the scientific study of the relation of living organisms to each other and their surroundings (Gr)

Ecology

 life processes and adaptation

distribution and abundance of organisms

the movement of materials and energy through living

communities

the successional development of ecosystems, and

the abundance and distribution of biodiversity in

context of the environment

 Microbial ecology is concerned with the interrelationships

between microorganisms and their environment (ecosystem).

Ecosystem complex of organisms in a specified

environment and the nonmicrobial surroundings with which the organisms are associated.

Community organisms inhabiting a particular site. Habitat site at which a community grows, reproduces and

survives

Niche role of an organism in a habitat.

Single cell

Community

Ecosystem

 Oral cavity presents numerous

surfaces for colonization

Various habitats are available Bathed always by bulk fluid,

saliva, leading to biofilm formation.

Varying complexities

depending on location, genetic background and environmental factors.

Cyntia Rodrigues et al (2010)

 Complex of microbiota may

appear of 700 species being detected (Paster et al ).

Inter and intra individual

variations seen.

Paster et al (2001)

 Microbes exist as biofilm in

the oral cavity and various factors control their establishment, composition and their re-establishment once they get disturbed

 are relatively undefinable microbial community associated

with a tooth surface or any other hard, non-shedding material. (Wilderer and Charalkis 1989).
first identified with marine bacteria by Zobell. have community life style, differ physiologically from those

growing in a planktonic state.

Community life style ????

1. broader habitat range for growth, (e.g.) oxygen-consuming species create environmental conditions suitable for obligate anaerobes 2. Increased metabolic diversity and efficiency molecules that are normally toxic can be used by other bacteria 3. Enhanced resistance to stress, antimicrobial agents etc

4. Enhanced ability to cause disease organisms that cannot cause disease are able to do so when they are present as a consortium (pathologic synergism)
Marsh 2005

 Surface Bulk fluid Microbial community

 Surface influenced by the nature of the surface, genetic

background (which might alter the surface receptors), possible introduction of artificial surfaces, hygiene practices etc

Bulk fluid passes over the biofilm, providing nutrients to

the colonizing organisms, removal of waste products, and transport of cells to new colonizing sites.

 Over 700 species detected, many remain non cultivable Differ from one habitat to another, intra and inter

individually
Different in health and disease Community based living Communication exist

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)

Healthy gingivae
Streptococcus Actinomyces and Corynebacterium Veillonella and Neisseria

Constitute early colonizers Can withstand high oxygen concentrations and removal mechanisms Enables adhesion of subsequent bacterial species

Gingivitis
Strptococcus spp, Actinomyces spp Fusobacterium spp, Prevotela spp Campylobacter, Capnocytophaga

Anaerobes predominate due to decrease in oxygen levels

created by increased biofilm thickness

Periodontitis
Red complex

Fusobacterium, Campylobacter,Aggregatibact er,E.corrodens Streptococcus, Actinimyces spp

Viruses have also been detected

Increase in the amount of pathogenic organisms

Gingivitis
Health Gram positive Coccus Non motile Aerobes Saccharolytic

Periodontitis Gram negative Rods and spirochetes Motile Anaerobes Proteolytic

Microbial succession

Hypothesized relationship between the addition of species during microbial succession leading to the development of gingival inflammation. In turn, the increased inflammation would result in increased growth of colonizing species.

Implications in Periodontal disease

Specific plaque hypothesis of the diverse organisms in

the microlora, 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) .

I. Formation of pellicle II. Initial adhesion and attachment of bacteria III.Plaque colonization and maturation

Step I. Formation of pellicle

Selective adsorption of host molecules forming a

conditioning film (<1 m thick) by van der Walls, electrostatic and hydrophobic forces.
Contains proteins, glycoproteins like statherin, proline rich

proteins etc.
Usually <1m thick, takes 90-120 min for the adsorption.
Intended to protect the tissues from desiccation, act as receptors favoring bacterial attachment.

 Step II. Initial adhesion and attachment of bacteria

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.

 Adhesion between a substrate and bacteria can

attractive or repulsive depending on the net forces acting, can be explained by

DLVO (Derjaguin, Landau, Verwey and Overbeek) or Extended DLVO theory

 The DLVO theory has been used to describe the net

interaction (VTOT ) between a cell and a surface as a balance between two additive factors: VA resulting from van der Waals interactions (generally attractive) and repulsive interactions (VR) from the overlap between the electrical double layer of the cell and the substratum
Cannot explain all molecular interactions

 extended DLVO theory has been suggested in which the

hydrophobic/hydrophilic interactions are included. So, the total adhesion energy can be expressed as:

G adh= GvdW+ Gdl+ GAB

 Initial colonizers include S.sanguinis, S.oralis, S.mitis

(within minutes) followed by Actinomyces and Neisseria spp (about 2 hours). Obligate anaerobes are very rare.
embedded in the matrix.

These bacteria multiply forming micro colonies and get

This step involves firm anchorage between the bacteria

and components of acquired pellicle.

Cellular phase

 Involves specific adhesins on the bacteria with salivary

receptors on the pellicle.

can be:

direct interaction between bacteria and pellicle proteins bacterial proteins interacting with pellicle involvement of cryptitopes

Direct interaction between bacteria and pellicle proteins

Pili, fimbriae present on the bacterial cell can act as adhesins and bind to components of pellicle.

type I and II fimbriae of A.viscosus binding to proline rich

proteins on the pellicle. binding of PRP-1 to S.gordonii and A.viscosus.

Bacterial proteins interacting with pellicle

Lectin like bacterial proteins interacts with oligosaccharide

or pellicle associated glycoproteins facilitating adhesion.

Binding of S.oralis by galactose binding lectin trisaccharide

structure containing sialic acid, galactose and N-acetyl galactosamine. Binding of Actinomyces via galactoside.

Involvement of cryptitopes

Molecules undergo conformational change when they

adsorb to the tooth surface, exposing their new receptors, called as cryptitopes.

Binding of A.naeslundii with acidic PRP, only when the

latter adsorbed to the surface.

Cleaving of terminal sialic acid residue from

oligosaccharide side chain of glycoprotein exposing galactosyl sugar by A.naeslundii, S.oralis and S.mitis utilised by bacteria having galactosyl binding lectins like A.naeslundii, F.nucleatum, L.buccalis.

Factors limiting colonization

Available physical space Preemptive colonization prior colonization by one species

excludes another (colonization resistance).

Environmental resistance restriction in number of

individual species or biomass imposed by physical, chemical or biological factors of the ecosystem.
Combination of all these factors determine the members in each habitat

Step III. Plaque colonization and maturation

Multitude of interactions occur between bacteria as plaque

matures.

Both intra and inter generic interactions seen between early and

secondary colonizers.

Cell to cell interactions seen between various organisms.

Coaggregation leads to unusual combination of bacteria like

corn-cobs' (Gram-positive filaments covered by Gram positive cocci), 'rosettes (coccal bacteria covered by small Gram-positive curved rods), or 'bristle brushes' (large filaments surrounded by Gram-negative rods or short filaments)

 Co-aggregation is the

interaction between planktonic microorganisms 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

Early Colonizers

Includes Streptococcus spp, Actinomyces spp,

Veillonella spp, Hemophilus spp and Propionibacterium spp.

Streptococcus , only genus that have extensive intra

and inter generic co aggregation.

They bind to the pellicle and provide site for

attachment of organisms that cannot colonize pellicle.

F. nucleatum and Late colonizers

F. nucleatum categorized as bridging organism : 1.more numerous in healthy sites and they are found in increased number in diseased sites

2.coaggregates well with all early and late colonizers

3. provide ideal anaerobic condition for the growth of P.gingivalis
Kolenbrander 2002

Kolenbrander et al 2001

test-tube brush found in subgingival plaque

Tannerella sp. (yellow) in a test-tube brush

test-tube brush with Lactobacillus sp. (red rods) as central structures. F. nucleatum (green) and Bacteroides cluster filaments radiating from the central structures.

Transversal view of Streptococcus sp. (green) aggregation around a central cell (not stained) in supragingival plaque

Longitudinal and transversal view of a testtube brush

Transversal view of the test-tube brush in the above panel, composed of Synergistetes group A species.

Transversal view of supragingival plaque with Streptococcus sp (green cocci) and Candida albicans (green hyphae) in the top layer of the biofilm and forming corn cob structures growing outwards

Microcolonies with their long axis parallel to tooth surface (mainly g + ve cocci with few filaments)

Filaments on top of columns (1 wk)

Predominantly filaments without evidence of cocci (3 weeks) Corn cobs can be seen

Spirochetes covered the film (2mnths)

Listgarten 1976

Nishihara and Koseki

Structure

 microcolonies of single or

multiple species, clearly delineated by extracellular matrix.

Each microcolony consists of

progeny of cells resulting in mound like or mushroom like colonies

Shape depends on the shear

force; low mushroom or tower high - elongated

 Anastomosing network of

water channels penetrating at all the levels- primitive circulatory system

Penetration of oxygen

through the water channels as far as the colonized surface

Wood et al 2000

open architecture in supragingival biofilms

Polymer containing channels or pores linking the biofilm and tooth surface Bacterial vitality varies throughout the film; most viable in the center and lining the voids Heterogeneity in pH over relatively short distance facilitating survival of fastidious bacteria

Wood et al 2000

 Sub gingival plaque not viewed directly by confocal

microscopy due to limited access.

Light microscopy revealed a complex organization of

attached microorganisms in which there exist distinct tooth associated and epithelial associated biofilms.

Socransky & Haffajee (2002)

Supra and sub gingival biofilm

One surface (tooth)

3 surfaces (tooth, tissue

and zone intermediate)

Saliva: bulk fliuid GCF :bulk fluid

Microbes: increased

Microbes : increased

number of Actinomyces and S.sanguis

number of orange complex organisms

Socransky et al 1998

 Mean count of supra and sub gingival plaque increases

in periodontitis.

Increased number of

taxa in both the sites Actinomyces, green complex in supragingival films orange and red complex in subginigival films

 Bulk consist of matrix mixture of water (80-90%) and

solutes
Dry material - exopolysaccharides, proteins, salts and cell

material (bacteria).
Bacterial colonies (70%) more than 500 species have

been identified.
Non bacterial organisms like mycoplasma, yeasts, viruses

also detected.

Exopolysaccharides (EPS)
Exopolysaccharides (back bone)produced by the bacteria,

are the major components, making up 5095% of the dryweight .

EPS includes insoluble glucans, fructans and

heteropolymers.
can produce several different polysaccharides depending

on the physiological state and the presence of specific substrates.

 Glucans are synthesized by glucosyltransferase (GTF). GTFs

can be secreted or adsorbed onto other bacteria, acquired pellicle.

Fructans are produced by fructosyltransferases (FTF)

which are short lived and act as nutrient storage compounds for use by other bacteria.

 Some exopolysaccharides are neutral, such as the mutan

whereas others are highly charged polyanionic macromolecules.

Different ionic charge and concentrations will alter the

confirmation and cause rapid changes in the threedimensional gel network of polysaccharides.
Exopolysaccharides can exist in both ordered or

disordered forms. At high temperatures at very low ionic concentrations, the disordered form predominates,

 The exopolysaccharides can be degraded and utilized by

bacteria within the biofilm.

One distinguishing feature of oral biofilms is that many of

the microorganisms can both synthesize and degrade the exopolysaccharides

Functions of EPS

Maintain integrity of the biofilm Adhesive Confers protection Prevents dessication bind essential nutrients such as cations to create a local

nutritionally rich environment favoring specific microorganisms. act as a buffer and assist in retaining extracellular enzymes (and their substrates), enhancing substrate utilization by bacterial cells.

Biofilm Properties
Metabolic communication
Bacterial competitive interactions Barrier function

Genetic exchange
Cell-cell communication Detachment

Biofilm Properties
Metabolic communication
Bacterial competitive interactions Barrier function

Genetic exchange
Cell-cell communication Detachment

1. through the excretion of a metabolite by one organism that can be utilized by another organism 2. breakdown of a substrate by the extracellular enzymatic activity of one organism that creates biologically available substrates for different organisms.

S.oralis

hydrolysis of host glycoproteins

monosaccharides
utilization by other bacteria

gram positive rods

Desialylation of immunoglobulin A1

facilitate the proteolytic activities of other oral bacteria.

3. Formation of food chains

Food Chain (Helderman 1976)

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 o

ammonia by F.nucleatum elevating pH favorable for the growth of P.gingivalis

 Utilization of metabolic products for maintaining structural

integrity (eg) succinic acid produced by T.denticola integrated onto the cell wall 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

Biofilm Properties
Metabolic communication
Bacterial competitive interactions Barrier function

Genetic exchange
Cell-cell communication Detachment

 Both synergistic and antagonistic interactions seen

Antagonism can be mediated by metabolites or through

bacteriocins
Can influence localization of residents in the film

Bacterial synergism

Bacterial antagonism

Metabolic products

Streptococci produce hydrogen per oxide which are

toxic to many bacteria

S.oligofermentus

peroxide

lactic acid toxic to S.mutans

hydrogen

Short chain fatty acids like lactic acid lowers pH,

having a disadvantageous effect n 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

lantibiotics

 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

 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

Biofilm Properties
Metabolic communication
Bacterial competitive interactions Barrier function

Genetic exchange
Cell-cell communication Detachment

 In collaboration with each other, bacteria may confront

oxygen, host immunity and antimicrobial agents through dental biofilm.

Metabolism of aerobic and oxygen tolerant species reduce

the concentration of oxygen to levels that can be detoxified by obligate anaerobes (eg) F.nucleatum make the environment suitable for P.gingivalis.

 BIC of chlorhexidine and amine fluoride was 300 and 75

times greater respectively, when S.sobrinus was grown in biofilm compared to planktonic cells metronidazole than planktonic cells

Biofilms of P.gingivalis tolerated 160 times the MIC of

Scaling and root planing cornerstone of periodontal therapy

Mechanisms

 Restriction by ECM Slower growth thereby different phenotypes Drug inactivating enzymes itself or by neighbors
Increased biofilm mass

less effective concentration

altered phenotype Cells at the biofilm surface and the extracellular polymer matrix interact with antibacterials, with active groups reducing their effect Induction or repression of certain genes upon attachment to surface Expression of biofilm specific resistance genes (efflux pumps)

Biofilm Properties
Metabolic communication
Bacterial competitive interactions Barrier function

Genetic exchange
Cell-cell communication Detachment

 Interaction that could have major consequence for the

physiology of biofilms.
Can occur through conjugation, transduction and

transformation collectively called as horizontal gene transfer.

Mechanisms by which antibiotic resistance genes can be

transferred.

Mobile genetic elements

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.

Conjugation bacterium

plasmid

bacterium

Transduction bacterium bacteriophage bacterium Transformation DNA outside the cell is fragmented and combined with bacterial DNA

Biofilm Properties
Metabolic communication
Bacterial competitive interactions Barrier function

Genetic exchange
Cell-cell communication Detachment

 Also called as quorum sensing is a process of chemical

communication between bacteria.

Gene regulation in response to cell density, which influence

various function.
Most commonly quorum sensing molecules include CSP

and AI 2.

involves the regulation of expression of specific genes through the accumulation of signaling compounds that mediate intercellular communication (Prosser 1999)

 Quorum sensing is dependent on cell density. Less cell density

molecules

low level of signalling

increased level of molecules

reaching quorum cell density activation of gene expression

Auto Inducer-2

Autoinducer 2 (AI-2) was initially identified in Vibrio harveyi and

subsequently identified in other species.

Synthesis is catalyzed by LuxS, an enzyme encoded by luxS gene. Present in wide rage of gram positive and negative bacteria. Involved in protecting the organisms from environmental

stresses like temperature, hydrogen peroxide and pH; might have different functions in different organisms.

LuxS deficient P.gingivalis show decreased iron and hemin

uptake, decreased gingipain production.

CSP

Competence-stimulating peptide (CSP) is a small soluble

peptide having from 14-23 amino acid residues and is potentially produced by many species of streptococci.
Implicated in bacteriocin production, virulence and biofilm

formation.
Can also induce bacteriocin production.

Hojo 2009

Biofilm Properties
Metabolic communication
Bacterial competitive interactions Barrier function

Genetic exchange
Cell-cell communication Detachment

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

Dental plaque a biofilm????

 Model systems related to dental plaque biofilm culture

models
1. Non-biofilm culture systems 2. Microbial biofilm biochemical reactors 3. Intra-oral models plaque growth on plastic strips, under bands, on enamel attached to teeth,and to intra-oral appliances in different configurations

 Major oral biofilm culture system technologies

1. Chemostat-based systems 2. Growth-rate-controlled biofilm fermenter (GRBF) 3. Artificial mouths 4. The constant-depth film fermenter (CDFF)

 Biofilm mainly commensal with limited number of

pathogens
Selective inhibition and modulation of microbial

composition to be followed
Many under experiments

 Inhibiting adherence with antagonists

aim is to prevent the incorporation of potentially pathogenic organisms into biofilm. reducing the colonization by S.mutans can limit formation of biofilm to some extent. a dodecapeptide analogue of active binding site of SpaP protein of S.mutans available which inhibits attachment of S.mutans synthesized.

 Regulating the levels of non pathogenic bacteria to influence

virulence

incorporation of some organisms into biofilms is dependent upon other antecedent biofilm residents. identification of such dependencies of known pathogens and targeting these antecedent organisms. targeting strains of F.nucleatum to avoid the colonisation of late colonizers.

 Probiotic approaches

originally developed by Hillman to prevent dental caries. uses organisms that naturally occur in plaque and considered to be safe (GRAS). implantation of S.sanguis in plaque as they are associated with reduced periodontopathic organisms. implantation of lactobacillus spp.

 Targeted antimicrobial therapy

Eckert et al developed a novel technology for a new class of antimicrobials STAMPs (specifically targeted antimicrobial peptides). fusion peptide: killing moiety, non specific and targeting moiety, specific. pheromone produced by S.mutans, namely CSP as a STAMP targeting domain to mediate S.mutans specific delivery of antimicrobial peptide domain (Eckert 2006).

 Dental biofilms: difficult therapeutic targets.

Socransky &Haffajee Periodontology 2000, Vol. 28, 2002 Periodontal microbial ecology. Socransky &Haffajee Periodontology 2000, Vol. 38, 2005 Subgingival biofilm formation. Kuboniwa & Lamont. Periodontology 2000, Vol. 52, 2010 Microbial ecology of dental plaque and its significance in health and disease. Marsh. Adv Dent Res 8(2):263-271, July, 1994 Overview of microbial biofilms. Costerton. Journal of Industrial Microbiology (1995) 15, 137-140