biofilm in periodontics - a tool in diagnosis.pptx

CONTENTS
• Introduction
• The Development of the Biofilm Era
• Biofilms in nature
• Techniques to study biofilms and their components
• The Architecture of Biofilms
• Advantages of being in a biofilm
• Formation of biofilms
• Antibiotic resistance

• The Evolution of Biofilms
• Communication between bacteria
• Uses of biofilms
• Harmful effects of biofilms
• Biofilms in dental unit waterlines
• controlling biofilms
• References

INTRODUCTION
• Prokaryotes are one of the most primitive organisms on
earth characterized by having extreme capabilities to
thrive and survive in any kind of environment. Various
prokaryotes such as bacteria and fungi have the capacity
to form biofilms in natural settings

BIODIVERSITY OF BACTERIA
There exist three major variations
in bacteria
• Ultra micro bacteria (UMB)
• Planktonic form
• Biofilms
UMB and biofilms are designed
for survival mechanism and
planktonic form for transmission

THE DEVELOPMENT OF THE BIOFILM
ERA
• The first observation of surface
associated bacteria was made by
Anthony van Leeuwenhoek (1684).
He observed "animals" in the scurf of
the teeth.
• Concept of “microbial aggregation”
• Henrici (1933): "It is quite evident that
for the most part water bacteria are
not free floating organisms, but grow
upon submerged surfaces." And
given a chance 99.9% of bacteria
prefer biofilm to planktonic form

"The pure culture period" :
• All this time bacteria were viewed as being purely
free floating single cells, also referred to as the
planktonic state. Most work on characterizing
bacteria was done by propagating the bacteria in a
liquid media in test tubes, or on agar plate.
• This is very peculiar with the present day's
knowledge, since we estimate that less than 0.1% of
the total microbial biomass lives in the planktonic
mode of growth (Costerton et al.1995)

LIMITATIONS OF CULTURES
• not all bacteria from a given environment will grow on
laboratory media
• the number of cells that were observed microscopically
far outweighed the number of colonies that grow on a
petri plate
• Observes the characteristics of only planktonic cells
which have different genotypic and phenotypic variations
from biofilm bacteria as well as they express different
metabolome, proteome and transcriptome

• In 1978 Costerton defined the term
biofilm for the first time and
described it as a matrix-enclosed
mode of growth.
• Father of biofilm
• (1999) defined a biofilm as “a
structured community of bacterial
cells enclosed in a self-produced
polymeric matrix, adherent to a
surface.“
• (1989)Lappin Scott : biofilm as
aggregates of microcolonies
attached to a surface and embedded
in exopolymeric substances.

BIOFILMS IN NATURE
• Biofilms are everywhere
Pristine alpine lakes Polluted areas

4.5 billion year stromatolites Deep sea biofilms near hydrothermal vents

BIOFILMS IN HEALTH AND THE HUMAN
BODY
• About 90 % of the cells in a human body are not
human In fact, the human body is heavily colonized
by microbes
• They live on all mucous surfaces ,teeth, digestive
tract, as well as on and in layers of our skin. We
depend on some of our gut biofilms to help with
digestion.

PLAQUE AS A BIOFILM
• It has been estimated that about 700 species of all
bacteria have been identified. (Moore WE 1994 ) 0f
which about 200 species are culturable.
• It is also known that >40% of the bacteria present in the
oral cavity and 99% in the environment are unculturable.
• VBNC (viable but not culturable) describes the fact that
most scientists believe less than 0.5% of the microbial
world has been cultured or is culturable. Part of this may
be due to biofilm.

TECHNIQUES TO STUDY BIOFILMS
AND THEIR COMPONENTS
• Confocal scanning laser microscopy (CSLM)
• 16s rRNA directed fluorescence in situ
hybridization(FISH probe): FISH probes designed to
react with their 16 S rRNA sequences (whether they
have ever been cultured or not) to locate them precisely
in the system or the infected tissue
• Non-invasive Biofilm Characterization Using Acoustic
Microscopy

• Lectin binding analysis for detection of biofilm
polysaccharide components of biofilm
• Denaturing gradient gel electrophoresis (DGGE)
technique and high-performance liquid chromatography
(HPLC): We can identify the predominant biofilm
organisms in any ecosystem

CONFOCAL SCANNING LASER
MICROSCOPY (CSLM)
• Confocal micrograph of biofilm
formed on gold foil carrier
placed in gingival crevice of
patient with controlled
periodontitis.
• confocal probes and
fluorophore-tagged lectins
shows an arboreal community
of linear organisms bearing
well-defined bacterial
microcolonies, while
amorphous EPS material
anchors the community

• Confocal image, showing
the presence of matrix-
enclosed biofilms of
Porphyromonas
gingivalis (green) and
Tanerella forsythensis
(orange) in the sulcus of a
patient with controlled
periodontitis.
• (PMNs), with blue nuclei,
can be seen to be
mobilized from tissues that
are mounting an
inflammatory
response to the presence of
these biofilms

• Three-dimensional (3-D) confocal laser scanning
microscopy images of an actively growing biofilm of
Shewanella oneidensis

FLUORESCENCE IN SITU
HYBRIDIZATION(FISH)

SCANNING ACOUSTIC MICROSCOPE
Three-dimensional
topographic display of a
biofilm surface. The data
to construct this image
was acquired using a
scanning acoustic
microscope.

THE ARCHITECTURE OF BIOFILMS
1. Attachment Surface
• Non Shedding Surface
• Shedding Surface
2. Biofilm Community
• Micro colonies of bacterial cells (15 - 20%)
• Matrix of glycocalyx (75 -80%)with open fluid filled channels
3. Bulk Fluid
• Stationary sub layer
• Layer of fluid in motion

COMPOSITION OF THE EPS
Extracellular polymeric substances are defined as organic
polymers of biological origin, which in biofilm systems are
responsible for the interaction with interfaces
• Polysaccharide -1-2% (neutral & Polyanionic)
• Proteins : <1-2%
• DNA & RNA : <1-2%
• HOST : Fibrin, RBCs, WBCs

STRUCTURE
In biofilm systems, one can expect two types of polymeric
carbohydrate structures:
• Those located on cell surfaces and
• Those located extracellularly throughout the biofilm
matrix.

• biofilms are composed of micro-colonies of these matrix-
enclosed cells and that the community is intersected by a
network of open water channels.
• primitive circulatory system that one could imagine being
responsible for delivery of nutrients and removal of
wastes
• The micro colonies were seen to take the form of simple
towers, or of mushrooms.

• Cells in biofilms are located in genetically determined
positions much like organelles are located within
eukaryotic cells.
• Sessile cells are arranged in patterns in which they are
separated by standard distances (4 to 10 μm).
• Type IV pili - motility

ADVANTAGES OF BEING IN A
BIOFILM
• Organization
• Protection
• communication
• Adequate facilities

ADVANTAGES OF BEING IN A
BIOFILM
Kryst (2001) and Massalha et al. (2007),
• An increase in the concentration of nutrients at the liquid-
biofilm interfaces, since the polymeric matrix favours the
adsorption of nutrient molecules;
• Protection against harsh environmental factors such as
fluctuation in pH, salt and heavy metal concentrations,
dehydration, shearing forces, aggressive chemical
substances, bactericides, antibiotics and predators.
• Enhanced virulence (‘pathogenic synergism’)
• possibility for the exchange of genetic material due to the
long microorganism retention times; and

ANTIBIOTIC RESISTANCE
• Antimicrobial agents penetrate biofilm matrices with ease
• Why, then, were biofilm cells resistant to antibiotics at
hundreds of times the concentrations that would kill
planktonic cells if biofilms simply consisted of planktonic
cells trapped in permeable matrices?

David Davies in 1995
• biofilm cells expressed their genes in a pattern that differed
from that expressed by planktonic cells, of the same species.
• Expression of algC gene
• Proteins produced (genes expressed) by cells in biofilms
differed profoundly from those produced by planktonic cells of
the same strain and these gene products differ by as much as
70% in location and intensity
Stewart and Costerton 2001
• Biofilm bacteria lack the targets for conventional antibiotics, all
of which were selected for their efficacy against planktonic
bacteria, and that this lack of cognate targets may account for
the inherent resistance of sessile bacteria to these agents

Pradeep Singh 2004
• In addition to the physiological variability inherent in
communities that contain fast and slow-growing cells,
engaged in both aerobic and anaerobic metabolic
processes
• This adds programmed genomic diversity to the
background of metabolic diversity, so that no two cells in
a biofilm are truly identical, and the “job” of any single
antibiotic or combination of conventional antibiotics is
made almost impossible
High rates of horizontal gene transfer and accelerated
recombination rates give biofilms a very high level of
genomic diversity

• The invading cells can delay the production of
compounds that will trigger the hosts defense
mechanisms until a substantial population of well-
protected cells has matured the pathogen can in
essence “fly below the host’s radar”.
• An attack launched once the biofilm has been formed
will be resistant not only to host defences, but to
antibiotic therapy as well.

ANTIMICROBIAL RESISTANCE IN
PLAQUE
• Bacteria growing in dental plaque also display
increased resistance to antimicrobial agents,
including those used in dentifrices and mouth rinses
• Examples:
Shani et al., 2000
• The biofilm inhibitory concentration for chlorhexidine
and amine fluoride was 300 and 75 times greater,
respectively, when S.sobrinus was grown as a
biofilm compared with the minimum bactericidal
concentration of planktonic cells

Larsen and Fiehn, 1996:
• Similarly, it was necessary to administer 10–50 times the
minimum inhibitory concentration of chlorhexidine to
eliminate S. sanguinis biofilms within 24 h.
Millward and Wilson 1989:
• The age of the biofilm can also be a significant factor;
older biofilms (72 h) of S. sanguinis were more resistant
to chlorhexidine than younger (24 h) biofilms .

Zaura-Arite et al - 2001
• Confocal microscopy of in situ established natural
biofilms showed that chlorhexidine only affected the
outer layers of cells in 24- and 48-hour plaque biofilms.
Larsen, 2002;
• Biofilms of oral bacteria are also more resistant to
antibiotics (e.g. amoxycillin, doxycycline, and
metronidazole).

• Confocal micrograph
showing dental biofilm
treated by an antibacterial
solution.
• majority of the bacterial
cells in the shallow biofilm
are dead (red), while almost
all of the cells in the raised
“towers” have survived
(green).
• Culture methods show a >
99% kill of this biofilm, by
this agent, because the
large aggregates do not
grow when plated on agar

THE EVOLUTION OF BIOFILMS
• At the zenith of the natural association of bacteria with
humans, before the development of antibacterial agents
in the past 200 years, some microbes had evolved the
very successful strategy of lurking in protected biofilms
and attacking with highly evolved planktonic cells.
• These bacteria exploited human ecosystems, without
wiping out this nutrient-rich niche, by attacking with
planktonic “missiles” that homed in on specific tissue
targets and killed selected individuals before they could
mount an acquired immune response.

COMMUNICATION BETWEEN
BACTERIA
Quorum sensing
• Hastings and Nealson 1977
• “quorum sensing” molecules (Fuqua et al. 1994)
• In biofilms, the matrix material (EPS) that holds cells in
close proximity allows concentrations of signal molecules
to build up in sufficient quantity to effect changes in
cellular behavior. Bacterial populations will activate some
genes only when they are able to sense, via cell
signaling, that their population is numerous enough to
make it advantageous and/or "safe" to initiate that
genetic activity.

QUORUM SENSING MOLECULES
• Fuqua et al. 1994: acyl homoserine lactone (AHL)
signals of Gram-negative bacteria
• Dunny and Leonard1997 : cyclic polypeptide signals of
Gram-positive bacteria
• Autoinducer II

QUORUM QUENCHING
• Quenching microbial quorum sensing
• also known as antipathogenic or signal interference,
which abolishes bacterial infection by interfering with
microbial cell-to-cell communication
Ex:
• AHL-lactonase
• AHL-acylase
• synthetic AIP-II
• furanone

USES OF BIOFILMS
• Water and wastewater treatment: pseudomonas putida
• Microbial leaching
• Remediation of contaminated soil and groundwater
• They live on all mucous surfaces ,teeth, digestive tract,
as well as on and in layers of our skin and some of our
gut microbes help with digestion.

COMMENSAL INTEGRATION WITH
EUKARYOTES
• Rhizobia and nitrogen-
fixing plants (Long 2001)
• “captive” cellulose-
digesting bacteria and
their insect (Bresnak and
Brune 1994) and
mammalian hosts,
Lactobacillus sp on vaginal
epithelium

• S.epidermis integrated into deeper parts of the skin

MEDICAL BIOFILM PROBLEMS
Costerton et al. 1999:
• Medical devices
• Orthopaedic devices
• Transcutaneous devices
• Urinary catheters
• Artificial heart valves
• Pacemaker leads
• Biofilms on host tissues
• Chronic wounds,Osteomyelitis,Cystic fibrosis
• Otitis media with effusion
• Hospital equipment
• Hemodialysis machines
• Mechanical ventilators
• Shunts, etc.
• Contact lenses and other optical devices

BIOFILMS IN DENTAL UNIT
WATERLINES
DUWLs (Dental unit water lines) provide an environment
conductive to rapid proliferation of biofilm. Due to
convergence of 3 factors
• Surface Colonization
• Laminar Flow
• Surface: Volume ratio

INTRAORAL
On the basis of physical and morphologic criteria, the oral cavity can be
divided into six major ecosystems (also called niches)
• The intraoral, supragingival, hard surfaces (teeth, implants,
restorations, and prostheses)
• Subgingival regions adjacent to a hard surface, including the
periodontal/periimplant pocket (with its crevicular fluid, the root
Cementum or implant surface, and the pocket epithelium)
• The buccal, palatal epithelium, and the epithelium of the floor of the
mouth
• The dorsum of the tongue
• The tonsils
• The saliva

• Most species (with the exception of spirochetes) are able
to colonize all of them. Some periodontopathogens
(Fusobacterium nucleatum) and Prevotella intermedia
are involved in the etiology of tonsillitis and most
pathogens are able to colonize the maxillary sinus.

HARD TISSUES
Teeth and implants are unique for two reasons:
• (1) they provide a hard non-shedding surface that allows
the development of extensive structured bacterial
deposits and
• (2) they form a unique ectodermal interruption. There is
a special seal of epithelium (junctional epithelium) and
connective tissue between the external environment and
the internal parts of the body.

QUORUM SENSING IN DENTAL
BIOFILMS
• Mainly through AI2
• Various pathogens exhibiting AI2 activity (frias et al
1994)
• Pg
• Aa
• Pi
• Fn
Pg requires mature plaque to colonize

CONTROLLING BIOFILMS
(a) Reduction of “Bacterial Loads” and
Colonization Rates
• The development of inhibitors and antiplaque agents
that are more effective against surface-associated
micro-organisms, coupled with more effective delivery
systems for targeting specific bacteria and for
improving the retention of agents in the mouth;

• Interference with communication networks that
coordinate or regulate microbial activities within biofilms;
in other areas of microbiology, attempts are being made
to block signalling molecules that induce a shift in the
host to a more virulent phenotype;
Ex: furanones,quorum quenching molecules
(b) Direct Manipulation of Biofilm Formation by
Signal Inhibition

FURANONES
• Blocks receptors that receives N-acyl homoserine
lactone that normally induce surface colonization.
• Prevents bacteria from forming groups and becoming
virulent, but does not physically kill the bacteria.
Therefore, the bacteria cannot become resistant. The
bacteria simply are unable to communicate with other
bacterial cells. Thus no bacterial film can form on the
surface of the seaweed.

(c) Preventing colonization of selected organisms
• (e.g.by interfering with attachment by modifying the
conditioning film – eg: Delmopinol
• or by ‘replacement therapy’, whereby organisms are
deliberately implanted to prevent subsequent colonization by
more pathogenic species) – probiotics, GPR
(d) Affecting biofilm architecture,
• for example, by the use of enzymes that can degrade the
exopolymers that comprise the plaque matrix;

(e) The neutralization of parameters that select for
the species that are implicated in disease;
• thus, strategies that reduce the pH response to
dietary carbohydrates will help prevent the enrichment
of acidogenic and aciduric bacteria;
(f) The identification of pathogenic clones could
also improve diagnosis and might predict sites that
are more susceptible to disease.

CONCLUSION
• Change in concepts of microbial culture and diagnosis
• Change in antibiotic treatment regimens
• Change in treatment modalities

REFERENCES
• The Biofilm Primer:J. William Costerton
• Biofilm highlights:flemming
• Oral biofilms and plaque control – H.J.Busscher and L.V.
Evans; FIRST EDITION, 1998.
• Clinical periodontology and implant dentistry, 5th edition
– JAN LINDHE, THORKILD KARREY and NIKLAUS P.
LANG.
• Anne.D.Haffajee and Sigmund.S.Socransky. Introduction
to microbial aspects of periodontal biofilm, communities,
development and treatment. Perio 2000;2006 42:7.

• Carranza, Newman, Takei Clinical Periodontology 11th
edition
• Dental biofilms: difficult therapeutic targets.SIGMUND S.
SOCRANSKY & ANNE D. HAFFAJEE Periodontology
2000, Vol. 28, 2002, 12–55
• Molecular genetics analyses of biofilm formation in oral
isolates. MARY E. DAVEY & JOHN W. COSTERTON
Periodontology 2000, Vol. 42, 2006, 13–26
• Genetic basis of horizontal gene transfer among oral
bacteria, ADAM P. ROBERTS & PETER MULLANY
Periodontology 2000, Vol. 42, 2006, 36–46

• Bacterial interactions and successions during plaque
development. PAUL E. KOLENBRANDER, ROBERT J.
PALMER JR, ALEXANDER H. RICKARD, NICHOLAS S.
JAKUBOVICS, NATALIA I. CHALMERS & PATRICIA I.
DIAZ Periodontology 2000, Vol. 42, 2006, 47–79
• Periodontal microbial ecology. SIGMUND S.
SOCRANSKY & ANNE D. HAFFAJEE Periodontology
2000, Vol. 38, 2005, 135–187

BIOFILMS IN MANUFACTURED
MATERIALS AND SYSTEMS
• Biofilm contamination and fouling occur in nearly every
industrial water-based process, including water
treatment and distribution, pulp and paper manufacturing
and the operation of cooling towers.
• Biofilms are responsible for billions of dollars in lost
industrial productivity, as well as product and capital
equipment damage each year. Biofilms are notorious for
causing pipe plugging, corrosion and water
contamination.

• In flowing systems with a low Reynolds’s number (Re),
laminar flow may exist which implies that a given "plug"
of liquid moves through the system (e.g. a pipe) with
little or no mixing.
• In turbulent flow the liquid in the plug is rapidly mixed.
Laminar flow is usually characterized by low fluid
velocity (Re < 2100) while high fluid velocity generally
leads to turbulent flow (Re > 4000).
• Biofilms grown under laminar flow, tend to be thicker,
but less dense than those grown under turbulent flow
conditions.

NUTRITION
• It has been shown that biofilms will grow on surfaces in
nutrients solutions so dilute that they will not support the
growth of planktonic cells.
• This observation supports the contention that biofilm acts
as an adsorbent layer concentrating organic materials
and other nutrients from the bulk fluid.
• High nutrient concentrations tend to produce biofilms
that are thicker and denser than those grown in low
nutrient concentrations. The nutritional requirement for
biofilm formation appears to be rather species specific.

biofilm in periodontics - a tool in diagnosis.pptx

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