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ORAL MICROBIAL FLORAPRESENTED BY : DR. Priya sathyamurthy1CONTENTSIntroductionTaxonomy terminologiesClassificationEcosystem Oral microfloraOral econichesEcological relationshipsMicrobial complexesColonization of micro-organisms in the form of biofilmSpecific bacteria in periodontal diseasesAssociation of plaque microorganisms with periodontal diseaseConclusionReferences

Micro organism A microorganism (from the Greek: mikros, "small" and organisms, "organism") is a microscopic organism, which may be a single cell or a multi-cellular organism.Single-celled microorganisms were the first forms of life to develop on Earth, approximately 34 billion years ago.The possibility that microorganisms exist was discussed for many centuries before their discovery in the 17th century.

Taxonomy TaxonomyClassification of living organisms into groupsPhylogenetic Classification System:Groups reflect genetic similarity and evolutionary relatednessPhenetic Classification System:Groups do not necessarily reflect genetic similarity or evolutionary relatedness. Instead, groups are based on convenient, observable characteristics.

Terminologies Species: A collection of microbial strains that share many properties and differ significantly from other groups of strainsStrain: A culture derived from a single parent that differs in structure or metabolism from other cultures of that species

Type strain: One of the first strain among the species, studied and fully characterised than other strains.Biovars - prokaryotic variant strains characterized by biochemical or physiological differencesMorphovars - prokaryotic variant strains characterized by morpholigical characteristicsSerovars - prokaryotic variant strains characterized by distinctive antigenic properties

Symbioses: both partners benefitCommensalism : one partner benefits, other is neutralParasitism: one partner benefits while the other is harmedMutualism: different organisms living together

7Mechanism of pathogenesis

TransmissionAdherenceInvasionColonizationDamageExitSurvival

Prokaryotes

Prokaryotes are organisms that lack a cell nucleus and the other membrane bound organelles. They are almost always unicellular.Consisting of two domains, bacteria and archaea.Cell structure OF PROKARYOTE1.- flagella 2.- pili (fimbriae)3.- capsules 4.- slime layer

BACTERIAThey lack a nucleus and other membrane-bound organelles, and can function and reproduce as individual cells, but often aggregate in multicellular colonies. Their genome is usually a single loop of DNA.

ARCHAEA Archaea differ from bacteria in both their genetics and biochemistry. For example, while bacterial cell membranes are made from phosphoglycerides with ester bonds, archaean membranes are made of ether lipids.EUKARYOTESMost living things that are visible to the naked eye in their adult form are eukaryotes, including humans.Unlike bacteria and archaea, eukaryotes contain organelles such as the cell nucleus, the Golgi apparatus and mitochondria in their cells.

Shapes

GRAM (+) OR (-) ?PEPTIDOGLYCAN LAYER :

EXCEPTIONS TO GRAM STAINING1.- MYCOBACTERIUM : ACID FAST2.- SPIROCHETES : DARK FIELD3.- MYCOPLASMA : NO CELL WALL

ATMOSPHEREAEROBIC : That can survive and grow in an oxygenated environment.ANAEROBIC : That does not requireoxygenfor growth.

Amicroaerophileis amicroorganismthat requiresoxygento survive, but requires environments containing lower levels of oxygen than are present in theatmosphere.Capnophiles, require an elevated concentration ofcarbon dioxide to survive. Eg:Campylobacter spp.AnaerobicObligate anaerobes, which are harmed by the presence of oxygen. Bacteroides, Clostridium, Fusobacterium,, Porphyromonas, Prevotella,

Aerotolerant organisms, which cannot use oxygen for growth, but tolerate its presence

Facultative anaerobes, which can grow without oxygen but use oxygen if it is present. Staphylococcus spp., Streptococcus spp., Escherichia coli, Listeria spp.

TEMPERATUREPSYCHROPHILIC: Temperatures below 15 degrees.MESOPHILIC: temperatures between 20 -45 degrees.THERMOPHILIC : temperatures above 60 degreesBACTERIAL METABOLISMpH :Neutrophil 6 8Acidophil 3Alkalophil 10.5AEROBIC GRAM (+) COCCISTAPHYLOCOCCUSSTREPTOCOCCUS AEROBIC GRAM (-) COCCINEISSERIASALMONELLAESCHERICHIAVIBRIOHELICOBACTERBRUCELLA

29Lancefield groups : A to V without I and T.29Ecological niche & ecosystemAnicheis a term describing the way of life of a species. Each species is thought to have a separate, unique niche. The ecological niche describes how an organism or population responds to the distribution of resources and competitors and how it in turn alters those same factors for its benefit. The ecosystem is composed of microbial communities living on specific sites surrounded by a different physical and chemical elements.

Definitions of Hutchinson: The set of biotic and abiotic conditions in which a species is able to persist and maintain stable population sizes."

Ways of obtaining and using nutrientsNutritionAutotrophic nutritionHeterotrophic nutritionHolozoic nutritionSaprophytic nutritionParasitic nutritionOral microfloraOral microflora refers to the community of microorganisms coexisting in the oral cavity as its primary habitat; These strains of bacteria colonize the various different surfaces present in the oral cavity, and communicate between each other through complex cell signaling processes; Over time as the individual is further exposed to external sources of bacteria, the biodiversity of the oral cavity increases, to a point where stability is reached. This is termed the climax community.

The source of microorganismsAt birth the oral cavity is sterile but rapidly becomes colonized from the environment, particularly from the mother in the first feeding.Streptococcus salivariusis dominant and may make up 98% of the total oral flora until the appearance of the teeth (6 - 9 months in humans). Majority of children obtain their resident microflora from their mothers, as they often possess identical strains of bacteria; This is known as vertical transmission; Horizontal transmission also takes place as children interact with their peers, and later in life between spouses and partners. Bacteria during the life cycleOral colonization begins in the birth canal:Populations on the tongue and mucosa;Established during infancy - include anaerobes;Tooth eruption provides non-shedding surfaces:The window of infectivity concept;Colonization from source sites and caregiver saliva;

Hormonal shifts - puberty and pregnancy:Can alter proportions of Gm- anerobes;Complete loss of teeth shifts flora towards infant state:Dentures restore supragingival non-shedding sites;Implants restore supra- and subgingival sites.

Oral flora changes with ageTime during a lifetimeMAJOR COMPONENTS & CHANGES IN ORAL FLORANewborn

Oral cavity sterile. Soon colonised by facultative and aerobic organisms; esp S. salivarius

6 months

Flora becomes more complex & includes anaerobic orgs eg. Veillonella sp. & Fusobacteria Tooth eruption

Increase in complexity. S sanguis, S mutans and A viscosus appear. New habitats include hard surfaces and gingival crevice.

Child to adult

Various anaerobes frequently found inc. Members of the Bacteroidaceae. Spirochaetes isolated more frequently

Loss of teeth

Disappearance of S mutan, S sanguis, spirochaetes and many anaerobes

Dentures etc

Reappearance of bacteria able to grow on hard surfacesFormation of the ecosystemThe development of microbial community comprises an alternation of populations;The process starts with the colonization of the environment of the first microbial population;It fills the new space, modify it and makes it convenient for resettlement of new microbial population.Growing microbial community was enriched with different species living in complex;The process of alternation of populations and enrichment of the community stops only when niches for colonization of new microbial species are unavailable.

Diverse ecological niches in the oral cavityThe heterogeneity of tissue types in the oral cavity, such as teeth, tongue and mucosa, means that a variety of sites are available for colonization by oral microorganisms;Each site has unique characteristics and allows those microorganisms best suited to the environment to inhabit the site. Dynamic Equilibrium:

(1) Swallowing, Mastication, Or Blowing The Nose(2) Tongue And Oral Hygiene Implements(3) The Wash-out Effect Of The Salivary, Nasal, And Crevicular Fluid Outflow(4) Active Motion Of The Cilia (Nasal And Sinus Walls).

Most organisms can only survive in the oropharynx when they adhere to either the soft tissues or the hard surfaces (teeth, dentures, and implants).40From an ecologic viewpoint, the oral cavity, which communicates with the pharynx, should be considered as an open growth system with an uninterrupted ingestion and removal of microorganisms and their nutrients.

40Oral cavity divided into 5 major niches41Oral ecological zonesMostly the same species are present, but proportions may differ;High biomass sites:Non-shedding surfaces:Supragingival tooth surfaces;Subgingival tooth surfaces;Shedding surface:The tongue;Low biomass (reservoir) sites;Shedding oral mucosal surfaces:Buccal, palate, external gingiva, floor of mouth;Saliva as a transitional zone.Oral econichesHard structures teeth, providing various locations for colonization:Subgingival;Supragingival;Soft structures mucosa:Cheeks, lips, tongue, gingiva, palate;Keratinized and nonkeratinized mucosa;Epithelium of the gingival sulcus.Mucosal Surfaces:These include the palate, cheek and tongue, which have cells which are constantly replaced due to the normal wear-and-tear of the mouth; The different mucosal surfaces also have different properties which contribute to the presence of different types of bacteria:

The presence of the numerous crypts on tongue allows for bacteria to be protected from the normal shedding and removal by saliva flow, Hence species not found elsewhere, such as obligate anaerobes, can be found on the dorsum of the tongue.

Mucosal reservoir sitesSome oral species can invade epithelial cells:This requires communication between bacteria and cells;Bacteria subvert the cell to take them in:Take control of the cytoskeleton;Can live and grow inside;Can direct the cell to export them to other cells;Multi-species intracellular flora resembles mixed biofilm.

Mucosa of the gingiva, palate, cheek, floor of the mouthStreptococcus:S. oralis, S. sanguis;Neisseria;Haemophilus;Veillonella.

Surface of the tongueStreptococcus salivarius, S. mitis;Veillonella spp.;Peptostreptococcus spp.Gram + rods - Actinomyces spp.;Gram - rods - Bacteroides spp.;Obligate anaerobes black - rods and spirochetes associated with periodontal diseases.Dental econichesSUPRAGINGIVAL PLAQUE:Gram positive MO;Facultative anaerobes:Streptococcus;Actinomyces;Gram negative:Veillonella, Haemophilus, BacteroidesOn approximal surfaces;On occlusal surfaces - fissures, pits and grooves;Cervical;

47Subgingival plaqueFrom healthy gingival sulcus isolate:Gram negative rods:Porphyromonas gingivalis;Porphyromonas endodontalis;Prevotella melaninogenica;Prevotella intermedia;Prevotella loesheii;Prevotella denticola.Subgingival tooth surfacesThey are the narrow crevice between gingival epithelium and cementum

They have low oxygen tension which is favorable for Gm- anaerobes;Major site for interaction between bacteria and host tissues;Species mix varies between each side and the center which form distinct microenvironments.Oral fluidsOral surfaces washes of two liquids:Saliva;Gingival fluid;They are the basis for maintaining oral ecosystem:Provide water;Nutrients;Adhesion;Antimicrobial factors.Microorganisms in salivaMicrobial composition of saliva is the most similar to the tongue.By acting as a buffer, saliva maintains the pH of the mouth, ensuring the optimal growth of the resident colonies;Most of the microbes present in the mouth utilize the glycoproteins and proteins in the saliva as their main source of nutrition; Proteins and glycoproteins of saliva are responsible for the formation of the pellicle.

Salivary flowIt is responsible for the removal of non-endogenous bacteria which is unable to adhere to specific sites in the mouth; Areas which receive markedly less saliva flow, such as deep gingival crevices, proximal spaces and occlusal fissures, tend to have significantly higher levels of bacterial buildup;Saliva is carrying bacteria - the main source of microbial transmission between individuals;Saliva also circulates bacteria within the oral cavity, resulting in re-colonization of oral surfaces where the microflora might be removed via mechanical forces such as cleaning.

Gingival crevicular fluid:The flow of this fluid removes foreign microbes which do not adhere to these surfaces;For the resident population, the flow of crevicular fluid provides these organisms with a source of nutrition.Normal - resident floraIn a healthy body, the internal tissues - blood, brain, muscle, etc., are normally free of microorganisms;However, the surface tissues - skin and mucous membranes, are constantly in contact with environmental organisms and become readily colonized by various microbial species;The mixture of organisms regularly found at any anatomical site is referred to as thenormal flora or resident flora, some researchers prefer the term "indigenous microbiota".

Resident microfloraTypical microflora of a econiche;Microorganisms are separated and grouped according to the different conditions of life;Resident microflora has an important function in host:Digestive and nutritional;Competition with pathogenic microflora.PATHOGENICITY OF MICROFLORAMicroflora usually is non-pathogenic and form an integral part of the host;The presence of friendly resident microorganisms on oral surfaces contributes to the bodys defense against foreign pathogens, which are generally transient and can give rise to harmful infections.This is known as colonization resistance;

Function of MicrofloraColonization resistance is firstly achieved through the saturation of oral surfaces with preexisting resident bacteria, which reduces the available sites left for the attachment of exogenous organisms;Essential nutrients derived from the saliva and various proteins in the oral cavity are also more effectively utilized by the resident microflora, which inhibits infections via competition for resources;

The normal bacterial flora of the oral cavityBenefit from the host who provides nutrients and habitat; Occupy available colonization sites which makes it more difficult for other microorganisms to become established; Contribute to host nutrition through the synthesis of vitamins, and they contribute to immunity by inducing low levels of circulating and secretory antibodies that may cross react with pathogens; Exert microbial antagonism against exogenous species by production of inhibitory substances such as fatty acids, peroxides and bacteriocins.TRANSIENT MICROBIOTA

Transient microbes are just passing through;Although they may attempt to colonize the same areas of the body as do resident microbiota, transients are unable to remain in the body for extended periods of time due to:Competition from resident microbes;Elimination by the bodys immune system;Physical or chemical changes within the body that discourage the growth of transient microbes.

OPPORTUNISTIC MICROBES

Under normal conditions, resident and transient microbes cause the host no harm; However, if the opportunity arises, some of these microbes are able to cause disease and become opportunistic pathogens. This can happen due to a number of different conditions:When the immune system is compromised or suppressed, normal flora can overpopulate or move into areas of the body where they do not normally occur;When the balance of normal microbes is disrupted, for example when a person takes broad spectrumantibiotics, microbes that are normally crowded out by resident microbes have an opportunity to take over; Disease can result when normal flora are traumatically introduced to an area of the body that they do not normally occur in.

ENDOGENOUS MICROFLORA

That is microflora already present in the body, but has previously been inapparent or dormant.EXOGENOUS MICROFLORAExogenous bacteriaare microorganisms introduced to closedbiological systemsfrom the external world;They exist in aquatic and terrestrial environments, as well as the atmosphere;Exogenous bacteria can be either be benign or pathogenic.Ecological relationshipsIndependenceLife free from influences, management or control of other organisms.DependenceMICROBIAL RELATIONSHIPSThere are a complex of relationships between different microbial species:Neutral;Antagonistic microbial interactions;Synergistic.SynergyRelationships between the two microbial species living together, wherein both are mutually favored, leading to enhancement of the effects of each of these.Example of synergyVarious microorganisms may cooperate to use substances which they are not able to metabolise themselves;P. gingivalis and F. Nucleatum both can hydrolyze casein.NEUTRALRelationship between two species living together without affecting each other - neither favorable nor unfavorable.AntagonismRelationship of two microbial species living together, yielding inhibition of their function, and less effect on the independent operation of each one of them. Mechanisms of microbial antagonismCompetition for adhesion receptors or for nutrients;Production of inhibitory substances:Organic fatty acids;Hydrogen peroxide:Dairy complex;Antibiotics;Enzymes;Bacteriocins.An example of microbial antagonismS. mutans and Lactobacillus produce lactic acid thereby producing an acidic environment;It inhibits the growth of S.sanguis and S. oralis, and gram negative microorganisms;Separation by bacteriocins inhibit gram positive microorganisms.Influence of external factorsDiets : Food high in carbohydrates increases the development of Str. mutans and Lactobacillus;A diet high in protein inhibits the development of Lactobacillus.

Oral hygiene and antimicrobial agents : Good oral hygiene:Facultative aerobes;Acidogenious microorganisms;

Neglected oral hygiene:Anaerobes;Proteolytic microorganisms.Drugs and diseases.Antimicrobial agentsFluorides have depressing effect on microbial attachment.Reducing sugar transport;Reduce their glycolytic activity;Suppress the acid tolerance of gram + microorganisms,DRUGS AND DISEASESPatients with reduced salivation have reduced capacity to remove sugars, reduced buffering capacity.Antibiotics suppress the resident microflora, leading to over-development of antibiotic resistant species - Candida and allow colonization of exogenous pathogens such as Enterobacteriaceae.Beneficial effect of the microflora on the bodyPrevent exogenous and endogenous infections;Stimulate an immune response;Help in renewal and healing of the epithelium;Deliver certain biological factors to the host.ADVERSE EFFECTSSource of endogenous infection;Change the local environment to facilitate exogenous microbial overdevelopment.Create hypersensitivity in organism to microbial agents.HUMAN ORAL FLORA GRAM-POSITIVE FACULTATIVE COCCI GRAM-NEGATIVE FACULTATIVE RODSStaphylococcus epidermidis Staph. aureusStreptococcus mutansStrep. sanguis Strep. MitisStrep. Salivarius Strep. FaecalisBeta-hemolytic streptococci EnterobacteriaceaeHemophilus influenzaeEikenella corrodensActinobacillusActinomycetemcomitans GRAM-POSITIVE ANAEROBIC COCCI GRAM-POSITIVE ANAEROBIC RODSPeptostreptococcus sp Actinomyces israelii A. odonotolyticusA. ViscosusLactobacillusGRAM-NEGATIVE ANAEROBIC COCCI GRAM-NEGATIVE AEROBIC OR FACULTATIVE COCCI DiphtheroidsCorynebacterium Eubacterium Neisseria siccaN. Flavescens

SPIROCHETESYEASTSTreponema denticolaT. Microdentium

Candida albicansGeotrichum sp.

ProtozoaMycoplasmaEntamoeba gingivalisTirchomonas tenaxMycoplasma oraleM. pneumoniaeImportant Oral Bacteria1. Gram Positive organisms: Rods (bacilli), cocci or irregular shape (pleomorphic); Oxygen tolerance varies from aerobes to strict anaerobes; Most are fermentative; Cell wall has thick peptidoglycan layer (penicillin has effect by interfering production of this layer).Three important genera:Actinomyces is facultative anaerobe; Lactobacillus, produce lactic acid, facultative anaerobe, role in dentine caries rather than enamel caries; Streptococcus is facultative anaerobic cocci which produces lactic acid Some are implicated in caries.ImportanCE of Streptococci in the oral and their propertiesImportant Oral Species Growth on hard surfaces

Pro-duction of Insol. Extra-cellular Polysa-ccharide

Pro-duction of Acid Cariogenic

Endo-carditis isolates

S mutans +++++++S sanguis +++++++S mitis+_+_++_+++S milleri+_+++S salivarius__+__Distribution of Streptococci in the oral cavitySpecies Cheek Tongue Saliva Tooth

S.mutans __+/-++S. mitis++++++++++++S. salivarius _++++_2. Gram Negative organismsMany Gram-negative bacteria are found in the mouth, especially in established/subgingival plaque; Mainly consist of Cocci, rods, filamantous rods, spindle shaped or spiral shaped; Range of oxygen tolerance but most important strict or facultative anaerobes; Some fermentative, produce acids which other organisms use acids as an energy source, others produce enzymes which break down tissue;

Most important Gram negative bacteria: Porphyromonas: P. gingivalis Prevotella: P. intermedia Fusobacterium: F. nucleatum periodontal pathogen; Actinobacillus/Aggregatibacter: A.actinomycetemcomitans associated with aggressive periodontitis; Treponema: group important in acute periodontal conditions i.e ANUG; Neisseria; Veillonella.Flora of normal, healthy dentate mouth % (approx) Bacteria 85% Streptococci Veillonella Gram positive Diptheroids Gram negative anaerobic rods

5-7% Neissaeria

2% Lactobacilli

1% Staphylococci & Micrococci Remainder

Other bacteria, fungi, protozoa & viruses FUNGI IN THE ORAL CAVITYMost commonly found :- candida species (C.albicans, C. tropicalis, C. stellatoidea, C. parapsilosis, C. guilliermondi)Other rhodotorula & torulopsis ( denture wearer)Conditions thrush ,erythematous candidiasis,hyperplastic candiasis,angular chelitisMedium used sabouraands agar(peptone-glucose)Indentification psuedohyphae, septate hyphae and germ tubes

77PARASITES IN THE ORAL CAITYENTAMOEBA GINGIVALISTRICHOMONAS TENAX

E. gingivalis found in soft calculus,periodontal pockets and infection of tonsilsCan become opportunistic pathogen T.tenax only parasitic flagellate in oral cavity --number increases in periodontitis

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Factors Affecting Growth of Microorganisms in the oral cavity

1. Temperature; 2. REDOX Potential / Anaerobiosis; 3. pH; 4. Nutrients (endogenous & exogenous (diet); 5. Host Defences (Innate & Acquired immunity); 6. Host genetics (changes in immune response etc); 7. Antimicrobial agents & inhibitors. TEMPERATURERelatively constant - 34-36 ;The temperature is variable on the teeth and mucosa;When microorganisms colonize they are exposed to extreme temperatures;PH, OR HYDROGEN ION CONCENTRATIONIn the mouth pH varies between 6.7 and 7.3; It is maintained by saliva through salivary flow and buffer systems;In an acid medium in the eco niches following are developed:Lactobacillus;Str. mutans;In the gingival sulcus pH is alkaline -7.5 - 8.5; In the gingival fluid pH is 7.5 7.9 . Redox potential and anaerobiosisUnder the action of the enzymes some of the components are subjected to oxidation, and others to reduction;These processes depend on the oxygen and their redox potential;In a predominance, reduction processes have a negative redox potential and develop anaerobic microorganisms;Positive redox potential are seen in buccal and palatal mucosa and the posterior part of the tongue;Negative redox potential are seen in approximal surfaces, fissures and gingival sulcus.NUTRIENTSDesquamated epithelial cells;Gingival fluid;Saliva;Residues from host`s food;Products of metabolism of other microbial species.HOST FACTORSHost defense mechanisms;Hormonal changes;Stress;Genetic factors.Relationships with the hostHost defenses in the mouth:Epithelial cells:Barrier function;Innate immunity - sensors (Toll-like receptors);Inflammatory mediators, antimicrobial peptides;Salivary antimicrobial factorsMucosal antibodies (secretory IgA);Cell-mediated immunity (T-cells);In most cases, host defenses tolerate oral bacteriaThe predominant relationships are commensal.

Host defense mechanismsRemove the microorganisms through stimulation of salivary flow;Specific protection:Salivary Ig A;Nonspecific protection:Mucin;Antimicrobial factors:Lysozyme;Lactoferrin;Salivary peroxidase;Histatine-rich peptides;Cistatin;Leukocytes;Complement.Removal of the microorganismsThe majority of microorganisms in the mouth is removed by washing action of saliva;Salivary flow is stimulated by muscle activity of lips and tongue.The soft tissue surfaces employ a variety of mechanisms to prevent adhesion of pathogenic organisms. One of the most important mechanisms is shedding. The high turnover rate of the intraoral epithelial cells, especially of the gingiva, prevents the permanent accumulation of large masses of microorganisms on these surfaces. In essence, this is a natural cleansing mechanism. However, bacteria can adhere to host cells and form a commensal relationship, beneficial for both parties.Specific protection - Secretory IgA systemSIgA-antibodies reduce microbial adhesion to enamel epithelium and through:Neutralizing enzymesNeutralize toxins Synergy with other antibacterial agents such as lysozyme, lactoferrin, peroxidase and mucin;Protects the mucosa of penetration of antigens;Helps complement activation.MucinProvides a protective coating of enamel and mucosa;Catches microorganisms and antigens like in a trap;Limits microorganisms penetration into tissues;Eliminates microorganisms with continuous updating of mucin layer combined with washing action of saliva flow;As part of pelicle protects teeth from demineralization.Colonization of micro-organisms in the oral cavitymicrobial adhesinsConstructed of:Polysaccharides;Lipoteichoic acid;Glycosyltransferase;Carbohydrate-binding proteins;They are located in the cell wall in the form of: Fimbriae; Fibrils; Capsules.ADHESIONAdhesins can be of the microbial surface and receptors on oral surfacesThe association of bacteria within mixed biofilms is not random, rather there are specific associations among bacterial species.Socransky et al (1998)Adherence mechanisms of oral bacteria are essential to bacterial colonization of the oral cavity; In their absence, bacteria become part of the salivary ow and are swallowed; Consequently, oral bacteria have evolved several mechanisms to fulll this role.

89Receptors on the oral structuresSALIVARY COMPONENTSMucin;Glycoproteins;Amylase;Lysozyme;IgA, IgG;Proline-rich proteins.BACTERIAL COMPONENTSGlycosyltransferase;Glucans.Bacterial attachment to a surface can be divided into several distinct phasesPrimary and reversible adhesion;Secondary and irreversible adhesion;Biofilm formation.Co-aggregation Coaggregation interactions are believed to contribute to the development of biolms by two routes:The rst route is by single cells in suspension specically recognizing and adhering to genetically distinct cells in the developing biolm; The second route is by the prior coaggregation in suspension of secondary colonizers followed by the subsequent adhesion of this coaggregate to the developing biolm; In both cases, bacterial cells in suspension (planktonic cells) specically adhere to cells in the biolm in a process known as coadhesion.Ecological significance of bacterial coaggregation Specic coaggregation processes are likely to have an important ecological role as an integral process in the development and maintenance of mixed-species biolm communities;Strengthens bacterial attachment;Increases the stability of the plaque matrix.ADHESION IS DEPENDENT ON:A contact: Start of interaction;A dose: There is a certain amount of micro-organisms;Frequency of exposure: Partial colonization;Adsorption:By electrostatic forces to the surface of pellicle;With specific receptors on the cell surfaces;With fibrillere coupling end.Bacterial cells within biofilms can produce enzymes such as beta-lactamase against antibiotics, catalases, and superoxide dismutases against oxidizing ions released by phagocytes.These enzymes are released into the matrix producing an almost impregnable line of defense. Bacterial cells in biofilms can also produce elastases and cellulases which become concentrated in the local matrix and produce tissue damage.94 Dental BiofilmBiofilm is a highly organized structure and consists of microcolonies of bacterial cells randomly distributed in a shaped matrix or glycocalyx.Biofilms can facilitate processing, uptake of nutrients, cross feeding, removal of harmful metabolites and development of appropriate physicochemical environment.Lower plaque layer are dense in which microbes are bound together in polysaccharide matrix with other organic and imorganic materials.On top of lower layer, loose layer can be seen which can extend into surrounding medium (for teeth and saliva)The fluid layer bordering the biofilm has a stationary sublayer and a fluid layer in motionNutrient components penetrate this fluid medium by molecular diffusionBiofilms act as primitive circulatory system. Microcolonies occur in different shapes according to shear forces.

95Biofilm models : heterogenous mosaic & dense biofilm model

At low forces : tower or mushroom likeAt high shear force : elongated & capable of rapid oscillation.

95BiofilmThe classic biofilm that involves components of the normal flora of the oral cavity is the formation of dental plaque on the teeth; it has open fluid filled channels running across the plaque mass Plaque is a naturally-constructed biofilm, in which the consortia of bacteria may reach a thickness of 300-500 cells on the surfaces of the teeth; These accumulations subject the teeth and gingival tissues to high concentrations of bacterial metabolites, which result in dental disease.High rate of reproduction and physiological adaptation to environmental resources help biofilms to survive and sustain.Changes in shear forces, mass transfer, nutrient concentrations and quorum sensing influence biofilm properties

Biofilm formationBiotic surfaces themselves act as nutrient bases. The presence of this base triggers genetic changes in bacteria nad attract them. The bacteria loosely attach to the surface and lose flagella. They grow in size and release chemical signals, quorum sensing, and form microcolonies.They secrete exopolysaccharides carbohydrate slime (exopolymer) that imbeds the bacteria and attracts other microbes to the biofilm for protection or nutritional advantages.97Exopolysaccharides The Backbone of BiofilmsThe bulk of the biofilm consists of the matrix or glycocalyx and is composed predominantly of water and aqueous solutes. The dry material is a mixture of Exopolysaccharides, proteins, salts, and cell material.Exopolysaccharides (EPS), produced by the bacteria in the biofilm, are the major components of the biofilm making up 5095% of the dry weight. They play a major role in maintaining the integrity of the biofilm as well as preventing desiccation and attack by harmful agents.In addition, they may also bind essential nutrients such as cations to create a local nutritionally rich environment favouring specific microorganisms.

9898They can be neutral or charged polyanionic macromolecules and different concentrations can alter the conformation of the gel network.They can exist in ordered and disordered forms.The density of fibrillar masses and the flexibility and configuration changes could affect accessibility to nutrients and solutes.The EPS matrix could also act as a buffer and assist in the retention of extracellular enzymes (and their substrates) enhancing substrate utilization by bacterial cells. The charge can be changed with sucrose or other sugars. The EPS can be degraded and utilized by bacteria within the biofi m.One distinguishing feature of oral biofilms is that many of the microorganisms can both synthesize and degrade the EPS.

disOrdered : at high temp and low ionic concentrations.

The quantity does not reflect proportion of bacteria!!

99Quorum sensing tells bacteria when to grow, and when its time to go;Bacteria at the outer surface of mature biofilms are signaled to detach and become planktonic;Different genes are active in planktonic and biofilm states.

Ecological succession1 colonizers (Gram+)Streptococci bind pellicle proteins from saliva - DENT 5302;

2 colonizers (Gram-)Bridge species - F. nucleatumBind other bacteria

3 colonizers (Gram-)Porphyromonas gingivalis

Inter-species communicationStreptococci ferment CHO;Excrete lactic acid;Veillonella use lactate made by Strep for nutrition;They are biofilm buddies.

Strep can make amylase;Starch-digesting enzyme;Enhances lactate excretion;

Veillonella send a chemical signal to activate transcription of Strep amylase gene;Bacteria sense other species.

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POTENTIAL NUTRITIONAL INTERACTIONS BETWEEN PLAQUE BACTERIALactic acid can be used by veillonella which transforms it to a midler acid, that reduces the likelyhood of carries cdevelopment for example. By having differnet species, can have protective environment so acid production can be buffered by presence of other microorganisms. Community of microorganisms, as long as you have the proper ones, it can be usefull.Interspecies collaboration - O2Streptococcus cristatus:Facultative species:

Fusobacterium nucleatum:Robust anaerobe;Binding to strep improves survival when O2 is present;

Porphyromonas gingivalis:Sensitive anaerobe;Coaggregation essential to survival when O2 is present.

Collaborative invasionF. nucleatum invades epithelial cells;

S. cristatus does not invade cells;

After coaggregation, S. cristatus is carried inside by F. nucleatum.

Streptococcus cristatus thus coaggregates with F. nucleatumFusobacterium nucleatum is considered a bridge species because it is a promiscuous coaggregator.

Primary ColonizersStreptococcus gordoniiStreptococcus intermediusStreptococcus mitisStreptococcus oralisStreptococcus sanguinisActinomyces gerencseriaActinomyces israeliiActinomyces naeslundiiActinomyces orisAggregatibacter actinomycetemcomitans serotype aCapnocytophaga gingivalisCapnocytophaga ochraceaCapnocytophaga sputigenaEik enella corrodensActinomyces odontolyticusVeillonella parvula106SecondaryColonizersCampylobacter gracilisCampylobacter rectusCampylobacter showaeEubacterium nodatumAggregatibacter actinomycetemcomitans serotype bFusobacterium nucleatum ssp nucleatumFusobacterium nucleatum ssp vincentiiFusobacterium nucleatum ssp polymorphumFusobacterium periodonticumParvimonas micraPrevotella intermediaPrevotella loescheiiPrevotella nigrescensStreptococcus constellatusTannerella forsythiaPorphyromonas gingivalisTreponema denticola107STRUCTURE AND COMPOSISTION OF DENTAL PLAQUE1108Dental plaque is composed primarily of microorganisms. One gram of plaque (wet weight) contains approximately 1011 bacteria.In a periodontal pocket, Healthy crevice - 103 bacteria. Deep pocket - 108 bacteria.Nonbacterial microorganisms that are found in plaque include Mycoplasma species, yeasts, protozoa, and viruses.

109Subgingival Tissue associatedSt. oralis, St. intermedius Peptostreptcoccus micros P. gingivalis, P. intermedia T. Forsythis, F. NucleatumDifference between mature supra & sub-gingival plaqueCharacteristicSupra gingival Sub gingivalGrams stain Gram + or -ve Mainly Gram veMorphotypes

Cocci, branching rods, filaments & spirochaetes

Mainly rods & spirochaetes

Energy Metabolism

Facultative, some anaerobic

Mainly anaerobic

Energy source

Mainly ferment carbohydrate

Many proteolytic forms

MotilityFewManyPathologyCaries & gingivitis Gingivitis & periodontitis Attached plaque: tooth associated and predominantly composed of gram +ve rods and cocciCause subgingival calculus and root cariesUnattached plaque: extends to frontier of apical plaque and have gram ve organisms. Form the bioactive area and cause large amount of inflammatory GCFEpithelium associated: loosely attached to gingival epithelium and contain gram ve and motile rods. Most important group in periodontal pathgenesis112

Are there true oral pathogens?Classic concept of a pathogenNot normally presentProduces virulence factorsDamage host directly (e.g. toxins)Induce host to damage itself (immune responses)Presumed oral pathogens dont quite fit that modelNormally present throughout lifeDamage requires presence in large numbersEcological concept of oral microbial diseasesEcological shifts lead to changes in proportionsBalance shifts in favor of pathogens/diseaseMICROBIOLOGY OF PERIODONTAL DISEASES113Similarity between Periodontal Diseases and Other Infectious DiseasesIndividuals may be colonized continuously by periodontal pathogens at or below the gingival margin and yet not show evidence of ongoing or previous periodontal destruction.In spite of the presence of periodontal pathogens, periodontal tissue damage does not take place.This phenomenon is consistent with other infectious diseases in which it may be observed that a pathogen is necessary but not sufficient for a disease to occur.114In acute infections, an agent is acquired by exposure to an individual harbouring that agent or from the environment. The agent establishes within tissues or on mucous membranes or skin. Within a short period of time, signs or symptoms of a disease appear at the site of introduction or elsewhere in the individual. A battle occurs between the parasite and the host resulting in increasingly obvious clinical signs and symptoms. This hostpathogen interaction often is resolved within a short period of time, usually, but not always, in favour of the host. Thus, daily experience suggests that colonization by a pathogen is rapidly followed by expression of disease. While certain infections follow this pattern, more commonly, colonization by a pathogenic species does not lead to overt disease, at least immediately114Unique Features of Periodontal InfectionThe major reason for this uniqueness is the unusual anatomic feature that a mineralized structure, the tooth passes through the integument, so that part of it is exposed to the external environment while part is within the connective tissues.

The tooth provides a surface for the colonization of a diverse array of bacterial species.

Bacteria may attach to the tooth itself, to the epithelial surfaces of the gingiva or periodontal pocket, to underlying connective tissues, if exposed, and to other bacteria which are attached to these surfaces.

Thus, a situation is set up in which microorganisms colonize a relatively stable surface, the tooth and are continually held in immediate proximity to the soft tissues of the periodontium.

115Although periodontal diseases have certain features in common with other infectious diseases, there are several features of these diseases that are quite different. In certain ways, periodontal diseases may be among the most unusual infections of the human.In contrast to the outer surface of most parts of the body, the outer layers of the tooth do not shed and thus microbial colonization (accumulation) is facilitated.115The organisms that cause periodontal diseases reside in biofilms that exist on tooth or epithelial surfaces.

The biofilm provides a protective environment for the colonizing organisms and fosters metabolic properties that would not be possible if the species existed in a free-living (planktonic) state.116Historical PerspectiveInvestigators in the period from 18801930 suggested four distinct groups of microorganisms as possible etiologic agents; amoeba, spirochetes, fusiforms, and streptococci. Periodontal infections and another biofilm-induced disease, dental caries, are arguably the most common infectious diseases affecting the human.116

AmoebaThey found higher proportions of amoeba in lesions of destructive periodontal diseases than in samples taken from sites in healthy mouths or mouths with gingivitis.The role of amoeba in periodontal disease was questioned by some authors because amoeba were found in sites with minimal or no disease and could not be detected in many sites with destructive disease and because of the failure of emitin to ameliorate the symptoms of the disease.118SpirochetesInvestigators reported higher proportions of spirochetes and other motile forms in lesions of destructive disease when compared with control sites in the same or other individuals.

FusiformsThe third group of organisms that were frequently suggested to be etiologic agents of destructive periodontal diseases, including Vincents infection, were the spindle-shaped fusiforms. These organisms were originally recognized on the basis of their frequent appearance in microscopic examination of subgingival plaque samples. The organisms were first related to periodontal disease by Plaut (1894). Vincent (1899) distinguished certain pseudomembranous lesions of the oral cavity and throat from diphtheria and recognized the important role of fusiforms and spirochetes in this disease. 119StreptococciThese microorganisms were proposed on the basis of cultural examination of samples of plaque from subgingival sites of periodontal disease.The selection of the streptococci may have been predicated upon the fact these were the only species that could be consistently isolated from periodontitis lesions using the cultural techniques of that era.

The important role of spirochetes and fusiforms in Vincents infection was widely recognized in the succeeding 2 decades.

119ACTINOBACILLUS ACTINOMYCETEMCOMITANS120 Aclinobaciltus" refers to the internal star-shaped morphology of its bacterial colonies on solid media (Colebrook 1920) and to the short rod or bacillary shape of individual cells (Lieske 1921, Slots 1982).This is a small, non-motile, Gram-negative, saccharolytic, capnophilic, round-ended rod.This species was first recognized as a possible periodontal pathogen in lesions of localized juvenile periodontitis. (Newman et al 1976, Slots 1980, Chung et al 1989) .Subjects harboring A. actinomycetemcomitans with the 530 bp deletion were 22.5 times more likely to convert to LJP (Bueno et al 1998).

121It was originally named Bacterium actinomyeetum comilans (Klinger 1912) which was changed to Bacterium comitans (Lieske 1921) and finally to Actinobacilius actinomycetemcomitans (Topley & Wilson 1929), Recently, Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus segnis to a new genus Aggregatibacter gen. nov. as Aggregatibacter actinomycetemcomitans comb. nov. The species of the genus Aggregatibacter are independent of X factor and variably dependent on V factor for growth in vitro. Norskov-Lauritsen N, Kilian M (2007)

122

PORPHYROMONAS GINGIVALIS:123It is a Gram negative, anaerobic, non motile, asaccharolytic rods that usually exhibit coccal to short rod morphologies.P. gingivalis is a member of the much investigated black pigmented Bacteroides group. Initially they were grouped into a single species, B. melaninogenicus (Bacterium melaninogenicum, Burdon 1928).

TANNERELLA FORSYTHIA124Third consensus periodontal pathogen, B. forsythus, was first described in 1979 (Tanner et al. 1979) as a fusiform Bacteroides.The organism is a Gram negative, anaerobic, spindle shaped, highly pleomorphic rod.B. forsythus was detected more frequently and in higher numbers in active periodontal lesions than inactive lesions (Dzink et al 1988).This species has been shown to produce trypsin like proteolytic activity (BANA test positive, Loesche et al 1992) and induce apoptotic cell death (Arakawa et al 2000).SPIROCHETES125These are Gram negative, anaerobic, helical shaped, highly motile microorganisms that are common in many periodontal pockets. Clearly, a spirochete has been implicated as the likely etiologic agent of acute necrotizing ulcerative gingivitis (Listgarten & Socransky 1964). The organism has been considered as possible periodontal pathogens since the late 1800s and in the 1980s. Difficulty in distinguishing individual species. At least 15 species of subgingival spirochetes have been described.126T. denticola was found to be more common in periodontally diseased than healthy sites (Haffajee et al 1998, Yuan et al 2001).These were the most frequently detected spirochetes in supra and subgingival plaques of periodontitis patients.Pathogen related oral spirochetes (PROS) were also shown to have the ability to penetrate a tissue barrier in in vitro systems (Riviere et al 1991).

PREVOTELLA INTERMEDIA/PREVOTELLA NIGRESCENS127P. intermedia is the second black pigmented Bacteroides to receive considerable interest in pathogenesis of chronic periodontitis.It is a Gram negative, short, round-ended anaerobic rod. They have been shown to be particularly elevated in acute necrotizing ulcerative gingivitis (Loesche et al 1982), and also in certain forms of periodontitis (Herrera et al 2000, Papapanou et al 2000).128This species appears to have a number of virulence properties exhibited by P. gingivalis and was shown to induce mixed infections. (Hafstrom & Dahlen 1997).It has also been shown to invade oral epithelial cells in vitro (Dorn et al 1998).Strains of P. intermedia that show identical phenotypic traits have been separated into two species, P. intermedia and P. nigrescens (Shah & Gharbia 1992).

FUSOBACTERIUM NUCLEATUM129F. nucleatum is the type species of the genus Fusobacterium, which belongs to the family Bacteroidaceae.. The name Fusobacterium has its origin in fusus, a spindle; and bacterion, a small rod: thus, a small spindle-shaped rod.Gharbia and Shah (1990) divided Fusobacterium species into four subspecies: subspecies nucleatum, polymorphum, fusiforme, and animalis.

This species is the most common isolate found in cultural studies of subgingival plaque samples comprising app. 7-10% of total isolates. (Moore et al 1985).The species can induce apoptotic cell death in mononuclear and polymorphonuclear cells (Jewett et al 2001).Acts as microbial bridge facilitating coaggregation between early and Late colonizers.CAMPYLOBACTER RECTUS130C. rectus is a Gram negative, anaerobic, short, motile vibrio. The organism is unusual in that it utilizes H2 or formate as its energy source.It was first described as a member of the vibrio corroders, eventually shown to include members of a new genus Wolinella and Eikenella corrodens.It was found in higher numbers in disease sites as compared with healthy sites and more in sites exhibiting active periodontal destruction.(Rams et al 1993).Like A. actinomycetemcomitans, C. rectus has been shown to produce a leukotoxin. These are the only two oral species known to possess this characteristic (Gillespie et al 1992). EIKENELLA CORRODENS131E. corrodens is a Gram negative, capnophilic, asacharolytic, regular, small rod with blunt ends.This species was found more frequently in sites of periodontal destruction as compared with healthy sites and higher levels in active sites (Tanner et al 1987).In tissue culture system, E. corrodens has been shown to stimulate the production of matrix metalloproteinase (Dahan et al 2001) and IL-6 and IL-8 (Yumoto et al 1999).

PEPTOSTREPTOCOCCUS MICROS132P. micros is a Gram positive, anaerobic, small, asaccharolytic coccus.Two genotypes can be distinguished with the smooth genotype being more frequently associated with periodontitis lesions than the rough genotype (Kremer et al. 2000).P. micros was found to be in higher numbers at sites of periodontal destruction as compared with healthy sites (Papapanou et al 2000, Riggio et al 2001).SELENOMONAS SPECIES133The selenomonas spp. are Gram negative, curved, saccharolytic rods and may be recognized by their curved shape, tumbling motility and, in good preparations, by the presence of a tuft of flagella inserted in the concave side.Moore et al (1987) described six genetically and phenotypically distinct groups isolated from oral cavity and found at a higher proportion of shallow sites (PD>4mm) in chronic periodontitis.

EUBACTERIUM SPECIES134Certain Eubacterium species have been suggested as possible periodontal pathogens due to their increased levels in disease sites. (Moore et al 1985). E. nodatum, Eubacterium brachy and Eubacterium timidum are Gram positive, strictly anaerobic, small somewhat pleomorphic rods.Some of these species elicited elevated antibody responses in subjects with destructive periodontitis. (Martin et al 1988)

PATHOGENS STRENGTH OF RELATIONSHIP WITH DISEASE135

GINGIVITIS136 The microbial load at diseased sites is greater, with approximately 104 to 106 bacteria. The microbiota of chronic gingivitis (plaque induced) consist of app. Equal proportions of gram positive (56%) and gram negative (44%) species, as well as facultative (59%) and anaerobic (41%) microorganisms. Pregnancy associated gingivitis is accompanied by increases in steroid hormones in crevicular fluid and dramatic increases in levels of P. intermedia, which uses the steroid as growth factorsCHRONIC PERIODONTITIS137Cultivation of plaque microorganisms from sites of chronic periodontitis reveals high percentages of anaerobic (90%) and gram negative (75%) bacterial species.Certain gram-negative bacteria with pronounced virulence properties have been strongly implicated as etiologic agents (e.g. P. gingivalis and Tannerella forsythensis).LOCALIZED AGGRESSIVE PERIODONTITIS138The microflora of subgingival biofilms from patients with LAP is similar to that of patients with chronic periodontitis and is predominantly composed of gram negative, capnophilic, and anaerobic rods.On a percentage basis, the most numerous isolates are several species from the genera Eubacterium, A. naeslundii, F. nucleatum, C. rectus, and Veillonella parvula. Actinobacillus actinomycetemcomitans plays 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 A. actinomycetemcomitans, and in still others P. gingivalis may be etiologically more important.

GENERALIZED AGGRESSIVE PERIODONTITIS139The subgingival flora in patients with generalized aggressive periodontitis resembles that in other forms of periodontitis.The predominant subgingival bacteria in patients with generalized aggressive periodontitis are P. gingivalis, T. forsythensis A. actinomycetemcomitans, and Campylobacter species.

NECROTIZING ULCERATIVE GINGIVITIS/PERIODONTITIS140The majority of the spirochetes (treponemes) associated with necrotizing ulcerative gingivitis are uncultivable, but it is clear that they constitute a very large and diverse group.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 ABSCESSES141The 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. forsythensis (47.1%).Enteric bacteria, coagulase-negative staphylococci, and Candida albicans have also been detected.

PERIIMPLANTITIS142Studies have shown that microbiota associated with periimplantitis are comparable to that of periodontitis (high proportion of anaerobic gram negative rods, motile organisms, and spirochetes).Species such as A. actinomycetemcomitans, P. gingivalis, T. forsythia, 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.

VaccinesThree types of vaccines were employed for the control of periodontal diseases. These included vaccines prepared from pure cultures of streptococci, and other oral organisms, autogenous vaccines, and stock vaccines such as Van Cotts vaccine, Goldenbergs vaccine or Inava Endocorps vaccine. These vaccines were administered systemically or locally in the periodontal tissues. Autogenous vaccines were prepared from the dental plaque of patients with destructive periodontal diseases. Plaque samples were removed from the diseased site, sterilized by heat, and/or by immersion in iodine or formalin solutions, then re-injected into the same patient, either in the local periodontal lesion or systemically.Proponents of all three techniques claimed great efficacy for the vaccination methods employed, while others using the same techniques were more sceptical.143Specific Bacterial behaviour in Biofilm: Antibiotic resistanceMicroorganisms in biofilm are 1000 to 1500 times more resistant to antibiotics than in their planktonic stageThe mechanism of this increased resistance differs from species to species, from antibiotic to antibiotic, and for biofilm growing in different habitats

144Resistance of bacteria to antibiotics is affected by their

Nutritional statusGrowth rateTemparaturepHPrior exposure to subeffective concentration of anti microbial agents

mmmm144145Also slower growth of bacterial species in biofilm is another important mechanism of antibiotic resistanceBiofilm matrix although not significant barrier in itself to diffusion of antibiotics but have certain properties to resist diffusion Biofilm act as ion-exchange resin removing antibiotics from solutionSome antibiotics such as Macrolide which are positive charged but hydrophobic are unaffected by this process146Also extracellular enzymes such as lactamases, formaldehyde lyase and formaldehyde dehydrogenase may become trapped and concentrated in the extracellular matrix thus inactivating some antibiotics(especially positive charged hydrophilic antibiotics)

Super-resistant bacteria have been identified within a biofilm and these cells have multidrug- resistant pump that can extrude antimicrobials from the cell

147Translocation and Mechanical DebridementTo reduce chance of intraoral transmission, one stage, Full mouth disinfection has been introduced by Leuven group in the 1990sThis strategy attempts to eradicate, or atleast suppress, periopathogens in short time not only from the periodontal pockets, but also from all their intraoral habitats(mucous membrane, tongue, and saliva)148References Textbook of Microbiology- AnantnarayanCarranzas clinical periodontology. 9th editionClinical Periodontology and Implant dentistry, Jan Lindhe, 4th edition. Blackwell munksgaard.PD Marsh: Plaque as a biofilm: pharmacological principles of drug delivery and action in the sub- and supragingival environment. Oral Diseases (2003) 9 (Suppl. 1), 1622Anne. D. Haffajee & sigmund.S. Socransky. Microbial etiological agents of destructive periodontal diseases. Periodontology 2000, Vol. 5, 1994, 78-1Tatsuj Nishihara & Takeyoshi koseki. Microbial etiology of Periodontitis. Periodontology 2000, Vol. 36, 2004, 1426.