PROPERTIES OF BIOFILM

Presented by
Dr Krishna Das
PG Student
Concept Of Biofilm

In nature, bacteria have to struggle to obtain sufficient
nutrients to support growth and have to compete with
other microbial communities containing many different
species, sometimes a hundred or more, sharing the
same site.

Nutrients tend to concentrate at interfaces, and
consequently the densest bacterial populations are
located at interfaces of various kinds.

Eg. Solid-liquid boundaries, solid-gas interface (needs
moisture or dies of desiccation)
The human body provides several interfaces
that support microbial populations, of which
the most important in healthy individuals are
the skin , gut , mouth , and female urogenital
tract.

The oral cavity is somewhat unique in this
regard since it provides hard, non shedding
surfaces (teeth) that are accessible for
microbial colonization.

The first description for microbial colonization in
oral cavity dates back to the 1683 when Anton
Von Leeuwenhoek - the inventor of the
Microscope, saw microbial aggregates on
scrapings of plaque from his teeth.

The importance of surfaces for microbial growth
was recognized as early as the 1920s, when a
number of workers independently noted that
bacteria growing on glass slides submerged in soil
were different from those that could be cultured in
broth.

However, it was not until around 50 years later
that sessile microbial populations were considered to
be sufficiently different from free-living
microorganisms to merit their own name, and the
term BIOFILM was coined. (Bill Costerton in 1978).

Biofilms are composed of microbial cells encased
within a matrix of extracellular polymeric substances
(EPS) such as polysaccharides, proteins, and nucleic
acids.
The term Biofilm (Wilderer and Charaklis 1989)
describes the relatively indefinable microbial
community associated with a tooth surface or any
other hard non-shedding material, randomly
distributed in a shaped matrix or glycocalyx.*

In 2002, Donlan and Costerton offered the most salient
description of a biofilm. They stated that biofilm is a
microbially derived sessile community characterized by
cells that are irreversibly attached to a substratum or
interface or to each other, embedded in a matrix of
extracellular polymeric substances that they have
produced, and exhibit an altered phenotype with
respect to growth rate and gene transcription.*
*Socransky SS, Haffajee AD. Dental biofilms: difficult therapeutic targets. Periodontol. 2000;2002 (28):1255.
On basis of its location
Supragingival - Present coronal to the gingival
margin and primarily made up of gram-positive
saccharolytic bacteria.

Subgingival - Present apical to the gingival margin
where environmental conditions and the
different composition of the host defense
elements select a different microbiota, largely
gram-negative proteolytic bacteria

CLASSIFICATION OF BIOFILMS
FORMATION OF A BIOFILM
Biofilm formation is a survival strategy for
bacteria, because it gives them certain
advantages over planktonic bacteria:
-sheltered environment
-better uptake of nutrients
-better communication (quorum sensing)
-resistance and the ability to adapt


HETEROGENEITY
Biofilms are heterogenous: variations in biofilm
structure exist within individual biofilms and
between different types of biofilms.

Environmental conditions, such as pH,
temperature, nutrient concentration, etc. can be
highly variable, which causes bacterial species
with different physiological requirements
(anaerobic, aerobic, microaerobic) or bacteria of
the same species to have very different
physiological states to coexist while separated by
just 10 m.



STRUCTURAL HETEROGENEITY

A wide variety of markedly heterogeneous
microniches or microenvironments that are close
together can exist within a biofilm where
microorganisms compete for space under varying
conditions.

Coaggregation : Is a process by which genetically
distinct bacteria becomes attached to one another
via specific molecules while it is referred to as
coadhesion when one partner of the pair is
attached to a surface.

Examples of coaggregation are:

Corncob formation- Streptococci adheres to
filaments of Cornybacterium matruchotti or
Actinomyces species.

Test tube brush- composed of filamentous bacteria
to which gram negative rods adhere.
All oral bacteria possess surface molecules that
foster some sort of cell cell interaction
through the highly specific steriochemical
interation of protein adhesin and carbohydrate
saccharide receptors molecules located on the
bacterial cell surface.

Adhesin -receptor interactions are mediated by
the fundamental physicochemical forces
(hydrophobic, electrostatic, and Van der Waals),
but they are highly specific.

Mediated by lectin like adhesins and can be
inhibitted by lactose and other galactosides.


Different species, or even different strains of a single
species, have distinct sets of coaggregation partners.
Fusobacteria coaggregate with all other human oral
bacteria while Veillonella spp., Capnocytophaga spp. and
Prevotella spp. bind to streptococci and/or actinomyces.

Well -characterized interactions of secondary colonizers
with early colonizers include the coaggregation of F.
nucleatum with S.sanguinis, Prevotella loescheii with A.
oris, and Capnocytophaga ochracea with A. oris.

Streptococci show intrageneric coaggregation.

Secondary colonizers, such as Prevotella intermedia,
Prevotella loescheii, Capnocytophaga spp. , F. nucleatum,
and P. gingivalis do not initially colonize clean tooth
surfaces but adhere to bacteria already in the plaque
mass.
Thus coaggregation interactions are believed
to contribute to development of biofilm by
two routes:

1. By single cells in suspension specifically
recognizing & adhering to genetically distinct
cells in the developing biofilm.

2. Second route by prior colonization in
suspension of secondary colonizer followed
by subsequent adhesion of this coaggregate
to the developing biofilm.
Diagram illustrating the possible roles of coaggregation in the development of multi-
species biofilms. (a) Primary colonization of a substratum covered in a conditioning film
composed of polysaccharides and proteins; (b) cell growth, division and production of
extracellular polysaccharide (EPS) leading to the development ofmicrocolonies; (c)
coadhesion of single cells, coaggregated cells and groups of identical cells into the young
multi-species biofilm; and (d) maturation and the formation of clonal mosaics within the
multi-species biofilm.
Using a DNA-hybridization methodology , defined color-coded
complexes of periodontal microorganisms that tend to be
found together in health or disease have been identified.

The composition of the different complexes was based on the
frequency with which different clusters of microorganisms were
recovered, and the complexes were color-coded for easy
conceptualization.

Socransky et al1998
The early colonizers are either independent of defined
complexes (A. naeslundii , A. oris) or members of the
yellow or purple complexes.

The secondary colonizers fell into the green, orange, or
red complexes

The green and orange complexes include species
recognized as pathogens in periodontal and non
periodontal infections.

The red complex is of particular interest because it is
associated with bleeding on probing.

The existence of complexes of species in plaque is a
reflection of bacterial interdependency in the biofilm
environment .

Physiological heterogeneity

Cells of the same microbial species can exhibit
extremely different physiological states in a biofilm.

Xu et al. (2000) grew Pseudomonas aeruginosa in an
aerated continuous flow reactor and measured various
physiologica properties by dyes and indicators.

DNA indicating the presence of bacterial cells was
detected throughout the 110-mm-thick biofilm.
Protein synthesis could be detected in the outer 30 mm.
Respiratory activity in the outer 24mm.

The authors suggest that antibiotics that kill actively
growing cells would affect the outer layer of the
biofilm, but the remaining cells would not be affected.
Water channels
Water channels are commonly
found in biofilms, and these
can form a primitive circulatory
system.

Function:

1. Provide nutrients and oxygen
for the bacterial micro colonies
2. Facilitate movement of bacterial
metabolites, waste products,
and enzymes within the biofilm
structure
3. Also create an appropriate
physicochemical environment
such as a properly reduced
oxidation reduction potential
Interbacterial communication
Bacteria that live together in a biofilm are able to
communicate with each other either
-by using chemical signals (Quorum sensing)
or
-by transferring genetic material through
conjugation and transformation.

Quorum sensing in bacteria "involves the regulation
of expression of specific genes through the
accumulation of signaling compounds that mediate
inter cellular communication" (Prosser 1999).
It depends on cell density. With few cells,
signaling compounds may be produced at low
levels; however, autoinduction leads to
increased concentration as cell density
increases.


Once the signaling compounds called the
autoinducers reach a threshold level (quorum
cell density), gene expression is activated in
surrounding bacteria.

Quorum sensing can provide biofilms with
some of their characteristic properties, both in
terms of their development, and of their
greater resistance to antimicrobials.

It can promote the expression of genes that encode
for resistance to a certain antibiotic from a certain cell
density; it can also have the ability to affect biofilm
structure, stimulating the growth of beneficial species
and inhibiting the growth of competitor species.

Quorum-sensing signaling molecules produced by
putative periodontal pathogens such as P. gingivalis, P.
intermedia and F. nucleatum have been detected (Frias
et al., 2001).

In general, autoinducers used by gram-negative
bacteria are acylated homoserine lactones and those
used by gram-positive bacteria are processed
oligopeptides (Miller et al., 2001).
Autoinducer-2 regulates iron (hemin) acquisition in the oral
pathogens A. actinomycetemcomitans and P. gingivalis.

Autoinducer-2 quorum sensing is closely linked to the
ability of A. actinomycetemcomitans to grow in a biolm.

Autoinducer-2-dependent quorum sensing was also shown
to be important for mutualistic dual-species biolm
formation by Streptococcus oralis and Actinomyces
naeslundii (Rickard et al., 2006).

Autoinducer-2 is produced by many other oral organisms,
including species of Prevotella, Fusobacterium and
Streptococcus (Frias et al., 2001).

The luxS gene and autoinducer-2 have also been identied
in the periodontal pathogen Eikenella corrodens (Azakami
et al., 2006).

Resistance to antimicrobial agents

Biofilm bacteria shows 1000 to 1500 times
greater resistance than planktonically grown
cells. (Costerton-1999)

The mechanisms of increased resistance in
biofilms differ from species to species, from
antibiotic to antibiotic and for biofilms growing
in different habitats.

1. Slower rate of growth of bacterial species
(biofilm persister cells) in biofilms, which makes
them less susceptible to many but not all
antibiotics. The cells deep in the biofilm
experience different conditions such as hydrogen
ion concentration or redox potentials than cells at
the periphery of the biofilm or cells growing
planktonically.

In addition, the slower-growing bacteria often
overexpress nonspecific defense mechanisms
including shock proteins and multi-drug efflux
pumps and demonstrate increased exopolymer
synthesis. (Gilbert et al.,1999)
2. The exopolymer matrix of a biofilm, although not a
significant barrier in itself to the diffusion of
antibiotics, does have certain properties that can
retard diffusion eg., strongly charged or chemically
highly reactive agents fail to reach the deeper zones
of the biofilm because the biofilm acts as an ion-
exchange resin removing such molecules from
solution. (Gilbert et al., 1999)

3. In addition, extracellular enzymes such as -
lactamases, formaldehyde lyase and formaldehyde
dehydrogenase may become trapped and
concentrated in the extracellular matrix, thus
inactivating some antibiotics.



5. Alteration of genotype and/or phenotype of the
cells growing within a biofilm matrix possibly due
to quorum sensing. Cells growing within a biofilm
express genes that are not observed in the same
cells grown in a planktonic state, and they can
retain this resistance for some time after being
released from 23 the biofilm.

For example, Brooun et al. (2000)
demonstrated that cells of Pseudomonas
aeruginosa liberated from biofilms were
considerably more resistant to tobramycin than
planktonic cells, suggesting that the cells
became intrinsically more resistant when growing
in a biofilm.

6. Recently, the notion of a subpopulation of cells within
a biofilm that are super-resistant was proposed.
Such cells could explain remarkably elevated levels of
resistance to certain antibiotics that have been
suggested in the literature. Brooun et al. (2000)
examined the contribution of multi-drug resistance
pumps to antibiotic resistance of organisms grown in
biofilms. These pumps can extrude chemically
unrelated antimicrobial agents from the cell. Since
extrusion places the antibiotics outside the outer
membrane, the process offers protection against
antibiotics that target cell wall synthesis.

Mechanism of Bacterial Resistance to Antimicrobial Agents
Oral biofilm has pathogenic potential and its
presence is associated with the development
of
1. Caries,
2. Gingivitis,
3. Periodontitis,
4. Peri-implant mucositis
5. Peri-implantitis.

Pathogenic Potential of Oral
Biofilm

Treatment of periodontal biofilms
Of concern to the therapist in treating dental biofilm infections is
the fact that the pathogenic species exist in very large numbers, are
widely distributed within the oral cavity and exist in community
structures that provide protection against host defense mechanisms
and antimicrobial agents.

Further, the organisms can multiply very fast and have ability to
attach to new surfaces of the host or other organisms that are
already attached to the host; thus, spread and re-colonization are a
persistent threat.

Periodontal therapies at the present time can be grouped in three
broad categories:
-mechanical debridement
-antiseptics and antibiotics
-those that affect the environment of the organisms
Fortunately, biofilms in the oral cavity, unlike many
other biofilms, are readily accessible allowing their
physical removal. Indeed, the most common form of
periodontal therapy is the removal of supra and
subgingival plaque by procedures such as self
performed oral hygiene, scaling and root planing or
periodontal surgery.

Systemically administered antibiotics do have certain
effects on segments of the subgingival microbiota, but
usually do not completely eliminate the sensitive
bacterial species.

The systemically administered antibiotics may have
affected, primarily, the organisms in the epithelial cell
associated biofilms and the loosely adherent adjacent
cells.

The species in the tooth-associated biofilms may
have been more resistant to the antibiotics due
to mechanisms described earlier, and thus the
potential for re-growth from this source was
perpetuated.

It might be surmised that physical removal of the
tooth-associated biofilms prior to or during
antibiotic administration might minimize this re-
growth.

Diagrammatic representation of the effect of therapy on colonizing
bacteria, the host and the habitat. Treatment can affect the
composition of the bacterial plaque directly, can affect the host
response or alter the habitat. Alterations of any of these factors can
impact on the remaining factors in this triad. As indicated,
treatment effects are influenced by the genetic background of the
subject, environmental influences such as smoking and the systemic
well-being of the patient.
CONCLUSION
Dental plaque is a naturally occurring biofilm that has
the potential to cause disease. Dental plaques have
many properties in common with biofilms found in
other locations.

However, they have certain characteristics that are
important in terms of control of disease. They are
easily accessible and thus allow direct removal and
application of antimicrobial agents.

Changes in thinking about the structure of dental
plaque have improved our understanding of why
periodontitis is so difficult to treat and will affect the
strategies used to prevent and control periodontitis in
the future.