BIOFILM

Dr. KRITIKA JANGIDMDS- Periodontics

What is biofilm..??

• The self-produced matrix of extracellular polymeric substance, which is also referred to as slime, is a polymeric conglomeration generally composed of extracellularbiopolymers in various structural forms.

Where do we find them..??

• One or more communities of micro-organisms

• Embedded in a glycocalyx that is attached to a solid surface.

• Allows micro-organisms to stick to and multiply on surfaces

STRUCTURE OF BIOFILM

Bacterial Microcolonies

• Mushroom shaped

• Diversity– Microbes– Environment• O2

• pH• Temperature

Extra Cellular Layer of Mucous

• Tight protective barrier that surrounds bacterial microcolonies

• Protects bacterial microcolonies from antibiotics, antimicrobials, and the body's immune system

Fluid Forces

• Saliva• Extensions from the main body of biofilms.• Can break free and to be swallowed, expectorated,

or form new biofilm colonies in other areas of the mouth.

• Cells clash to bacteria in biofilms.– Rapid spread of genes among bacteria– Leads to increased virulence of the bacteria and

antimicrobial resistance– Means that the bacteria are in constant evolution

Fluid Channels

• Direct the fluid in and around biofilms,– Nutrients and oxygen– Bacteria – Carry away bacterial waste

• Liquids include everything from saliva that any drinks consumed.

COMPOSITION OF BIOFILM

• Bacterial community- 20%

• Glycocalyx membrane- 80%

Exopolysaccharides

• Matrix made up of water and aqueous solutes• Produced by bacteria• Function– Integrity of biofilm- Adhesiveness– Affect the hydrophilic or hydrophobic nature of the

surface.– Aid in protecting microbial cells within the biofilm by

preventing desiccation and attack by harmful agents– Binds essential nutrients like cations to create a locally

rich environment favoring a specific microorganism

DENTAL PLAQUE

• Dental plaque can be defined as the soft deposits that form the biofilm adhering to the tooth surface or other hard surfaces in the oral cavity, including removable and fixed restorations

Carranza 10th ed.

TYPES OF PLAQUE

• SUPRA GINGIVAL

• SUB GINGIVAL

SUPRA GINGIVAL PLAQUE

• Originate from saliva• On the tooth surface: Gram

+ cocci and short rods predominate

• On the outer surface of matured plaque: Gram –ve rods and filaments and spirochetes predominate.

SUB GINGIVAL PLAQUE

TOOTH ASSOCIATED PLAQUE

• Predominated by gram +ve rods and cocci (S. mitis, A. viscosus, A. naslundi, Eubacterium)

• At the cementum: filamentous micro-organisms predominate

• At the apical border near junctional epithelium: Filamentous micro-oragnisms absent. More of gram –ve rods.

• Gram –ve rods and cocci• Filamentous bacteria• Flagellated rods• Spirochetes• Predominant are S. oralis, S. intermedius,

Peptostreptococcus micros, P.g, P. intermedia, T. forsythia, F. nucleatum)

• Host tissue cells

TISSUE ASSOCIATED PLAQUE

COMPOSITION OF DENTAL PLAQUE• 80% water• 20% solids, includes cells mainly bacteria making up 35%

of the dry weight and extra cellular components making 65% of the dry weight

• Other than bacteria, non bacterial organisms include:– Mycoplasma– Yeast– Protozoa– Viruses

• Host cells– Epithelial Cells– Macrophages– Leukocytes

Intercellular Matrix of Dental Plaque

Organic constituents

Inorganic constituents

Materials from Saliva, GCF and bacteria

ORGANIC CONSTITUENTS

• Polysaccharides: Dextran 95% (adhesion), levan 5%, Sialic acid, fructose

• Proteins: Albumin

• Glycoproteins: Saliva

• Lipid materials: Membrane remnants of bacteria and host cells

• Primarily: Calcium and Phosphates

• Traces: Sodium, Potassium and Fluoride

• Fluoride is derived from external sources like toothpaste, mouth washes

INORGANIC CONSTITUENTS

For

PLAQUE FORMATION AT ULTRASTRUCTURAL LEVEL

Formation of pellicle on the tooth surface

Initial adhesion and attachment of bacteria

Colonization and plaque maturation

FORMATION OF PELLICLE

• Within nanoseconds after vigorously polishing the teeth, a thin, saliva derived layer called the acquired pellicle covers the tooth surface.

• Consists of more than 180 peptides, proteins, glycoproteins, including keratins, mucins, proline rich proteins and other molecules which function as adhesive sites for bacteria.

Ultrastructure of Dental Pellicle

• Thickness- 30-100nm• 2 hr pellicle: Granular structures which form

globules, that connect to hydroxyapatite surface via stalk like structures

• 24 hrs later: Globular structures get covered up by fibrillar particles: 500-900 nm thick

• 36 hrs later: Pellicle becomes smooth and globular

• Studies of early (2 hr) enamel pellicle reveals that amino acid composition differs from that of saliva, indicating that the pellicle forms by selective adsorption of the environmental macromolecules. (Scannapieo FA et al, ‘Saliva and dental pellicles’ Complementary Periodontics, 1990

• Mechanisms involved are– Electrostatic forces– Van der Waals– Hydrophobic forces

Transport to surface

Initial adhesion

Attachment

Colonization of the surface and biofilm formation

Initial Adhesion and Attachment of Bacteria

Transport to the surface

• Random contacts occur through:

– Brownian motion (40µm/hr)– Sedimentation of organisms– Liquid flow– Active bacterial movement (chemotactic activity)

Transport to surface

Initial adhesion

Attachment

Colonization of the surface and biofilm formation

Initial Adhesion

• Reversible adhesion of the bacterium and the surface

• Initiated by interactions between bacterium and surface through long range and short range forces, including Van der Waals attractive forces and electrostatic repulsive forces

Transport to surface

Initial adhesion

Attachment

Colonization of the surface and biofilm formation

• First step involves transportation of bacteria. Few are mobile and the majority of the organisms are transported by the bulk fluid.

• Long range, between bacteria and pellicle coated enamel. The strength of this interaction is weak and span around 10-20 nm.

Physical phase

• Next step involves the closer movement of bacteria so that specific short range interaction occur.

• This is to happen if water is removed between the two

surfaces, brought about by bacterial cell components.

This initial attachment of bacteria to surfaces is the initial part of

adhesion, which makes the molecular or cellular phase of adhesion possible.

DLVO theory

• Derjaguin,Landau, Verwey & Overbreak (DLVO) have postulated that above a separation distance of 1nm, the summation of previous two forces describes total range interaction also called as total Gibbs Energy (GTOT).

• The result of summation (GTOT= GA+GE) is function of a separation distance between negatively charged particle and a negatively charged surface in a medium ionic strength suspension medium.

Three stages

1. Secondary minimum (reversible attraction)2. Positive maximum (energy barrier)3. Primary minimum (irreversible attraction)

• Extended DLVO theory has been suggested in which the

hydrophobic/hydrophilic interactions are included. So, the

total adhesion energy can be expressed as:

ΔG adh= ΔGvdW+ ΔGdE+ ΔGAB

• GEL is calculated from Zeta potentials of interacting surfaces.

• Calculation of G∆LW and G∆AB relies on contact angle

measurements.

• With various liquids on the interacting surfaces application of a

thermodynamic approach becomes essential.

• It considers free energy states of microorganisms in suspension

and in an adhering state.

• ∆Gadh is negative (nature tends to minimize free energy),

adhesion is thermodynamically favoured and will proceed

spontaneously.

ATTACHMENT• Firm anchorage

between bacterium and surface established by

specific interactions (ionic, covalent or hydrogen bonding)

Transport to surface

Initial adhesion

Attachment

Colonization of the surface and biofilm formation

• The bonding between bacteria and pellicle is mediated by specific extracellular protenaceous components (adhesions) of the organism and complementary receptors (proteins, glycoproteins, polysaccharides) on the surface (pellicle) and is species specific

These receptors are species specific .

Eg.1. Streptococci sangius ( early colonizer) binds to

acidic proline rich proteins and other receptors such as α amylase and saliac acid.

2. Actinomyces species posses fimbriae that contains adhesins that specifically bind to proline rich proteins of dental pellicle.

• CRYPTITOPES - They are hidden receptors for bacterial adhesins.-Some molecules from the pellicle undergoes a

conformational change when they adsorb to the tooth surface so that new receptors become available in adsorbed molecule .

Eg. Proline rich proteins undergo conformational changes A.viscous recognizes cryptic segment of proline rich proteins.

Colonization of the surface and biofilm formation

Colonization of the surface and biofilm formation

- Mainly by 2 mechanisms

COAGREGGATION.

COADHESION Transport to surface

Initial adhesion

Attachment

Colonization of the surface and biofilm formation

• Co-aggregation is the interaction

between planktonic micro-

organisms of a different strain or

species

• Co-adhesion is the interaction

between a sessile, already adhering

organism and planktonic micro-

oganisms of a different strain or

species

Co- Aggregation• Cell to cell recognition of a genetically distinct

partner cell type

• Occurs primarily through 1. Highly specific interaction of protiens and

carbohydrate molecules located on the bacterial cell surfaces.

2.Less specific interaction resulting from hydrophobic electrostatic & van der waals forces.

Primary Colonizers

• They provide new binding sites for adhesion by other oral bacteria

• Metabolic activity of primary colonizers modifies the local micro environment which influences the ability of other bacteria to survive in the dental plaque biofilm.

Late colonizers

• They do not initially colonize the clean tooth surface but adhere to the bacteria already in the plaque mass

• Co-aggregation -by lectin like adhesins.• Inhibition of co-aggregation- by lactose and

galactosides.

Eg. of co-aggregation between early colonizer & secondary colonizer:

• F.Nucleatum with Actinomyces viscous.• Capnocytophaga Ochraceus with A.Viscous

• Early colonizers like streptococci and actinomycetes are facultative anaerobes.

• These 2 groups of primary colonizers prepare a favorable environment for later secondary colonizers like P.intermedia, P.Loeschilli, Capnocytophaga spp , F.nucleatum, Porphymonas gingivalis which have a more fastidious growth requirements.

• During later stages of plaque formation co aggregation between different gram negative species is likely to predominate.

Eg. Coaggregation of F. nucleatum with P. gingivalis or T. denticola

Specificity of strains

• Streptococcal co-aggregation group 6 co-aggregates only with actinomyces co-aggregation group D.

Coaggregation Bridges:-A coaggregation bridge is formed when the common partner

bears two or more types of coaggregation mediators.-Mediators can be various types of receptor polysaccharides, or

various types of adhesins, or a mixture of the two.-Bridging is usually considered to be a cooperativeevent that brings three or more cell typesinto close proximity and fosters symbiotic relationships.-Bridging can also be an antagonisticevent which brings together organisms that competewith each other for nutrient or other needs.

COAGGREGATION COMPETITION: Competition occurs when multiple cell types recognize the

same coaggregation mediator on the common coaggregation partner.

Model depicting competition for binding sites on Streptococcus oralis .

Morphological types• Corncob formation:– Feature of plaque present on

teeth associated with gingivitis .

– A central gram negative filamentous core supports the outer coccal cells, which are firmly attached by interbacterial adherence or coaggregation.

– Detected between streptococcus and actinomyces

Described by Gibbsons and Nygaard

• Test tube brush: – Composed of a central axis

of a filamentous bacterium with perpendicularly associated short filaments.

– Commonly seen in the subgingival plaque of teeth associated with periodontitis

– Detected between filaments of bacteria to which gram –ve rods adhere.

Socransky et al (1998)

• Most periodontal sites either all or none of the species belonging to the same complex

• Red complex seldom detected in the absence of orange complex; higher the orange complex higher the red complex.

• Yellow and green cluster show similar preference for each other; weaker relation with orange and red complex.

• Purple complex, loose relation with all the other complexes.

• Yellow and green associated with sites with PD< 3mm and red orange complexes with sites having PD > 4mm

Distribution of different complexes in subgingival plaque sample Kigure et al (1995)

CO-ADHESION

• Some bacteria are unable to bind directly to the conditioning film,

but are able to interact with molecules on bacteria that are already

attached (co-adhesion), also by adhesin-receptor interactions.

• One bacterium, Fusobacterium nucleatum, can co-adhere with

almost all other bacteria found in dental plaque, and is considered

to be a key bridging organism between early and later colonisers.

• Adhesins can be subdivided into two main classes:– Fimbrial adhesins

• Fimbriae• Pili• Curli• Type IV pili

– Non fimbrial adhesins• Autotransporter• Outer membrane• Secreted adhesins

– Those attached with biofilm formation

Perio 2000, Vol 52, 2010, 12-37

Adhesins

• Fimbrial adhesins of gram negative are classified into 5 major classes based on the biosynthetic pathway.

1. Chaperone –Usher(CU )pili2. Curli3. Type IV pili4. Type III secretion pili5. Type IV secretion pili

Importance of food chains

• Provide metabolites which serve as energy source for other members. (eg) lactate utilization by Veillonella produced by streptococci

• Formation of symbiotic relationship

• Making the environment favorable (eg) generation of ammonia by F.nucleatum elevating pH

favorable for the growth of P.gingivalis

• Stimulation of growth of other bacteria (eg) stimulation of

growth of T.denticola by butyric acid produced by P.gingivalis

• Increasing the virulence of organisms (eg) more virulent

strains of P.gingivalis in the presence of S.gordonii.

• Removal of toxic metabolites (eg) protection from hydrogen

peroxide by A.neaslundii

• Utilization of metabolic products for maintaining structural

integrity (eg) succinic acid produced by T.denticola integrated

onto the cell wall of P.gingivalis

Bacterial competitive interactions

• Both synergistic and antagonistic interactions seen

• Antagonism can be mediated by metabolites or through

bacteriocins

• Can influence localization of residents in the film

Bacterial competitive interactions

Bacterial synergism

Bacterial antagonism

Metabolic products

• Streptococci produce hydrogen per oxide which are toxic to many bacteria

• S.oligofermentus lactic acid hydrogen peroxide toxic to S.mutans

• Short chain fatty acids like lactic acid lowers pH, having a disadvantageous effect and less aciduric bacteria.

Bacteriocins

• Proteinaceous bactericidal substances produced by bacteria to inhibit

the growth of closely related bacterial species or strains (Hojo et al

2009)

• Regulated by genetic and environmental factors

• Enable bacteria to select their neighbours, promote the

establishment of a community with specific bacterial species.

• Usually narrow spectrum with few exceptions like antibiotics

• Streptococcus produces mutacins (mutacin I – V) active against

S.sanguinis

• Inhibition of growth of P.gingivalis, T.forsythia, S.salivarius,

S.sanguinis by bacteriocin produced by L.paracaesi

• Nigrescin, produced by P.nigrescens display bactericidal effect

against P.gingivalis, P.intermedia, T.forsythia, Actinomyces spp.

• Bacteriocin production also reported by P.intermedia, A.a,

C.ochracea, F.nucleatum, E.corrodens, H.influenzae

Clinical significance

• Bacteria determine their neighbours

• Prevention of pathogenic biofilm formation bacteriocins produced by S.pyogenes and S.salivarius are structurally similar, antagonize each other when colonise at the same time, inhibit the growth of other via antagonizing growth dependent signaling, prevent the biofilm formation of the former by the latter.

• Maintain ecological balance

CHANGING VIEWS OF PLAQUE

• Specific plaque hypothesis– of the diverse organisms in the microflora,

only a very limited number are actively involved in the disease (Loesche

1976) .

• Non specific plaque hypothesis– many organisms play a role, and the

disease is a result of overall interaction of plaque and the host (Theilade

1986) .

• Ecologic plaque hypothesis– change in the key environmental factor(s)

will trigger a shift in the balance of resident flora, and this might

predispose to the disease (Marsh 1991)

• Microbial shift hypothesis - due to decrease in the no: of beneficial

symbionts and/or increase in the number of pathogen (dysbiosis)

De Novo Supragingival Plaque Formation: Clinical Aspects

• During 1st 24 hrs, starting from a clean tooth surface, plaque growth is negligible from clinical view point

• Following 3 days, plaque growth increases at a rapid rat, then slows down

• After 4 days, on average 30% of total crown area will be covered with plaque. Plaque does not seem to increase substantially after 4th day

Topography of Supragingival Plaque

• Initial along gingival margin and interdental space, then coronally

• Grooves, cracks, perikymata or pits

Surface micro-roughness

• Threshold: 0.2 micrometers

Variation within dentition

• Lowers• Buccal• Molars• Interdental

Impact of gingival inflammation

• Increased GCF- favours initial adhesion and colonization

Impact of Pts age

• No influence

De Novo Subgingival Plaque Formation

• Early studies, using culturing techniques examined changes in subgingival microbiota during 1st week after mechanical debridement, partial reduction followed by fast regrowth to almost pre treatment levels within 7 days.

• This reveals that a high proportion of treated tooth surfaces still harbored plaque & calculus after scaling, these remaining bacteria were considered primary source for subgingival recolonization.

Ageing & Microflora

• Following tooth eruption the isolation frequency of spirochetes & black pigmented anaerobes increases.

• Increased prevalence of spirochetes & black pigmented anaerobes is found in teenagers, this is due to hormones entering gingival crevice & acting as a novel nutrient source.

• Rise in P. intermedia in plaque during 2nd trimester of pregnancy has been ascribed due to elevated levels of oestradiol & progesterone which supplies napthoquinone for growth of this microorganism.

Signaling and biofilms

Definition

• It is defined as the cell density dependent regulation of gene expression in response to soluble signals called autoinducers (Bassler 1999)

• Quorum sensing can occur within a single bacterial species as well as between diverse species, and can regulate a host of different processes, essentially serving as a simple communication network.

Why the name quorum??

• Accumulation of a stimulatory concentration of an

extra-cellular autoinducer can only occur when a

minimum number of cells i,e critical cell density called

a “quorum,” is present.

Quorum sensing is dependent on cell density

Low level of signalling molecules

Increased level of molecules

Activation of gene expression

Less

cel

l den

sity

HOW BACTERIA TALK TO EACH OTHER:

Bacteria

Inducer Receptor

AI

Transcription of genes

• Quorum sensing-controlled behaviors are those that only occur when bacteria are at high cell population densities.

• These behaviors are ones that are unproductive when undertaken by an individual bacterium but become effective by the simultaneous action of a group of cells.

ROLE OF QS

There is an increase in

• Virulence and pathogenicity• Secondary metabolite production• Motility• Conjugation• Biofilm formation• Growth inhibition

Key players in quorum sensing

Autoinducers

•AHL•Autoinducer 2•Cyclic dipeptides•Bradyoxetin•Other types

Autoinducersynthases

•AHL synthases•AI2 synthases•Synthases of other types of autoinducers

Quorum sensingRegulators

•Lux R type•Lux P/Q type

Types of Quorum sensing molecules

Autoinducer 11st detected in Vibrio fisheri - by Nealson et al 1979

Chemically – N – Acyl Homoserine Lactone(AHL)Proteins involved are designated as Lux I & Lux R Lux I - Catalyses the synthesis of AHL Lux R - transcriptional regulatorAutoinducer 1 is not common in oral biofilm.It usually regulates gene expression in genetically identical cells.

Autoinducer 2

First observed by Schauder et al 2001

• Collection of molecules formed from spontaneous

rearrangement of 4,5 dihydroxy-2-3 pentanedione

(DPP)

• Produced by both gran +ve & -ve organism

• Gene responsible for it production - lux S - protein -

LuxS

• In the absence of two component response circuit (receptor

protein)

auto inducer does not function in cell-cell communication but

functions in basic metabolism - catalyses methyl cycle.

Autoinducer 2 mediate gene expression in mixed communities.

It is also density dependent

Commensal bacteria respond to low levels and pathogenic bacteria

respond to high levels of autoinducer 2

Other functions of autoinducer 2

1. Regulate iron uptake in Aa

2. Regulate hemin (iron source) acquisition in Pg.

3. Regulate enzymes involved in stress related function.

4. Control the formation of multi species biofilm.

5. Induces expression of leukotoxon in Aa and modulate protease activities in Pg.

• Transposons – elements capable of excision from the chromosome of the donor genome, transfer to recipient cell and get integrates its genome.

• Integron – gene cassette system – mechanism that allows bacteria to accumulate diverse genes at a common locus, useful in acquiring antibiotic resistance, are site specific recombinase of Intl family .

• Genomic islands – regions of genome acquired horizontally.

• Combination of these.

Mobile genetic elements

• Plasmid - It is an extra chromosomal genetic element consisting of DNA situated in the cytoplasm in free state and reproducing independently.

• They are grouped into incompatibility groups (inc groups) based on their inability to co-exist in the same cell.

• Bacteriophage - viruses that parasitize bacteria and consist of nucleic acid core and a protein coat.

Genetic exchange

Conjugation

Bacterium

Bacterium

Transduction

Bacterium

Bacterium

Transformation

DNA outside the cell is

fragmented and combined with

bacterial DNA

plasmid

bacteriophage

Detachment

Can be Movement of

Individual cells or Biofilm en masse

Individual Cell Transfer

• The detachment of cells from biofilm is essential to allow colonization of

new habitats by bacteria.

• Cells detach in different fashions.

Erosion - detachment of single cells in a continuous predictable fashion the

Sloughing - sporadic detachment of large groups of cells or

Intermediate process whereby large pieces of biofilm are shed from the

biofilm in a predictable manner, resulting in detached clusters

consisting of about 104 cells.

• The detachment rate was shown to be about six clusters per mm2 of surface per hour.

• Can be within the oral cavity and from subject to subject (bacterial translocation)

• Microbes show centers of spread called bacterial reservoir.

• Both horizontal (spouse to spouse for P.gingivalis) and vertical transmission (parent to child for A.a) has been demonstrated.

En masse transfer

• This possibility has been demonstrated in vitro studies of mixed biofilm that showed movement of intact biofilm structures across solid surfaces while remaining attached to them.

• Advantage in that formation of the biofilm is not reliant on planktonic cells, which are known to be more susceptible to antimicrobial agents .

Stoodley 1991

Negative regulators of QS

Anti- activator proteins• AHL degradation

enzymes• RNA dependant

regulation

Interference of QS e• Furanones• L-canavanine• Human

hormones

Bacterial components• Transgenic plants• Synthetic

analogues

Mechanism of small Quorum quenching inhibitors

synthetic AIPs

Interferes with the signal or decreases the receptor concentration

Triclosan and closantel

Inhibits enoyl-ACPreductase an

intermediate in AHLbiosynthesis

Structural mimics of QS inhibitors Enzyme inhibitors

Inhibitor of histidine kinase sensor

MICROORGANISMS ASSOCIATED WITH SPECIFIC PERIODONTAL

DISEASESPERIODONTAL HEALTH 102 to 103 bacteria. Certain bacterial species have been proposed to be beneficial to the

host, including S. sanguis, Veilonella parvula, and C. ochraceus(Carranza 10th)

Bacteria associated with periodontal diseases are often found in the subgingival microflora at healthy sites, although they are normally present in small proportions(Rose & Maeley, 6th)

Nonmotile nature.

GINGIVITIS 104 to 106 bacteria. Gram-negative bacteria. Compared with healthy sites, noticeable increase also occur in the

numbers of motile bacteria, including cultivable and uncultivable Treponemas (spirochetes).

Pregnancy associated gingivitis is accompanied by dramatic increases in levels of P. intermedia, which uses the steroid as growth factors(Carranza,10th )

CHRONIC PERIODONTITIS C. rectus, P. gingivalis, P. intermedia, F. nucleatum and T. forsythia

were found to be elevated in the active sites(Carranza,10th ) Sites with chronic periodontitis will be populated with greater

proportions of gram-negative organisms and motile bacteria. Certain gram-negative bacteria with pronounced virulence properties

have been strongly implicated as etiologic agents e.g. P. gingivalis and Tannerella forsythus.

LOCALIZED AGGRESSIVE PERIODONTITIS Gram -ve, and anaerobic rods. The most numerous isolates are several species from the

genera Eubacterium, A. naeslundii, F. nucleatum, C. rectus, and Veillonella parvula.

In some populations, a strong case can be made for Aa playing a causative role in LAP, especially in cases in which patients harbor highly leukotoxic strains of the organism.

However, some populations of patients with LAP do not harbor Aa, and in still others P. gingivalis may be etiologically more important.

GENERALIZED AGGRESSIVE PERIODONTITIS

The sub-gingival flora in patients with generalized aggressive peri odontitis resembles that in other forms of periodontitis.

The predominant subgingival bacteria in patients with generalized aggressive periodontitis are P. gingivalis, T. forsythis A. actinomycetemcomitans, and Campylobacter species.

REFRACTORY CHRONIC PERIODONTITIS

Unusually diverse and may contain enteric rods, staphylococci, and Candida.

Persistently high levels are found of one or more of P. gingivalis, T. forsythis, S. inter-medius, P. intermedia, Peptostreptococcus micros, and Eikenella corrodens.

Persistence of Streptococcus constellatus has also been reported.

NECROTIZING ULCERATIVE GINGIVITIS/PERIODONTITIS

More than 50% of the isolated species were strict anaerobes with P. gingivalis and F. nucleatum accounting for 7-8% and 3.4%, respectively.

PERIODONTAL ABSCESSES

The bacteria isolated from abscesses are similar to those associated with chronic and aggressive forms of periodontitis.

An average of approximately 70% of the cultivable flora in exudates from periodontal abscesses are gram-negative and about 50% are anaerobic rods.

Periodontal abscesses revealed a high prevalence of the following putative pathogens: F. nucleatum (70.8%), P. micros (70.6%), P. intermedia (62.5%), P. gingivalis (50.0%), and T. forsythis (47.1%).

Enteric bacteria, coagulase-negative staphylococci, and Candida albicans have also been detected.

PERIIMPLANTITIS High proportion of anaerobic gram negative rods, motile

organisms, and spirochetes). Species such as Aa, Pg, Tf, P. micros, C. rectus, Fusobacterium,

and Capnocytophaga are often isolated from failing sites. Other species such as Pseudomonas aeruginosa,

enterobacteriaceae, Candida albicans and staphylococci, are also frequently detected around implants.

FIGHTING ORAL BIOFILMS-ADJUNCTIVE TREATMENTS

• Scaling and root planing is the primary therapy of choice for most clinicians, and it is widely considered the gold standard for treating periodontitis.

• However,scaling and root planing alone often does not produce the clinical outcomes desired in severe cases.

OTHER ADJUNCTIVE THERAPY ARE:1. Antibiotics2. Antiseptics3. Host modulation therapy.4. Photodynamic therapy.

Antibiotic resistance - characteristic feature of biofilm

• BIC of chlorhexidine and amine fluoride was 300 and 75 times

greater respectively, when S.sobrinus was grown in biofilm

compared to planktonic cells

• Biofilms of P.gingivalis tolerated 160 times the MIC of

metronidazole than planktonic cells

Scaling and root planing cornerstone of periodontal

therapy

Mechanisms

.

•The biofilm matrix may restrict the penetration of a charged antimicrobial agent (diffusion-reaction theory)

• Agent may also bind to, and inhibit, the organisms at the surface of the biofilm, leaving cells in the depths of the biofilm relatively unaffected.

•The novel phenotype expressed in a biofilm may result in the drug target being modified or not expressed, or the organism may use alternative metabolic strategies

• Bacteria replicate only slowly in an established biofilm and, as a consequence, are inherently less susceptible than faster dividing cells.

• In addition, samples of gingival crevicular fluid (GCF) can contain sufficient ß lactamase to inactivate the concentrations of antibiotic delivered to the site

• A susceptible pathogen can be rendered resistant if neighbouring, non-pathogenic cells produce a neutralising or drug-degrading enzyme.

Can viruses form biofilms?

• The recent finding that the human T-cell leukemia virus type 1 (HTLV-1) encases itself in a carbohydrate-rich adhesive extracellular ‘cocoon’, which enables its efficient and protected transfer between cells, unveiled a new infectious entity and a novel mechanism of viral transmission.

• These HTLV-1 structures are observed at the surface of T cells from HTLV-1-infected patients and are reminiscent of bacterial biofilms.

• The virus controls the synthesis of the matrix, which surrounds the virions and attaches them to the T cell surface.

• Similar to bacterial biofilms, viral biofilms could represent ‘viral communities’ with enhanced infectious capacity and improved spread compared with ‘free’ viral particles, and might constitute a key reservoir for chronic infections.

Maria-Isabel Thoulouze, Andrés Alcover

Bacteriophage and Biofilms• Phages along with other

viruses can become trapped non-specifically within biofilm extracellular polymeric substances (EPS).

• Can degrade these polymers

• Can infect bacteria that can go on to making up biofilms

Conclusion

In order to adjust a complex microbial ecosystem to one

that is compatible to health, it is essential to define the

range of species that colonize the area, recognize their

relationship with each other and the host, and develop

effective strategies to guide these ecosystems to those

compatible with long-term oral health.

REFERENCES

-Clinical Peridontology 10th edition: Carranza, Neuman& Takei.

-Peridontology 2000 Vol 38 2005.-Peridontology 2000 Vol 42 2006.-Peridontology 2000 Vol 52 2010.-Peridontology 2000 Vol 55 2011.