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Periodontal microbiology
 

Periodontal microbiology

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Periodontal microbiology Periodontal microbiology Presentation Transcript

  • Periodontal Microbiology
  •  Human fetus is sterile but after passing through birth canal it acquires vaginal and fecal microorganisms  By 2nd day Anaerobic flora can be detected in infant edentulous mouth Dr Saif Khan 209/10/13
  •  Within 2nd week a nearly mature microbiota is established in the gut of newborn  After weaning(>2yrs), entire human microbial flora formed by complex collection of more than 400 types of bacteria. 09/10/13 Dr Saif Khan 3
  • Oral cavity is an “Open growth system” Dr Saif Khan 409/10/13
  • Oral cavity divided into 5 major niches Dr Saif Khan 509/10/13
  •  Most species colonize on all above described niches with the exception of spirochetes  Adherence of bacteria to oral epithelial cells is directly related to its virulence Dr Saif Khan 609/10/13
  • Teeth and Implants are unique from a microbiological point of view for two reason – Provide hard, non-shedding surface that allows for the development of extensive structured baterial deposit – Form a unique ectodermal interruption A special seal of epithelium and connective tissue exists between external enviroment and internal part of body Dr Saif Khan 709/10/13
  • Teeth are primary habitat for periopathogens Thus teeth can be considered as “Port of entry for Periopathogens Cariogenic species like S mutans remain restricted to solid surfaces and are called “obligate periphyte” Dr Saif Khan 809/10/13
  • Dental Plaque Dental plaque is a specific, amorphous, granular deposit which accumulates on the surface of teeth, dental restoration and dental calculus. - -Glickmann Dr Saif Khan 909/10/13
  • WHO defnition “Dental plaque is a specific but highly variable entity resulting from growth and colonization of micro-organisms on surfaces of teeth,restoration and soft- tissue consisting of various species microorganisms entangled in extracellular matrix.” Dr Saif Khan 1009/10/13
  • Dental plaque • Defined clinically as structured, reselient, yellowish-grayish substance that adhere tenaciously to the intraoral hard surface,including removable and fixed restoration. • Plaque is primarily composed of bacteria in matrix of salivary glycoproteins and extracellular polysaccharides • 1gm of dental plaque(wet weight) contains 1011 bacteria Dr Saif Khan 1109/10/13
  • 09/10/13 Dr Saif Khan 12 Thin supragingival dental plaque of a 32-year-old man who had not brushed his teeth for 7 days. A, Unstained plaque is not readily apparent. B, Extent of the plaque becomes apparent when stained with a disclosing solution (i.e.,erythrosine dye)
  • No of bacteria in Supragingival plaque on single tooth – 109 GingivalCrevice- 103 Deepperiodontalpocket- 108 More than 500 distinct microbial species are found in dental plaque Non bacterial microorganism found in plaque include Mycoplasma species, yeasts, Protozoa and Viruses Dr Saif Khan 1309/10/13
  • Dental plaque is broadly Classified into Dr Saif Khan 1409/10/13
  • Supragingival plaque visualized by Disclosing solution Dr Saif Khan 1509/10/13
  • Dr Saif Khan 1609/10/13
  • aque-bacteria interaction with tooth surface and Periodontal tissDr Saif Khan 1709/10/13
  • Dr Saif Khan 1809/10/13
  • Dr Saif Khan 1909/10/13
  • The enviromental paramaters of subgingival region is different from supragingival region – Gingival crevice is bathed by GCF which may contains many substances that bacteria may use as nutrients – Low oxygen tension in the subgingival area – Host inflammatory cells and mediators have considerable influence on establishment and growth of bacteria Dr Saif Khan 2009/10/13
  • The apical border of the plaque mass is separated from junctional epitheliun by a layer of host leukocytes and the bacteria of this apical-tooth associated region show an increased concentration of Gm-ve rods Dr Saif Khan 2109/10/13
  • Dental Plaque as a Biofilm • Biofilm is a highly organized structure • Consists of microcolonies of bacterial cells randomly distributed in a shaped matrix or glycocalyx. • Lower plaque layer are dense in which microbes are bound together in polysaccharide matrix with other organic and imorganic materials Dr Saif Khan 2209/10/13
  • • 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 motion • Nutrient components penetrate this fluid medium by molecular diffusion Dr Saif Khan 2309/10/13
  • • The dental plaque biofilm has open fluid filled channels running across the plaque mass • Act as primitive “circulating system” Dr Saif Khan 2409/10/13
  • Intercellular Matrix • Organic Constituent- Consists of – Polysacchrides, Proteins, Glycoprotein & Lipid – Albumin derived from GCF – Lipid material consists of debris from the membranes of disrupted bacterial & Host cells and possibly Food debris – Glycoprotein from saliva important component of pellicle – Polysacchrides produced by bacteria of which Dextran is most predominant form & play major role in maintaining integrity of Biofilm Dr Saif Khan 2509/10/13
  • Inorganic component • Predominantly Ca and P • Trace minerals Na, K and F • Source of inorganic constituent of supragingival plaque is primarily saliva • As mineral content increases plaque calcifies to Calculus • Inorganic portion of subgingival fluid are derived from crevicular fluid • F content of plaque is basically derived from external sources such as floridinated toothpaste, rinses, water etc Dr Saif Khan 2609/10/13
  • Plaque formation at Ultrastructural level 1. Formation of Pellicle – Thin, saliva derived layer – Consists of Glycoprotein (mucins), Proline rich protein, Phosphoproteins (Statherin), Histidine rich proteins,enzymes – Forms within seconds of prophylaxis – Forms by selective adsorption of enviromental macromolecules – Mechanism involved in pellicle formation include Vanderwall forces & Hydrophobic forces Dr Saif Khan 2709/10/13
  • Dr Saif Khan 2809/10/13
  • Dr Saif Khan 2909/10/13
  • 2. Initial Adhesion and attachment of bacteria Phase I : Transport to the surface •Initial contact of bacterium to tooth surface •Random contacts through Brownian movement (40μm/hr) •Through Active bacterial movement Phase II: Initial Adhesion •Reversible adhesion •Interaction b/w bacteria and surface at certain distance (50nm) through long range and short range forces •Including Vanderwall attractive and electrostatic repulsive forces •Gibbs total energy GTOT = GA + GE •GTOT is a function of separation distance between negatively charged particle and negatively charged surface in a medium ionic suspension (saliva) •For most bacteria reversible binding takes place 5-20 nm from surface Dr Saif Khan 3009/10/13
  • Schematic representation of interactions involved in bacterial adhesion to solid substrata Dr Saif Khan 3109/10/13
  • Phase III: Attachment •After intial adhesion firm adhesion is established by specific interactions (covalent,ionic or hydrogen •Rough surfaces are more conducive for attachment as bacteria are better protected against sheer force leading change from reversible to irreversible bonding •The bonding between bacteria and pellicle is mediated by by specific extracellular protein components eg; Streptococci (S sangius) early colonizer binds to acidic proline-rich proteins, also α-amylase and sialic acid Dr Saif Khan 3209/10/13
  • 3. Colonization and plaque maturation Primary Colonizers: Streptococci and Actinomycetes Secondary Colonizers: P intermedia, P loescheii, Capnocytophaga, F nucleatum, P gingivalis Dr Saif Khan 3309/10/13
  •  All oral bacteria possess surface molecule or receptor which foster cell to cell interaction Highly specific stereo chemical interaction of proteins and carbohydrate molecules located on bacterial cell surfaces leading to Coaggregation Most Co aggregation are among strains of different genera are mediated by Lectin like adhesins and inhibited by Lactose and other galactosides Dr Saif Khan 3409/10/13
  • Examples of Co-aggregation  Fusobacterium nucleatum with steptocooci sangius,Prevotella loescheii with A viscosus  Capnocytophaga ochraceus with A viscosus  Intrageneric co-aggregation in streptocooci Dr Saif Khan 3509/10/13
  • Dr Saif Khan 36 Fusobacterium nucleatum S sangius P loeschii A viscosus 09/10/13
  • Coaggregation 09/10/13 Dr Saif Khan 37
  • Dr Saif Khan 3809/10/13
  • Growth dynamics of Dental Plaque • First 2-8 hrs, adherent pioneering streptococci saturate the salivary pellicular binding sites and cover 3-30% of enamel surface • After 1 day the term Biofilm is fully deserved because organization takes place in it • After 3 days plaque growth increases at rapid rate and then slows down Dr Saif Khan 3909/10/13
  • • There is shift towards more anaerobic and gram- negative flora, including an influx of Fusobacteria, filaments, Spiral forms and spirochetes • In ecological shift within of the biofilm, there is a transition from the early aerobic environment characterized by Gm+ve facultative species to a highly oxygen- deprived environment in which Gm-ve anaerobic microorganism predominate Dr Saif Khan 4009/10/13
  • Topography of Supragingival Plaque • Early plaque formation follow typical topograhic pattern with initial growth along gingival margin and interdental space • Later further there is extension in coronal direction • This pattern changes when tooth contains surface irregularities such as grooves,cracks, perikymata, or pits • Surface irregularities can give rise to “individualized plaque growth pattern Dr Saif Khan 4109/10/13
  • Surface Microroughness • Rough intraoral surface (crown,implant abutments, denture bases)accumulate and retain more plaque and calculus in terms of thickness area and colony forming unit • [Ra=0.2µ] is threshold for surface roughness above which bacterial adhesion is facilitated Dr Saif Khan 4209/10/13
  • Individual variables influencing plaque formation Heavy(fast) plaque formers  Rapid plaque formers demonstrate higher proportion of Gm-ve rods (35% vs 17%) in 14-day old plaque Light (slow) plaque formers Dr Saif Khan 4309/10/13
  • Intersubject variation in plaque formation can be explained by factors such as • Diet • Food • Smoking • Presence of copper amalgam • Tongue& palate brushing • Colloid stability of bacteria in the saliva • Antimicrobial factors present in saliva • Chemical composition of the Pellicle • Retention depth of dentogingival area Dr Saif Khan 4409/10/13
  • Variation within the Dentition Early plaque formation occurs faster in •lower jaw compared to upper •Molar areas •Buccal tooth surfaces as compared to oral sites (esp in upper jaw) •Interdental region compared to buccal or oral surfaces Dr Saif Khan 4509/10/13
  • Impact of Gingival inflammation • Early invivo plaque formation is more rapid on tooth surfaces facing inflamed gingival margins than those adjacent to healthy gingival margins • Increase in crevicular fluid production enhances plaque formation Dr Saif Khan 4609/10/13
  • Impact of Age Recent studies show that subject’s age does not influence de novo plaque formation Dr Saif Khan 4709/10/13
  • Spontaneous tooth cleaning • Firm attachment between bacteria and surface this is unlikeky • Even occlusal part of molars, plaque remains after chewing fibrous food • Only negligible differences in plaque extension could be observed Dr Saif Khan 4809/10/13
  • De novo subgingival plaque formation Recent studies suggest that complex subgingival microbiota, including most periopathogens, is established within 1 week after abutment insertion Smooth abutments[Ra<0.2µ] were found to harbour less bacteria than less ones, with a slightly higher density of coccoids (i.e nonpathogenic) cell Dr Saif Khan 4909/10/13
  • Physiological properties of Dental Plaque  The transition from Gm+ve to Gm-ve microorganism observed in structural development of plaque is paralled by physiologic transition in the developing plaque  Early colonizers use oxygen and lower redox potential of the environment which then favors growth of anaerobic species Dr Saif Khan 5009/10/13
  •  Early colonizers use sugar as energy source and saliva as carbon source  Bacteria which predominate in mature plaque or late colonizers are Asaccharolytic and use amino acids and small peptides as energy source  Lactate and formate are by products of metabolism of streptococci and actinomycetes may be used by other microorganism Dr Saif Khan 5109/10/13
  • Hemin a breakdown product from host hemoglobin is important in metabolism of P gingivalis Increase in steroid hormone is associated with increase in proportions of Prevotella intermedia in subgingival plaque Dr Saif Khan 5209/10/13
  • Metabolic interaction among different bacteria species found in plaque and also between host and plaque bacteria Dr Saif Khan 5309/10/13
  • Ecological plaque Hypothesis  Given by Marsh & Co- Workers in 1990  Total amount of dental Plaque and the specific microbial composition of plaque contribute to transition from health to disease  Health –associated dental plaque microflora is considered to be relatively stable overtime and in state of dynamic equilibrium or “ microbial homeostasis ” Dr Saif Khan 5409/10/13
  • Ecological plaque Hypothesis  Change in nutrient status of a periodontal pocket or  Chemical and physical changes to habitat can lead over growth of pathogens Eg: Increase in GCF flow can lead to enrichment of proteolytic species(periopathogens) by providing essential nutrients such as heme containing molecules Dr Saif Khan 5509/10/13
  • Dr Saif Khan 56 Schematic representation of the ecological plaque hypothesis in relation to periodontal disease 09/10/13
  • Dr Saif Khan 57 Relationship between the microbial composition of dental plaque in health and disease. 09/10/13
  • Non- specific plaque hypothesis  Periodontal disease results from elaboration of noxious products from entire plaque flora  Large amount of plaque produces large amount of noxious product that would overwhelm host’s defense Dr Saif Khan 5809/10/13
  • Dr Saif Khan 59  Control of periodontal disease depend on control of amount of plaque deposit  The current standard treatment of periodontitis still focuses on the removal of plaque and its product founded on non-specific plaque hypothesis 09/10/13
  • Non-specific plaque hypothesis has been discarded because  Some individuals with considerable amount of plaque and calculus as well as gingivitis never developed destructive periodontitis  Individuals with periodontitis demonstrated considerable site specificity in pattern of disease  Individuals with very less plaque developed destructive periodontal disease as in Aggressive periodontitis Dr Saif Khan 6009/10/13
  • Specific plaque Hypothesis  States that only certain plaque is pathogenic  And its pathogenecity depends on presence of or increase in specific microorganism Dr Saif Khan 6109/10/13
  • 09/10/13 Dr Saif Khan 62 Plaque harboring specific bacterial pathogen results in periodontal disease because these organism produce substance that mediate the destruction of host tissue Eg: A actinomycetemcomitans as pathogen in localized aggressive periodontitis
  • Specific Bacterial behaviour in Biofilm: Antibiotic resistance Microorganisms in biofilm are 1000 to 1500 times more resistant to antibiotics than in their planktonic stage The mechanism of this increased resistance differs from species to species, from antibiotic to antibiotic, and for biofilm growing in different habitats Dr Saif Khan 6309/10/13
  • Resistance of bacteria to antibiotics is affected by their • Nutritional status • Growth rate • Temparature • pH • Prior exposure to subeffective concentration of anti microbial agents Dr Saif Khan 6409/10/13
  • 09/10/13 Dr Saif Khan 65 Also slower growth of bacterial species in biofilm is another important mechanism of antibiotic resistance Biofilm 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 solution
  • 09/10/13 Dr Saif Khan 66 Also extracellular enzymes such as β lactamases, formaldehyde lyase and formaldehyde dehydrogenase may become trapped and concentrated in the extracellular matrix thus inactivating some antibiotics(especially positive charged hydrophilic antibiotics)
  • 09/10/13 Dr Saif Khan 67  Some antibiotics such as Macrolide which are positive charged but hydrophobic are unaffected by this process
  • 09/10/13 Dr Saif Khan 68 “Super-resistant” bacteria have been identified within a biofilm and these cells have multidrug- resistant pump that can extrude antimicrobials from the cell
  • Quorum Sensing Bacteria in biofilm communicate with each other This involves the regulation of expression of specific genes through accumulation of signalling compounds that mediate intercellular communication When these signalling compounds reach a threshold level(quorum cell density) gene expression is activated Dr Saif Khan 6909/10/13
  • • Quorum sensing seems to play a role in expressing genes for antibiotic resistance and encouraging growth of beneficial species to the biofilm and discouraging the growth of competitors Dr Saif Khan 7009/10/13
  • Schematic representation of the types of interaction that occur in a microbial community, such as dental plaque Dr Saif Khan 7109/10/13
  • Dr Saif Khan 72 High density of bacterial cells in biofilm facilitates the exchange of genetic information among cell of the same species and genera through; 1. Conjugation (sex pilus) 2. Transformation (movement of small pieces of DNA from enviroment into bacterial chromosome) 3. Plasmid tranfer 4. Transposon transfer (DNA sequence which can change sequence within the genome) 09/10/13
  • Dr Saif Khan 7309/10/13
  • Translocation and Mechanical Debridement • To reduce chance of intraoral transmission, one stage, Full mouth disinfection has been introduced by Leuven group in the 1990s • This 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) Dr Saif Khan 7409/10/13
  • One stage, Full mouth disinfection  Full mouth scaling and root planning within 24 hrs to reduce number of subgingival pathogenic organisms  Subgingival irrigation of all pockets with 1% chlorhexidine gel to kill remaining bacteria  Tongue brushing with an antiseptic to suppress the bacteria in the niche  Mouth rinsing with antiseptic to reduce the bacteria in the saliva and on the tonsil Dr Saif Khan 7509/10/13
  • Benefits of the One stage, Full mouth disinfection • Pocket depth reduction • Gain in clinical attachment level • Microbiologic shift Dr Saif Khan 7609/10/13
  • The current concept on etiology of Periodontitis considers three factors that determine whether active periodontitis will occur 1. Susceptible Host 2. Presence of a Pathogenic species 3. Absence or small numbers of beneficial species Dr Saif Khan 7709/10/13
  • Dr Saif Khan 7809/10/13
  • Role of Beneficial species Role of Beneficial species Passively occupying niches otherwise occupied by pathogenic bacteria Passively occupying niches otherwise occupied by pathogenic bacteria Actively limiting pathogens ability to adhere to appropriate tooth surface Actively limiting pathogens ability to adhere to appropriate tooth surface Adversely affecting the vitality or growth of pathogens Adversely affecting the vitality or growth of pathogens Affecting the ability of pathogenic species to produce virulence factor Affecting the ability of pathogenic species to produce virulence factor Degrading Virulence factor produced by pathogen Degrading Virulence factor produced by pathogen Dr Saif Khan 7909/10/13
  • Dr Saif Khan 80 Beneficial species such as S sangius, Veillonella parvula and C ochreus are typically found in higher number at periodontal sites with no attachment loss where as lower in number where there is active periodontal destruction 09/10/13
  • Criteria for identification of Periodontal pathogen Kochs Postulates Given by Robert Koch as classic criteria by which microorganims are judged causative Must be routinely isolated from diseased individual Must be grown in pure culture in laboratory Must produce similar disease when inoculated in susceptible laboratory animal Must be recovered from lesions in diseased laboratory animal Streptococcus mutans has been shown to follow Koch’s postulate as an etiologic agent of dental caries Dr Saif Khan 8109/10/13
  • koch’s criteria are difficult to apply in periodontal disease because of 3 reasons 1. Inability to culture all the microorganism that have been associated with disease (eg: spirochetes) 2. The difficulties inherent in defining and culturing sites of active disease 3. Lack of good animal model for study of Periodontitis Dr Saif Khan 8209/10/13
  • Socransky criteria • Proposed criteria by which periodontal microorganism may be judged to be potential pathogens 1. Must be associated with disease, as evident by increase in the number of organisms at diseased sites 2. Must be eliminated or decreased in sites that demonstrate clinical resolution of disease with treatment 3. Must demonstrate a host response, in the form of an alteration in the host cellular or humoral immune response 4. Must be capable of causing disease in experimental animal model. 5. Must demonstrate virulence factors responsible for enabling the microorganism to cause destruction of periodontal tissue Dr Saif Khan 8309/10/13
  • Microorganism associated with specific periodontal disease  Fewer coccal cells and more motile rods and spirochetes are found in diseased state than healthy sites by means of phase-contrast or dark-field microscopy  All most all periodontal pathogens except Campylobacter rectus are immobile  Bacteria from healthy periodontal sites consists of gram +ve facultative rods and cocci Dr Saif Khan 8409/10/13
  • Periodontal Health  Gm+ve facultative species of genera Streptococcus and Actinomycetes(S sangius, S mitis, A viscosus, A naeslundi)  Small proportions of Gm-ve speciesa are also found (P intermedia, F nucleatum, Capnocytophaga, C ochareus) Dr Saif Khan 8509/10/13
  • Gingivitis Micro biota of dental –plaque induced gingivitis(chronic gingivitis) consists of equal proportions of  Gm+ve(56%) & Gm-ve(44%) species Facultative(59%) & anaerobic(51%) microorganisms Predominant Gm+ve microorganisms are S sangius, S mitis, S intermedius, S oralis, A viscosus, A naeslundii and P micros Dr Saif Khan 8609/10/13
  • Dr Saif Khan 87 The predominant Gm-ve microorganisms are F nucleatum, P intermedia, V parvula as well as Haemophilus, Campylobacter and Capnocytophaga Pregnancy associated gingivitis is acute inflammation of gingivae associatedin pregnancy. There is increase in steroid hormones in crevicular fluid and dramatic increase in Prevotella intermedia which uses steroid as growth factor 09/10/13
  • Chronic periodontitis  Microscopic examination of plaque from sites with chronic gingivitis consistently revealed elevated proportions of spirochetes  Cultivation of plaque microorganisms from sites chronic periodontitis have reveal high percentages of anaerobic(90%) and gram negative (75%) bacterial species Dr Saif Khan 8809/10/13
  • Dr Saif Khan 89  Bacteria most often cultivated include P. gingivalis , T. forsythus,,C rectus, E corrodens, F. nucleatum, A actinomycetemcomitans, P micros, Treponema and Eubacterium  C rectus, P gingivalis, P intermedia, F nucleatum & T forsythia are elevated in active sites09/10/13
  • Dr Saif Khan 90  Detectable levels of P gingivalis, P intermedia, T forsythia, C rectus and A actinomycetemcomitans are associated with disease progression and their elimination by therapy is associated with improve clinical outcome 09/10/13
  • Dr Saif Khan 91 Also recent studies have documented association between chronic periodontitis and viral microrganisms of Herpes group, most notably Epstein barr virus-1 (EBV-1) and Human cytomegalovirus (HCMV) are associated with putative pathogens P gingivalis, T forsythia, P intermedia and T denticola 09/10/13
  • Microbial Shift during disease Dr Saif Khan 9209/10/13
  • Localized Aggressive Periodontitis  A actinomycetemcomitans compose of 90% of total cultivable microbiota  P gingivalis, E corrodens, C rectus, F nucleatum, B capillus, Eubacterium brachy, Capnocytophaga sp and Spirochetes are also found in significant levels  Herpes viruses, including EBV-1 and HCMV have been associated with LAP Dr Saif Khan 9309/10/13
  • Necrotizing Periodontal disease  Microbiologic studies indicate high levels of Prevotella intermedia and especially Spirochetes in NUG lesions  Spirochetes penetrate deep into necrotic tissue and unaffected connective tissue Dr Saif Khan 9409/10/13
  • Microbial specificity in Periodontitis • There is no “black-or-white” situation; most pathogens might be present, but do not necessarily have to be present for specific form of periodontitis • Microbial composition can not be used to differentiate different forms of periodontal disease Dr Saif Khan 9509/10/13
  • Dr Saif Khan 96 Most pathogens can also be detected in healthy subjects with frequencies ranging from 10% to 85%. This automatically reduces the specificity of microbiologic testing in periodontology 09/10/13
  • Periimplantitis  Inflammatory process affecting the tissue around an already osseointegrated implant resulting in loss of supporting bone  Healthy periimplant pockets are characterised by high proportions of coccoid cells, low anaerobic/aerobic ratio, low number of gram anaerobic species and low detection frequency for periodontal pathogens Dr Saif Khan 9709/10/13
  • 09/10/13 Dr Saif Khan 98
  • Key characteristics of specific Periopathogens Dr Saif Khan 9909/10/13
  • Dr Saif Khan 10009/10/13
  • Dr Saif Khan 10109/10/13
  • Dr Saif Khan 10209/10/13