Disease Control and Prevention endorse
flushing water lines for several minutes prior to the
first patient visit and for 20 to 30 seconds between
patients, this infection control effect is transient.
• Flushing between patients will most likely reduce
levels of oral flora, which do not typically colonize
upstream tubing, but most species rapidly return to
pre-flush levels as pieces of biofilm are dislodged.
• Additionally, at room temperature, the concentration
of aqueous bacteria can double every twenty minutes.

Sterile water delivery system
• Separate sterile water delivery systems that bypass
the dental unit also available.
• These systems offer the option of providing sterile
irrigating solution through disposable or autoclavable
tubing, address biofilm formation by completely
preventing it.
• Apart from manual syringes, they are the only
devices that can deliver sterile solutions to the
patient.

Other approaches
• Ozone disinfction
• ultraviolet disinfection
• Electrolyzed Functional Water Filters
• Atmospheric Glow Technologies, Inc. (AGT) is
developing atmospheric plasma

Ozone Disinfection
• Ozone is created when oxygen (O2) is separated by
an energy source into oxygen atoms
• The oxygen atoms collide with each other to form a
more stable configuration (O2), which later forms
ozone (O3) gas.
• Ozone is a very strong purifier when used for primary
disinfection in water and wastewater treatment
plants.
• Because ozone gas does not have a stable chemical
residual, it is not used as a secondary disinfectant

• Ozone is more effective at inactivating organisms
than chlorine.
• The other advantages to using ozone treatment
include taste and odor control, oxidation of organic
substances in water, and the destabilization of
particles.

Ultraviolet Disinfection
• Ultraviolet disinfection is the transmission of
electromagnetic energy from a mercury arc lamp.
• As UV radiation enters the cell wall of microorganism,
the UV light damages the deoxyribonucleic acid
(DNA) or ribonucleic acid (RNA), thus preventing the
organism from reproducing.
• Pathogens are successfully killed at wavelengths
ranging from 245 to 285 nm.
• ultraviolet light was unpredictable and difficult to
control, so chlorine became the disinfectant of
choice.

• Very small concentrations of DBPs are formed when
UV disinfection is used.
• Halopropanones and chloral hydrates are some other
DBPs that are formed from disinfection with ozone.
• All of these DBPs are toxic

electrolyzed water generating system
• electrolyzed water generating system is based on a 3
compartment cell system, that produces high-purity
electrolyzed water, which offers sterilization and
cleaning applications, rapidly killing many types of
bacteria, fungi and viruses from the surface of many
kinds of material.
• Its extremely low chlorine gas causes no corrosion
and thus makes it possible to joint-assemble with
other devices.
• Unlike chemical reagents, it uses only water, and
achieves both sterilization and cleaning.

• System can generate water that is harmless to the
human body and kill most microorganisms within 30
seconds, e.g., mycobacterium, MRSA, pseudomonas,
salmonella, shigella, candida, bacillus, and virus.
• mechanism of action is intra-cellular; as it destroys
bacteria, fungi and viruses without destroying human
cells.
• Solution is non-toxic; therefore, is safe to the
target materials, staff, and the environment

Atmospheric Glow Technologies, Inc. (AGT)
is developing One Atmosphere Uniform Glow
Discharge Plasma (OAUGDP)
• an alternative method for the destruction of biofilms
on surfaces and within tubing
• the plasma treatment has been shown not only to
reduce the viability of biofilms, but to remove them
from surfaces as well
• feasible alternative for inactivating resistant biofilm
on surfaces and in water lines.
(106th General Meeting of the American Society
for Microbiology 2006 Aug in Orlando, Florida)

Prospects for Prevention
• Presently available microbial and human genomic
sequence data allow for the prediction of potential
targets that are unique to the micro-organisms
• For therapeutic purposes, it is necessary to attack
the established biofilm.
• Therefore, genes essential for viability represent the
traditional targets for anti-microbial drug design.
• Potential agents include, among others, microbial
fatty acid biosynthesis inhibitors, bacteriophages,
and anti-microbial peptides (Hancock, 1999; Payne et
al., 2001; Sulakvelidze and Morris, 2001

• For prophylactic purposes, it seems reasonable to
target processes involved in the actual biofilm
formation of single- or mixed-bacterial communities
that have the potential to cause or favor disease,
without perturbing the balance of the normal flora.
• In this respect, two-component systems and quorum-
sensing seem to represent promising future targets
• The facts that two-component systems are present in
most micro-organisms and that signal transduction in
mammalian cells is mediated through different
mechanisms provide the rationale for using signal
transduction systems as targets for prophylactic
purposes.

• It is likely that the mature oral biofilm is the result
of a well-regulated series of processes that could
represent potential targets for biofilm control

Surface modification
• The strategy of surface modification involves altering
the tooth surface or the salivary pellicle to impede
bacterial colonization
• Thus, the composition of the pellicle may modulate
bacterial adhesion events
• It is well-known that the salivary pellicle provides
binding sites for the oral bacteria through a complex
array of specific and non-specific binding mechanisms

• It has been shown in vitro that the combination of an
alkylphosphate and a non-ionic surfactant alters the
surface characteristics of the tooth, making it less
attractive for micro-organisms
• Unfortunately, the clinical efficacy of such coating
agents has been low, probably because of difficulties
in securing persistent coating with the active
component (Olsson, 1998)
• If these problems are resolved, a future approach to
reduce colonization could be to coat the tooth
surface with agents that interfere with two-
component signal transduction in oral micro-
organisms.

• One approach is to change the surface
characteristics by manipulating the protein film on
the enamel, thereby reducing bacterial adhesion
• Functional groups like phosphate and phosphonate may
be used to anchor water-soluble, protein-repelling
substances to the mineral surface (Olsson, 1998).

Replacement therapy
• Replacement therapy has been suggested as a
strategy to replace potential pathogenic micro-
organisms with genetically modified organisms that
are less virulent
• The requirements for this type of therapy are, that
there should be a definite pathogen to replace
The replacement organism
• must not cause disease itself,
• it must colonize persistently,
• it must replace the pathogen effectively, and
• it must possess a high degree of genetic stability

• DNA technology has made it possible to produce
potential candidates for replacement therapy in
caries prevention.
• These strains hydrolyze urea to ammonia, thereby
counteracting acidification of the environment
• Among those is a super-colonizing strain of S. mutans.
This strain produces mutacin, which enables it to
replace wild-type strains efficiently

• However, one cannot exclude the possibility that a
genetically modified replacement strain might later
undergo transformation in oral biofilms and then
become an opportunistic pathogenic strain.
• A possible future approach is to use genetically
modified micro-organisms to deliver tailored
molecules that could interfere with adaptive
pathways such as two-component signal transduction
or quorum-sensing systems

Immunization
• Immunization against oral diseases —particularly
dental caries, but also periodontal disease—has been
a central research topic in recent decades (Koga et
al., 2002; Smith, 2002).
• Several molecules involved in the various stages of
both dental caries and periodontal disease
pathogenesis would be susceptible to immune
intervention and could function as vaccine targets
• The aim is to inhibit adhesion or reduce the virulence
of putative microbial etiologic agents

• Efforts have been made to immunize both actively
and passively
• In passive immunization, the antibody itself is
administered.
• In active immunization, an antigen which will elicit a
protective immune response is administered.
• Micro-organisms could, for instance, be cleared from
the oral cavity by antibodies prior to colonization,
antibodies could block adhesins or receptors involved
in adhesion, or modify metabolically important
functions or virulence factors

• A major problem is that immunization approaches are
generally directed against single bacterial species
epitopes, whereas both dental caries and periodontal
disease are ecologically driven multi-microbial
diseases (Marsh, 1994).
• Furthermore, since micro-organisms have the ability
to form biofilms and to adapt and undergo
transformation that may lead to altered antigenicity,
it is questionable whether immunization will provide
lasting protection.

Concluding Remarks and Future Directions
Developing oral prophylactic strategies through interference with
two-component systems or quorum-sensing of biofilm micro-
organisms represents an interesting future challenge.
• While the stages of biofilm formation seem to follow basically
the same model in various micro-organisms, the biofilm
architecture and molecular mechanisms involved in biofilm
formation appear to differ
• . Information on the genetic regulation of oral biofilm
formation, however, is still lacking.
• A better understanding of these processes is necessary to the
development of novel strategies for oral disease prevention and
control based on interference of two-component signal
transduction systems or quorum-sensing.

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