Beginning of Microbes

Bacteria first appeared

on earth about 3.6
billion years ago, long
before the appearance
of Homo sapiens around
100,000 years ago..
Van Leeuwenhoek
was the first person to
visualize, graphically
illustrate, and label
"animalcules" (bacteria)
that he found in plaque
scraped from his own
teeth.

The term biofilm was introduced to designate the

thinlayered condensations of microbes
(e.g.bacteria, fungi, protozoa) that may occur on
various surface structures in nature. Free-oating
bacteria existing in an aqueous environment,socalledplanktonicmicroorganisms,are a
prerequisite for biofilm formation. Such films
may thus become established on any organic or
inorganic surface substrate where planktonic
microorganisms prevail in a water-based
solution.

Despite the discovery of microbial biofilms as far back as

the 17th
century, scientists have largely focused their attentions on
the solitary,or planktonic, forms of microorganisms.
In nature, however, most microorganisms live together in
large
communities attached to a surface
Here, bacteria free in saliva (planktonic organisms) serve as the
primary source for the organization of this specific biofilm (1

 Biofilms

were already discovered by one of the

first microbiologist, the Dutch Antonie van
Leeuwenhoek in 1650s, but the actual
breakthrough of the phenomenon had to wait
another 328 years until Costerton and
colleagues published their work on How
Bacteria Stick in 1978. Since 1978 the
research within biofilm bacteria has exploded.
The inherent high tolerance to antimicrobials
and immune cells explain its vast implication in
chronic infections.

 Bacteria

are found in at least two distinct states

either as planktonic or sessile cells. Planktonic
cells are classically defined as free owing
bacteria in suspension as opposed to the sessile
state (the so called biofilm): a structured
community of bacterial cells enclosed in a selfproduced polymeric matrix and adherent to an
inert or living surface as stated by Costerton and
coworkers (1999). In the above section sessile
growing bacteria were defined as an assemblage
of cells embedded in a self-produced polymeric
matrix

Definition of a Biofilm
Biofilm

can be defined as a sessile

multicellular microbial community
characterised by cells that are firmly
attached to a surface and enmeshed
in a self produced matrix of
extracellular polymeric substance
usually polyssacharide

Definition of a Biofilm

Biofilms are communities of

microorganisms in a matrix that joins them
together and to living or inert substrates
Biofilms are surface-attached communities
of bacteria, encased in an extracellular
matrix of secreted proteins,
carbohydrates, and/or DNA, that assume
phenotypes distinct from those of
planktonic cells

 Biofilm

are not merely passive

assemblage of bacterial cells that are
stuck to surface they are structurally
and dynamically organized complex
biologic system.

Biofilm formation

The first step is the attachment of the

bacterial cells to the selected abiotic or
biotic surface

Initial attachment is mediated through

weak reversible van der Waals interactions
between the cell surface and the
substratum, which can lead to stronger
adhesion receptor mediated attachment

 Bacterial

cell surface structures such as

agella, fimbriae, LPS, and
exopolysaccharides participate in
irreversible interactions

The

second step corresponds to the

development of micro-colonies
promoted by the growth and division of
the first attached cells (primary
colonizers)

 The

micro-colonies progressively
enlarge and coalesce to form the first
layer of cells covering the surface

When

multiple layers of cells pile up

on the surface, the third step of the
formation is obtained

indicated by the presence of a mature

biofilm characterized by the presence of
macro-colonies surrounded by water
channels that help distribute nutrients
and signaling molecules

Finally, to survive when nutrients

become limited, or simply to spread and
colonize to other niches, some biofilm
cells can detach individually or in clumps

composition
15%

bacterial microcolonie
85% extracellular polysaccharide
matrix
Both of these are separated by water
channels

Biofilm Structure

biofilms display a common attribute, the

biofilm matrix.

Contrary to free-oating planktonic cells,

biofilm cells are embedded in a self produced
extracellular matrix, the extracellular
polymeric substance (EPS), that holds them
together

Biofilms are composed of about 8085% EPS

(by volume) and only 1520% cells (by volume)

 Although

the EPS may vary in chemical

and physical properties, its major
components are polysaccharides,
proteins, and extracellular DNA

The

EPS plays a major role in

maintaining the integrity of the biofilm
and can confer other beneficial
properties.

 Since

the EPS is also highly hydrated, it

can prevent desiccation in some natural
biofilms

The

EPS can also act as a diffusion

barrier, preventing toxic substances
such as antibiotics and disinfectants
from reaching their target

Cell heterogeneity within

biofilms
Most

biofilms found in nature are

polymicrobials, where diverse
species expressing multiple
phenotypes are involved

The

most amazing fact is that even

in a mono-species biofilm,
phenotypic heterogeneity exists

 Cell

differentiation in biofilms may

depend on the local environmental
conditions surrounding the cells

Different

concentration gradients of
oxygen, nutrients, ions, and chemicals
create a wide variety of microhabitats
providing conditions suitable for
bacterial colonization

Cells located in the upper biofilm layers

consume all available oxygen and grow
aerobically, while an anaerobic micro-niche
developed underneath the aerobic layer

Oxygen- and nutrient depleted regions are

found at the bottom layers of the biofilm
structure and under these circumstances,
most of the sessile cells are metabolically
inactive or dead

Communication language in
biofilm
A

few years ago, Watnick & Kolter

proposed the interesting idea that
biofilms can be compared to cities

In

these cities of microbes,

microorganisms are considered
social organisms able to
communicate with one another

 Using

different chemical languages,

bacteria learn about their current cell
population and determine when they
have reached a critical mass

Using

that information, bacteria can

thus modify their behavior to carry out
processes that would require many cells
acting in conjunction to be effective

 Cell-to-cell

communication is generally
carried out by diffusible signal
molecules produced and released by
bacteria

When

bacteria are growing within a

biofilm, they secrete signaling
molecules (auto-inducers) that increase
in concentration as a function of
bacterial cell density

 In

a process called quorum sensing,

bacteria communicate with one
another by using auto-inducers to
regulate their gene expression in
response to uctuations in the cell
population density

Two

types of quorum-sensing systems

are recognized in bacteria:

 intra-species

communication and
inter-species communication

During

intra-species communication,
several auto-inducers have been
identified

Gram-negative bacteria usually use

acyl homoserine lactone (AHL) as
signal molecules, while Gram-positive
bacteria utilize small peptides

During inter-species communication,

bacteria use autoinducer-2 (AI-2)

Cells located in the upper biofilm layers

consume all available oxygen and grow
aerobically, while an anaerobic micro-niche
developed underneath the aerobic layer

Oxygen- and nutrient depleted regions are

found at the bottom layers of the biofilm
structure and under these circumstances,
most of the sessile cells are metabolically
inactive or dead

 In

some cases, quorum sensing does

not seem to be involved in biofilm
structural development

while

for other species, there is

evidence that quorum sensing is
important for the attachment of
bacteria to the surface, the maturation
of the biofilm, or the control of events
leading to the dispersion of cells

Multi-drug tolerance of
biofilms
A

significant characteristic of microbial

biofilms is their high-level drug
tolerance

Bacterial

biofilms have been shown to

have a 100- to 1,000- fold increased
tolerance toward antibiotics in
comparison to their free-swimming
counterparts

Diffusion barrier imparted by the EPS matrix

Phenotypic heterogeneity

Gene Transferring

Secretion of antibiotic degrading enzymes

Protective role of EPS matrix

A

primary function that has been

attributed to the biofilm matrix is
protection,

Several

studies have shown that the

EPS matrix can act as an impermeable
barrier to limit antimicrobial
penetration, thereby protecting the
biofilm cells

Such protection can be due either to

physical hindrance in antimicrobial
diffusion or to direct binding of the
antibiotics by the EPS matrix

Upon antibiotic treatment, cells at the top

of the liquid biofilm interface die due to
their closer exposure, while bacteria
embedded deep inside the biofilm are able
to survive

 Anionic

EPS matrix can bind and

sequester toxic cationic heavy
metals, cationic antimicrobial
peptides, and positively-charged
antibiotics (e.g. aminoglycosides)

Phenotypic heterogeneity

Phenotypic heterogeneity occurs within

biofilms notably due to different
concentration gradients

Cells localized at the bottom layers of the

biofilm are normally found in a growth
stage analogous to stationary-phase
planktonic cells, while the physiology of
cells localized at the top surface layers is
similar to exponentially growing planktonic
cells

Microbial cells, especially those in the

deeper layers of the biofilms where
nutrients and oxygen are limited, are
associated with a lower growth rate

This reduced metabolic activity might

account for the enhanced tolerance
toward antibiotics that target bacterial
cellular processes such as DNA
replication or translation

 Conventional

antibiotics used to treat

infections are mostly effective at killing
rapidly growing cells

The

decreased metabolic activity of

cells found within the deeper biofilm
layers may thus contribute to antibiotic
tolerance and the persistence of
biofilm infections

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

Secretion of antibiotic
degrading enzymes

Bio film constitutes

A

biofilm is an aggregate of
microorganisms in which cells are stuck
to each other and/or to a surface. These
adherent cells are frequently embedded
within a self-produced matrix of
extracellular polymeric substance (EPS).
Biofilm EPS, which is also referred to as
"slime," is a polymeric jumble of DNA,
proteins and polysaccharides.

Biofilm is a complex
substance.

A biofilm is a complex
aggregation of
microorganisms growing
on a solid substrate.
Biofilms are
characterized by
structural heterogeneity,
genetic diversity,
complex community
interactions, and an
extracellular matrix of
polymeric substances.

Biofilms found in Nature

everywhere where is there is
moisture

More

properly known as biofilm,

slime cities thrive wherever there is
water - in the kitchen, on contact
lenses, in the gut linings of animals.
When the urban sprawl is extensive,
bio films can be seen with the naked
eye, coating the inside of water pipes
or dangling slippery and green from
plumbing." (Coghlan 1996)

Biofilm supports the

Bacterial growth

Biofilm are a common mode of bacterial

growth in nature and their presence has an
enormous impact on many aspects of our
lives, such as sewage treatment, corrosion of
materials, food contamination during
processing, pipe collapse, plantmicroorganisms interaction in the biosphere,
the formation of dental plaque, the
development of chronic infections in live
tissue (mastitis, Otitis, pneumonia, urinary
infections, osteomyelitis) or problems related
to medical implants.

Formation of Biofilms
Biofilms

may form
on living or nonliving surfaces,
and represent a
prevalent mode of
microbial life in
natural, industrial
and hospital
settings

Biofilms increases Antibiotic

resitance
With
microorganisms
are highly
resistant to
antimicrobial
treatment and are
tenaciously bound
to the surface

Mechanisims of Biofilm
formation

Formation of a biofilm
begins with the
attachment of freefloating microorganisms
to a surface. These first
colonists adhere to the
surface initially through
weak, reversible van der
Waals forces. If the

colonists are not

immediately
separated from the
surface, they can
anchor themselves
more permanently
using cell adhesion
structures such as pili

Factors Inuencing Rate and

Extent of Biofilm Formation

Indwelling medical device when contaminated

with microorganisms, several variables
determine whether a biofilm develops. First
the microorganisms must adhere to the
exposed surfaces of the device long enough to
become irreversibly attached. The rate of cell
attachment depends on the number and types
of cells in the liquid to which the device is
exposed, the ow rate of liquid through the
device, and the physicochemical
characteristics of the surface

Technology understands
Biofilms better

Technological progress in
microscopy, molecular
genetics and genome
analysis has significantly
advanced our
understanding of the
structural and molecular
aspects of biofilms,
especially of extensively
studied model
organisms such as
Pseudomonas
aeruginosa.

Steps in Biofilm
Development

Biofilm development can

be divided into several
key steps including
attachment, micro
colony formation, biofilm
maturation and
dispersion; and in each
step bacteria may recruit
different components
and molecules including
agella, type IV pili, DNA
and exo polysaccharides.

Stages of biofilm
development.

Steps in Biofilm
formation

Bacteria associated with

Biofilms differ
Bacteria

living in a biofilm can have

significantly different properties from
free-oating bacteria, as the dense and
protected environment of the film allows
them to cooperate and interact in various
ways. One benefit of this environment is
increased resistance to detergents and
antibiotics, as the dense extracellular
matrix and the outer layer of cells protect
the interior of the community.

Biofilms major cause of

Nosocomial infections

Microbial biofilms,
which often are
formed by
antimicrobialresistant
organisms, are
responsible for
65% of infections
treated in the
developed world.

Biofilms a Great threat to

Implants
A

significant number of people are

affected by biofilm infections which
develop on medical devices implanted in
the body such as catheters (tubes used to
conduct uids in or out of the body),
artificial joints, and mechanical heart
valves. When implanted material
becomes colonized by microorganisms, a
slow developing but persistent infection
results.

Biofilms a Grwoing concern in

Modern Medicine
Prosthetic

device infections, such as

those involving artificial heart valves,
intravascular catheters, or prosthetic
joints, are prime examples of biofilmassociated infections. With the
increasing use of such devices in the
modern practice of medicine, the
prevalence of these infections is
expected to increase.

Dental plaque

Dental plaque is a
yellowish biofilm
that build up on
the teeth. If not
removed regularly,
it can lead to
dental caries.

Dental plaques

The formation of
dental plaque bio
films includes a
series of steps that
begins with the
initial colonization
of the pellicle and
ends with the
complex formation
of a mature bio film.

Formation of Dental
Biofilms

Additionally, through
the growth process of
the plaque bio film, the
microbial composition
changes from one that
is primarily grampositive and
streptococcus-rich to a
structure filled with
gram-negative
anaerobes in its more
mature state.

Cell-cell signaling (ex. quorum

sensing), and communication with
different bacteria enhance Biofilm
formation

Biofilms everywhere

They're everywhere:
on your shower
curtain, on medical
devices implanted in
patients, on rocks in
rivers and streams,
and in your nose.
While the sheer
number of different
organisms a biofilm
may contain makes it
a challenge to study,

CDC on Biofilms
Biofilms

form on the surface of

catheter lines and contact lenses.
They grow on pacemakers, heart
valve replacements, artificial joints
and other surgical implants. The CDC
(Centers for Disease Control)
estimate that over 65% of
Nosocomial (hospital-acquired)
infections are caused by biofilms.

Biofilms interfere in Antibiotic

Therapy

Bacteria growing in a
biofilm are highly
resistant to antibiotics,
up to 1,000 times more
resistant than the same
bacteria not growing in
a biofilm. Standard
antibiotic therapy is
often useless and the
only recourse may be
to remove the
contaminated implant.

Biofilm and Antibiotic

resistance

A key property of bio

films is that individual
microorganisms are
bound together by a
polymeric substance
excreted by the
microorganisms.. This
protective
encapsulation is
believed to play a role
in some antibioticresistant infection.

Bacterial resitance and

Biofilms
Another

area of great importance from a

public health perspective is the role of
biofilms in antimicrobial-drug resistance.
Bacteria within biofilms are intrinsically
more resistant to antimicrobial agents
than plank tonic cells because of the
diminished rates of mass transport of
antimicrobial molecules to the biofilm
associated cells or because biofilm cells
differ physiologically from plank tonic
cells

Biofilms in Cystic fibrosis

Biofilms are
involved in
numerous
diseases. In cystic
fibrosis patients
have Pseudomonas
infections that
often result in
antibiotic resistant
biofilms.

Endocarditis and Biofilms

Microorganisms may attach and develop

biofilms on components of mechanical heart
valves and surrounding tissues of the heart,
leading to a condition known as prosthetic
valve endocarditis. The primary organisms
responsible for this condition are S.
epidermidis, S. aureus, Streptococcus spp.,
gram-negative bacilli, diphtheroids,
enterococci, and Candida spp. These
organisms may originate from the skin, other
indwelling devices such as central venous
catheters, or dental work.

Eye and Biofilms

The presence of
bacterial biofilms has
been demonstrated on
many medical devices
including intravenous
catheters, as well as
materials relevant to the
eye such as contact
lenses, scleral buckles,
suture material, and
intraocular lenses. Many
ocular infections often
occur when such
prosthetic devices come
in contact with or are
implanted in the eye.

Biofilms and Contact lenses

Bacterial biofilm
formation on contact
lenses and contact lens
storage cases may be
a risk factor in contact
lens-associated corneal
infections. Studies
have shown that
contamination of lens
cases by bacteria,
fungi, and amoebae is
common with 20% to
80% of lens wearers
having a contaminated
lens case.

Urinary catheters and

Biofilms

Urinary catheters are tubular latex or

silicone devices, which when inserted may
readily acquire biofilms on the inner or
outer surfaces. The organisms commonly
contaminating these devices and
developing biofilms are
S.
epidermidis, Enterococcus faecalis, E.
coli, Proteus mirabilis, P. aeruginosa,
K. pneumoniae, and other gram-negative
organisms. The longer the urinary catheter
remains in place, the greater the tendency
of these organisms to develop biofilms and
result in urinary tract infections.

Biofilms and indwelling medical

devices

Biofilms on indwelling medical devices may

be composed of gram-positive or gramnegative bacteria or yeasts. Bacteria
commonly isolated from these devices
include the gram-positive Enterococcus
faecalis, Staphylococcus aureus,
Staphylococcus epidermidis, and
Streptococcus viridians; and the gramnegative Escherichia coli, Klebsiella
pneumoniae, Proteus mirabilis, and
Pseudomonas aeruginosa.

Indwelling catheters and

Biofilms*

Central venous catheters, the reference

method for quantification of biofilms on
catheter tips is the roll-plate technique, in
which the tip of the catheter is removed
and rolled over the surface of a
nonselective medium. Quantification of the
biofilm depends on the number of
organisms recovered by contact with the
agar surface. Biofilm-associated cells on the
inner lumen of the device are not detected
with this method, which has low diagnostic
sensitivity and low predictive value for
catheter-related bacteraemia.

Indwelling catheters and

Biofilms*

In addition, this method cannot detect

more than 1,000 colony-forming units
(CFU) per tip. A method that used
sonication plus vortexing as a means
of quantifying biofilms on catheter tips
showed that a level of 104 CFU per tip
is predictive of catheter-related
septicaemia

* Biofilms and Device-Associated Infections

Rodney M. Donlan Centers for Disease Control and Prevention
Atlanta, Georgia, USA

Antibiotic therapy alone may

not cure ?

Antimicrobial agents are

administered during valve
replacement and
whenever the patient has
dental work to prevent
initial attachment by
killing all microorganisms
introduced into the
bloodstream. As with
biofilms on other
indwelling devices,
relatively few patients
can be cured of a
biofilm infection by
antibiotic therapy alone

Biofilms a concern in
Antimicrobial Therapy

Microbial biofilms may pose a public health

problem for persons requiring indwelling
medical devices. The microorganisms in
biofilms are difficult or impossible to treat
with antimicrobial agents; detachment from
the device may result in infection. Although
medical devices may differ widely in design
and use characteristics, specific factors
determine susceptibility of a device to
microbial contamination and biofilm
formation.

Biofilms need higher

concentration of Antibiotics
Biofilms

are remarkably difficult to treat

with antimicrobials. Antimicrobials may
be readily inactivated or fail to penetrate
into the biofilm. In addition, bacteria
within biofilms have increased (up to
1000-fold higher) resistance to
antimicrobial compounds, even though
these same bacteria are sensitive to
these agents if grown under plank tonic
conditions.

Biofilms help Gene

transfer

Biofilms increase the

opportunity for gene
transfer
between/among
bacteria.. Gene
transfer can convert
a previous a virulent
commensals
organism into a
highly virulent
pathogen.

Biofilms Quorum
sensing

Certain species of
bacteria communicate
with each other within
the biofilm. As their
density increases, the
organisms secrete low
molecular weight
molecules that signal
when the population
has reached a critical
threshold. This process,
called quorum
sensing, is responsible
for the expression of
virulence factors.

Quorum sensing helps the

survival of pathogens

Biofilms contribute for new

phenotypes
Bacteria

express new, and sometimes

more virulent phenotypes when growing
within a biofilm. Such phenotypes may
not have been detected in the past
because the organisms were grown on
rich nutrient media under plank tonic
conditions. The growth conditions are
quite different particularly in the depths
of biofilms,

Biofilms protects from

Immune responses
Bacteria

embedded within biofilms are

resistant to both immunological and
non- specific defence mechanisms of
the body. Contact with a solid surface
triggers the expression of a panel of
bacterial enzymes which catalyze the
formation of sticky polysaccharides that
promote colonization and protection.

Biofilms Protects from

Phagocytosis
Phagocytes

are unable to effectively

engulf a bacterium growing within a
complex polysaccharide matrix
attached to a solid surface. This
causes the phagocyte to release
large amounts of pro-inammatory
enzymes and cytokines, leading to
inammation and destruction of
nearby tissues.

Current objectives on
Biofilm research

o Development of improved imaging of

biofilms in situ;
o Development of improved clinically
relevant in vitro and in vivo models of
biofilms under specific in vivo conditions such
as ow rate, nutrient content, and
temperature;
o
Development of better probes (genetic,
metabolic, and immunological) for real- time
analysis;
o
Studies of quorum sensing/signaling
molecules;

Current objectives on Biofilm

research

o Further characterization of biofilm-specific

gene expression;
o
Studies of the exchange of genetic material
within biofilms;
o
Studies of organic contaminants on substrata,
and their inuence on biofilm structure;
o Development
of novel approaches to control pathogenic
bacteria by, for example, devising strategies
to favour growth of non-pathogenic
microorganisms in biofilm communities;

Current objectives on Biofilm

research

o Studies of pathogenic
mechanisms of microbes
growing in biofilms;
o Elucidation of
mechanisms of
resistance of biofilms to
antimicrobial agents;
o Studies of
host immune
responses, both
innate and adaptive
to biofilms;

Current objectives on Biofilm

research

In studies of infectious lung disease in cystic

fibrosis;
o Studies on the potential of diagnostic
procedures such as Bronchoalveloar lavage
and bronchoscopy to disturb local biofilm
ora and inoculate distant locations;
o Development of
mathematical models and computer
simulations of biofilms;
o Development of the
methodology for the prevention and control
of biofilms from catheters, water unit lines,
and other clinically important solid surfaces;.

Searching for alternatives

Tissue engineering

Role of biofilms in multiple pathologies and the

difficulty in resolving these pathologies speaks
to the importance of developing means of
replacing or enhancing the therapies already
in use. The use of synthetic materials in the
body ranges from catheters to mesh to stents
to heart valves and beyond. Until the
development of viable and practical tissue
engineering, then number and types of
applications in which synthetic materials are
used will continue to increase.

Emerging Methods

Several researchers
are finding solutions
for the cure of Biofilms
, yet it is experimental,
with advances in
molecular biology
better model
treatments can be
identified to reduce
the problem of Biofilm
interference in
Antibiotic therapy.

Created for Dr.T.V.Rao MDs e

learning series.
Email
doctortvrao@gmail.com

Endodontic biofilms could be classified as follows: 1]

Intracanal biofilms that are generally present on the
root canal dentine of an endodontically infected tooth.
2] Extraradicular microbial biofilms that are formed on
the root (cementum) surface adjacent to the apex of
endodontically infected teeth. 3] Periapical microbial
biofilms that are isolated biofilms found in the periapical
region of an endodontically treated tooth. 4] Biomaterial
centered infection that is formed when bacteria adheres
to an artificial biomaterial surface such as root canal
obturating materials, thereby forming a biofilm. These
biofilms could be intraradicular or extraradicular,
depending on the apical extent of the obturation.3

Endodontic Microora:
Most of the bacteria in an endodontic infection are
strict anaerobes. The ora is usually polymicrobial,
dominated by obligate anaerobic bacteria. Grampositive organisms are found in approximately 75%
of the samples; the most predominant are
streptococci (28%), staphylococci (15%),
corynebacteria (10-25%), yeasts (12%), and others.
The Gram-negative bacteria (24%) include
spirochetes (9-12%), neisseria (4%), bacteroides
(7%), fusobacterium (3%), pseudomonas (2%),
coliform bacteria (1%), and others

Types of Endodontic
Infections
I] Intraradicular Infection
Primary intraradicular infections:
Microorganisms that initially invade and colonize the
necrotic pulp tissue cause primary intraradicular
infection. Primary infections are characterized by a
mixed consortium composed of 10 to 30 bacterial
species and 103 to 108 bacterial cells per canal. The
involved microbiota is conspicuously dominated by
anaerobic bacteria, but some facultative or
microaerophilic species can also be commonly found
in primary intraradicular infections.4

Secondary Intraradicular Infections:

Microorganisms that were not present in the
primary infection but that were introduced
into the root canal system at some time after
professional intervention cause secondary
intraradicular infections. The entry can be
during treatment, between appointments, or
even after root canal filling. Species involved
can be oral or nonoral microorganisms,
depending on the cause of infection.4

Persistent Intraradicular Infection:

Microorganisms that can resist
intracanal antimicrobial procedures
and endure periods of nutrient
deprivation in a prepared canal cause
persistent intraradicular infections.
This is also termed recurrent
infection. Involved microorganisms
are remnants of a primary or
secondary infection. E faecalis are
predominant and is a persistent
organism. It is commonly found in a
high percentage of root canal as a
single organism or as a major
component of the ora. Persistent

II] Extraradicular Infection:

Microbial invasion of the inamed
periradicular tissue is invariably a
sequel of interradicular infection.
Acute alveolar abscess is an example
of extraradicular extension or a
sequel to interradicular infection.
Sometimes extraradicular infection
can be independent of intraradicular
infections. For example, apical
actinomycosis caused by
Actinomyces species and P.
propionicum is a pathological disease
which can be treated only by
periapical surgery.

Strategies to prevent biofilm formation depend on context

IAN UNIVERS
gpp
Medical (small scale)
SITY OF LIFE
Chemical approaches:antibiotics, biocides etc.
Physicalapproaches:Useofacousticenergyultrasoundvibration
SCIENCES
Physical approaches:Use of acoustic energy, ultrasound, vibration, Piezo electric
elements Biological approaches:Lytic bacteriophages, interference with
Idtil(ll)
inter-bacterial signaling, interference with genetic programs that control biofilm
formation
Industrial (large scale) Chemical approaches:chlorine, ozone, ionization, hydrogen
peroxide, various biocides
Physical approaches:choice of material that inhibits attachment, vibration,
mechanical cleaning (e.g. high pressure flushing), UV
www.umb.no
Biological approaches:alteration of growth conditions?,

 Methods

for analysing microbial biofilms:

IAN UNIVERS
yg
Electron microscopy
SITY OF LIFE
Nanoscale imagingConfocal laser scanning microscopy Epiuorescence
microscopy Atomic force microscopy
SCIENCES
Nanoscale analysis
Tip enhanced Raman spectroscopy
Mass spectroscopy
Mllthi
Microarray analysis Molecular techniquesWhole genome sequencing
Fluorescent labeling techniques
Essential know howMicrobial physiology and metabolism Microbial genetics

 Possibly

the first identification of

biofilm structures in infected root
canals was carried out by Nair (10).
On the basis of transmission electron
microscopy (TEM) he examined the
root canal content of 31 teeth, which
had gross coronal caries and to which
the periapical inammatory process
was attached upon extraction

 he

bacterial condensations showed a

palisade structure similar to the one
for dental plaque on external tooth
surfaces, suggesting similar
mechanisms for bacterial attachment
as those for dental plaque

Molven et al. (11) noted by scanning

electron microscopic (SEM)
observations of the apical 2 mm of
infected root canals that cocci and
rods and/or filaments often formed
micro-colonies in this area into which
spirochetes were interspersed.
However, rarely did spirochetes
gather in clusters. Cocci were also
seen attached to filaments assembled
into the so-called corn-cob-like
structures, which are also described
for dental plaque (37)

In a study of cases resisting treatment, the socalled refractory endodontic cases, Tronstad et al.
(4) examined the surfaces of the root tips removed
during surgical intervention by SEM. They noted
that the apex of the roots adjacent to the apical
foramen was coated with a continuous, smooth,
structure-less layer containing a variety of
bacterial forms. In irregularities of the surfaces and
in crypts and holes, bacteria were seen held
together by an extracellular material. The
organisms were identified as cocci and rods with
some presence of fibrillar form

Noiri et al. (7) analyzed the presence of

biofilm formations on root tips of extracted
teeth with refractory periapical pathosis
and gutta-percha points removed during
endodontic treatment by SEM. Guttapercha
points sticking out through the apex were
almost completely covered with glycocalyxlike structures. Bacteria, mostly filaments
or long rods, were seen on the external root
surfaces in the extracted teeth