Properties of Biofilm

8/10/2019 Properties of Biofilm

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PROPERTIES OF BIOFILM

Presented by

Dr Krishna Das

PG Student


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Concept Of Biofilm

In nature, bacteria have to struggle to obtain sufficientnutrients to support growth and have to compete withother microbial communities containing many different

species, sometimes a hundred or more, sharing thesame site.

Nutrients tend to concentrate at interfaces, andconsequently the densest bacterial populations are

located at interfaces of various kinds.

Eg. Solid-liquid boundaries, solid-gas interface (needsmoisture or dies of desiccation)


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The human body provides several interfacesthat support microbial populations, of whichthe most important in healthy individuals are

the skin , gut , mouth , and female urogenitaltract.

The oral cavity is somewhat unique in this

regard since it provides hard, non sheddingsurfaces (teeth) that are accessible formicrobial colonization.

The first description for microbial colonization inoral cavity dates back to the 1683 when AntonVon Leeuwenhoek - the inventor of theMicroscope, saw microbial aggregates on

scrapings of plaque from his teeth.


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The importance of surfaces for microbial growthwas recognized as early as the 1920s, when anumber of workers independently noted that

bacteria growing on glass slides submerged in soilwere different from those that could be cultured inbroth.

However, it was not until around 50 years laterthat sessile microbial populations were considered tobe sufficiently different from free-livingmicroorganisms to merit their own name, and theterm BIOFILMwas coined. (Bill Costerton in 1978).

Biofilms are composed of microbial cells encasedwithin a matrix of extracellular polymeric substances(EPS) such as polysaccharides, proteins, and nucleicacids.


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The term Biofilm (Wilderer and Charaklis 1989)describes the relatively indefinable microbialcommunity associated with a tooth surface or anyother hard non-shedding material, randomlydistributed in a shaped matrix or glycocalyx.*

In 2002, Donlan and Costerton offered the most salientdescription of a biofilm. They stated that biofilm is amicrobially derived sessile community characterized bycells that are irreversibly attached to a substratum orinterface or to each other, embedded in a matrix of

extracellular polymeric substances that they haveproduced, and exhibit an altered phenotype withrespect to growth rate and gene transcription.*

*Socransky SS, Haffajee AD. Dental biofilms: difficult therapeutic targets.Periodontol.2000;2002 (28):1255.


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On basis of its location

Supragingival - Present coronal to the gingivalmargin and primarily made up of gram-positive

saccharolytic bacteria.

Subgingival- Present apical to the gingival marginwhere environmental conditions and thedifferent composition of the host defenseelements select a different microbiota, largelygram-negative proteolytic bacteria

CLASSIFICATION OF BIOFILMS


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FORMATION OF A BIOFILM


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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

roperties of oral biofilms


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HETEROGENEITY Biofilms are heterogenous: variations in biofilm

structure exist within individual biofilms andbetween different types of biofilms.

Environmental conditions, such as pH,temperature, nutrient concentration, etc. can behighly variable, which causes bacterial species

with different physiological requirements(anaerobic, aerobic, microaerobic) orbacteria ofthe same species to have very differentphysiological states to coexist while separated by

just 10 m.


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STRUCTURAL HETEROGENEITY

A wide variety of markedly heterogeneousmicroniches or microenvironments that are closetogether can exist within a biofilm wheremicroorganisms compete for space under varyingconditions.

Coaggregation : Is a process by which genetically

distinct bacteria becomes attached to one anothervia specific molecules while it is referred to ascoadhesion when one partner of the pair isattached to a surface.


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Examples of coaggregation are:

Corncob formation- Streptococci adheres tofilaments of Cornybacterium matruchotti or

Actinomyces species.

Test tube brush- composed of filamentous bacteria

to which gram negative rods adhere.


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All oral bacteria possess surface molecules thatfoster some sort of cell cell interactionthrough the highly specific steriochemicalinteration of protein adhesinand carbohydratesaccharide receptors molecules located on thebacterial cell surface.

Adhesin -receptor interactions are mediated bythe 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.


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

Well -characterized interactions of secondary colonizerswith 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 toothsurfaces but adhere to bacteria already in the plaquemass.


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Thus coaggregation interactions are believedto contribute to development of biofilm by

two routes:

1. By single cells in suspension specificallyrecognizing & adhering to genetically distinctcells in the developing biofilm.

2. Second route by prior colonization in

suspension of secondary colonizer followedby subsequent adhesion of this coaggregateto the developing biofilm.


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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.


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Using a DNA-hybridization methodology , defined color-codedcomplexes of periodontal microorganisms that tend to befound together in health or disease have been identified.

The composition of the different complexes was based on thefrequency with which different clusters of microorganisms wererecovered, and the complexes were color-coded for easyconceptualization.

Socranskyet al1998


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The early colonizers are either independent of definedcomplexes (A. naeslundii , A. oris) or members of theyellow or purple complexes.

The secondary colonizers fell into the green, orange, orred complexes

The green and orange complexes include speciesrecognized as pathogens in periodontal and nonperiodontal 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 areflection of bacterial interdependency in the biofilmenvironment .

h l l h


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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 anaerated continuous flow reactor and measured various

physiologica properties by dyes and indicators.

DNA indicating the presence of bacterial cells wasdetected 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 activelygrowing cells would affect the outer layer of the

biofilm, but the remaining cells would not be affected.


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Water channels Water channels are commonly

found in biofilms, and thesecan form a primitive circulatorysystem.

Function:

1. Provide nutrients and oxygenfor the bacterial micro colonies

2. Facilitate movement of bacterialmetabolites, waste products,and enzymes within the biofilmstructure

3. Also create an appropriatephysicochemical environmentsuch as a properly reduced

oxidation reduction potential


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Interbacterial communication

Bacteria that live together in a biofilm are able tocommunicate 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 regulationof expression of specific genes through theaccumulation of signaling compounds that mediate

inter cellular communication" (Prosser 1999).


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It depends on cell density. With few cells,

signaling compounds may be produced at low

levels; however, autoinduction leads toincreased concentration as cell density

increases.


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Once the signaling compounds called theautoinducers reach a threshold level (quorumcell density), gene expression is activated insurrounding bacteria.

Quorum sensing can provide biofilms withsome of their characteristic properties, both in

terms of their development, and of theirgreater resistance to antimicrobials.


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It can promote the expression of genes that encodefor resistance to a certain antibiotic from a certain cell

density; it can also have the ability to affect biofilmstructure, stimulating the growth of beneficial speciesand inhibiting the growth of competitor species.

Quorum-sensing signaling molecules produced byputative periodontal pathogens such as P. gingivalis, P.intermediaand F. nucleatumhave been detected (Friaset al., 2001).

In general, autoinducers used by gram-negativebacteria are acylated homoserine lactones and thoseused by gram-positive bacteria are processedoligopeptides(Miller et al., 2001).


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Autoinducer-2 regulates iron (hemin) acquisition in the oralpathogensA. actinomycetemcomitansand P. gingivalis.

Autoinducer-2 quorum sensing is closely linked to theability of A. actinomycetemcomitans to grow in a biofilm.

Autoinducer-2-dependent quorum sensing was also shownto be important for mutualistic dual-species biofilmformation by Streptococcus oralis and Actinomycesnaeslundii (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 identifiedin the periodontal pathogen Eikenella corrodens (Azakamiet al., 2006).


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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.


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1. Slower rate of growth of bacterial species(biofilm persister cells) in biofilms, which makes

them less susceptible to many but not allantibiotics. The cells deep in the biofilmexperience different conditions such as hydrogenion concentration or redox potentials than cells atthe periphery of the biofilm or cells growing

planktonically.

In addition, the slower-growing bacteria oftenoverexpress nonspecific defense mechanismsincluding shock proteins and multi-drug effluxpumps and demonstrate increased exopolymersynthesis. (Gilbert et al.,1999)


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2. The exopolymer matrix of a biofilm, although not asignificant barrier in itself to the diffusion of

antibiotics, does have certain properties that canretard diffusion eg., strongly charged or chemicallyhighly reactive agents fail to reach the deeper zonesof the biofilm because the biofilm acts as an ion-

exchange resin removing such molecules fromsolution. (Gilbert et al., 1999)

3. In addition, extracellular enzymes such as -

lactamases, formaldehyde lyase and formaldehydedehydrogenase may become trapped andconcentrated in the extracellular matrix, thusinactivating some antibiotics.


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5. Alteration of genotype and/or phenotype of thecells growing within a biofilm matrix possibly dueto quorum sensing. Cells growing within a biofilm

express genes that are not observed in the samecells grown in a planktonic state, and they canretain this resistance for some time after beingreleased from 23 the biofilm.

For example, Brooun et al. (2000)demonstrated that cells of Pseudomonasaeruginosa liberated from biofilms were

considerably more resistant to tobramycin thanplanktonic cells, suggesting that the cellsbecame intrinsically more resistant when growingin a biofilm.


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6. Recently, the notion of a subpopulation of cells withina biofilm that are super-resistant was proposed.Such cells could explain remarkably elevated levels ofresistance to certain antibiotics that have beensuggested in the literature. Brooun et al. (2000)examined the contribution of multi-drug resistancepumps to antibiotic resistance of organisms grown inbiofilms. These pumps can extrude chemicallyunrelated antimicrobial agents from the cell. Since

extrusion places the antibiotics outside the outermembrane, the process offers protection againstantibiotics that target cell wall synthesis.


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Mechanism of Bacterial Resistance to Antimicrobial Agents

P th i P t ti l f O l


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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


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Treatment of periodontal biofilms

Of concern to the therapist in treating dental biofilm infections isthe fact that the pathogenic species exist in very large numbers, arewidely distributed within the oral cavity and exist in communitystructures that provide protection against host defense mechanismsand antimicrobial agents.

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

Periodontal therapies at the present time can be grouped in threebroad categories:

-mechanical debridement

-antiseptics and antibiotics

-those that affect the environment of the organisms


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Fortunately, biofilms in the oral cavity, unlike manyother biofilms, are readily accessible allowing theirphysical removal. Indeed, the most common form of

periodontal therapy is the removal of supra andsubgingival plaque by procedures such as selfperformed oral hygiene, scaling and root planing orperiodontal surgery.

Systemically administered antibiotics do have certaineffects on segments of the subgingival microbiota, butusually do not completely eliminate the sensitivebacterial species.

The systemically administered antibiotics may haveaffected, primarily, the organisms in the epithelial cellassociated biofilms and the loosely adherent adjacent

cells.


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The species in the tooth-associated biofilms mayhave been more resistant to the antibiotics dueto mechanisms described earlier, and thus thepotential for re-growth from this source wasperpetuated.

It might be surmised that physical removal of thetooth-associated biofilms prior to or during

antibiotic administration might minimize this re-growth.


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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.


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CONCLUSION Dental plaque is a naturally occurring biofilm that has

the potential to cause disease. Dental plaques havemany properties in common with biofilms found inother locations.

However, they have certain characteristics that areimportant in terms of control of disease. They areeasily accessible and thus allow direct removal andapplication of antimicrobial agents.

Changes in thinking about the structure of dentalplaque have improved our understanding of whyperiodontitis is so difficult to treat and will affect thestrategies used to prevent and control periodontitis in

the future.


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Properties of Biofilm