DENTAL PLAQUE - PART 2 (BIOFILM)

Dental plaque part 2

1. DENTAL PLAQUE - II GUIDED BY: DR. RUPINDER KAUR DR. DIVYA JAGGI PRESENTED BY: DR.MALVIKA THAKUR PG II YEAR

2. CONTENTS Introduction Properties of biofilm Factors affecting biofilm development and behavior Microbial specificity of periodontal disease Biofims and Host in conflict Possible strategies to control oral biofilm Detection of dental plaque Conclusion References

3. M atrix embedded microbial populations, adherent to each other and/or to surfaces or interfaces ( Costerton et al. 1999) Biofilms consist of one or more communities of microorganisms, embedded in a glycocalyx , that are attached to a solid surface. (Sigmund S. Socransky & Anne D. Haffajee . 2001) INTRODUCTION 1/45

4. A microbial biofilm is considered a community that meets the following four basic criteria: 2/45

5. Schematic representation of the types of interaction that occur in a microbial community, such as dental plaque 30-Aug-2016 BENEFITS OF MICROBIAL COMMUNIY LIFESTYLE 3/45

6. PROPERTIES OF BIOFILM 4/45

7. 1. STRUCTURE OF A BIOFILM  Biofilms are composed of microcolonies of bacterial cells (15–20% by volume) that are non-randomly distributed in a matrix or glycocalyx (75–80% volume ). 5/45 The bacterial vitality varies throughout the biofilm, with the most viable bacteria present in the central part of plaque , and lining the voids and channels ( Auschill et al 2001)

8. The biofilm matrix is penetrated by fluid channels that conduct the flow of nutrients, waste products, enzymes, metabolites, and oxygen.  6/45 Structure of the Biofilm depends on environmental parameters under which they are formed. These include: • Surface and interface properties • Nutrient availability • Composition of the microbial community • Hydrodynamics

9. The bacteria in a biofilm use a communication system termed quorum sensing that involves sending out chemical signals .   These chemical signals trigger the bacteria to produce potentially harmful proteins and enzymes, virulence factors that help the intraoral biofilm bypass host defense systems 7/45

10. EXOPOLYSACCHARIDES – the backbone of the biofilm The 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), which are produced by the bacteria in the biofilm, are the major components of the biofilm making up 50–95% of the dry weight. 8/45

11. 2. ATTACHMENT OF BACTERIA   The key characteristic of a biofilm - the microcolonies within the biofilm attach to a solid surface. Many bacterial species possess surface structures such as fimbriae and fibrils that aid in their attachment to different surfaces . Fimbriae have been detected on a number of oral species including P. gingivalis , A. actinomycetemcomitans and some strains of streptococci. Oral species that possess fibrils include S. salivarius , the S. mitis group, Pr. intermedia, Pr. nigrescens , and Streptococcus mutans . Sigmund S. Socransky & Anne D. Haffajee . Dental Biofilms: Difficult Therapeutic Targets Periodontology 2000 2001;28:12–55. 9/45

12. VARIABLES IMPORTANT IN CELL ATTACHMENT AND BIOFILM FORMATION PROPERTIES OF THE SUBSTRATUM PROPERTIES OF THE BULK FLUID PROPERTIES OF THE CELL Texture or roughness Hydrophobicity Conditioning Flow velocity pH Temperature Cations Presence Cell surface hydrophobicity Fimbriae Flagella Extracellular polymeric substances of antimicrobial agents

13. 3 . PHYSIOLOGICAL HETEROGENEITY Cells of the same microbial species can exhibit extremely different physiologic states in a biofilm even though separated by as little as 10 μm . 11/45 Clinical Periodontology and Implant Dentistry by Jan Lindhe , 5 th Edition. Studies to date indicate that sessile cells growing in mixed biofilms can exist in an almost infinite range of chemical and physical microhabitats within microbial communities.

14. The residents in the microbial community display extensive interactions while forming biofilm structures , carrying out physiological functions, and inducing microbial pathogenesis . These interactions, include Competition between bacteria for nutrients Synergistic interactions which may stimulate the growth or survival of one or more residents Production of an antagonist by one resident which inhibits the growth of another Neutralization of a virulence factor produced by one organism by another resident Interference in the growth-dependent signaling mechanisms of one organism by another 4. MICROBIAL INTERACTIONS 12/45

15. NUTRIENTS AS THE BASIS FOR BACTERIAL INTERSPECIES INTERACTION WITHIN BIOFILM

16. GENERAL METABOLIC PRODUCTS WHICH INFLUENCE BIOFILM RESIDENT INTERACTIONS Antagonistic effect - S . sanguinis group are producers of H 2 O 2 , a nonspecific antimicrobial agent - an antagonistic effect on other coresidents , such as S. mutans . Synergistic effect - lactic acid produced by S. mutans can be readily metabolized by members of the Veillonella family. Co-operative metabolic interactions -

17. 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. 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 15/45

18. Bacterial co-aggregation influences localization within biofilms Gibbons and coworkers(1970 ) , demonstrated that strains of P. gingivalis aggregated with several oral streptococci, this might be an important mechanism - P . gingivalis could become incorporated into dental plaque that is composed primarily of the initial streptococcal colonizers . T. denticola does not appear to form biofilms on most inert surfaces, in contrast to P. gingivalis , which does so readily. However, in the presence of P. gingivalis , T. denticola is incorporated into the biofilm . T. forsythia is a weak colonizer of inert surfaces but will become incorporated into biofilms in the presence of F. nucleatum . 16/45

19. 5 . QUORUM SENSING It is defined as the cell density dependent regulation of gene expression in response to soluble signals called autoinducers ( Bassler 1999) It has been defined by Miller (2001) as “the regulation of gene expression in response to fluctuations in cell population density ”. Quorum sensing can occur within a single bacterial  species   as well as b/w diverse species. 17/45

20. Quorum sensing has been described in both G+ve & G- ve bacteria. Cell-cell communication may occur b/w and within bacterial species (Miller, 2001) Quorum sensing-controlled behaviors are those that only occur when bacteria are at high cell population densities. 18/45

21. Three types of molecules : Acyl- homoserine lactones (AHLs) - signaling molecules used by many G - ve bacteria, it synthesized by Lux-I family protiens . Autoinducer peptides (AIPs ) - signaling molecules used by G + ve bacteria Autoinducer-2 (AI-2) - used by both G- ve & G+ve bacteria, chemically it is furanosyl borate diester . Synthsized by protein Lux-S. Schauder , S. and B. L. Bassler (2001). "The languages of bacteria." QUORUM SENSING MOLECULES 19/45

22. 20/45

23. This communication controls various functions reflecting the needs of a specific bacterial species to inhabit a particular niche such as the production of virulence factors, or by the transmission and acquisition of the generic information needed to produce virulence factors from other species in the biofilm development ( Passador et al ., 1993; Reading et al ., 2006). Several strains of P. intermedia, T. forsythia, F. nucleatum and P. gingivalis were found to produce quorum sensing signal molecules (Frias et al ., 2001; Sharma et al., 2005 ). 22/45

24. 23/45

25. 26 Strategies for quorum sensing inhibition 3 strategies can be applied Targeting AHL signal dissemination Targeting the signal receptor Targeting signal generation Signal precursor Signal Signal receptor Signal precursor Signal precursor Signal Signal Signal receptor Signal receptor X X X 24/45

26. 6 . ANTIBIOTIC RESISTANCE Bacteria growing in a biofilm are highly resistant to antibiotics, up to 1,000-1,500 times more resistant than the same bacteria not growing in a biofilm. 25/45 MIC of chlorhexidine and amine fluoride was 300 and 75 times greater respectively, when S.sobrinus was grown in biofilm compared to planktonic cells Biofilms of P.gingivalis tolerated 160 times the MIC of metronidazole than planktonic cells

27. . 27/45

28. 7. EXCHANGE OF GENETIC INFORMATION Conjugation , transformation and transduction have all been shown to occur in naturally occurring mixed species biofilms. Clinical Periodontology and Implant Dentistry by Jan Lindhe , 5th Edition . 28/45

29. Cells also communicate and interact with one another in biofilms via horizontal gene transfer. Gene transfer between Treponema denticola and S. gordonii has also been demonstrated in the laboratory. Wang BY, Chi B, Kuramitsu HK. Genetic exchange between Treponema denticola and Streptococcus gordonii in biofilms. Oral Microbiol Immunol 2002: 17: 108– 112. The presence of “pathogenicity islands” in periodontal pathogens such as P. gingivalis is also indirect evidence for horizontal gene transfer having occurred in plaque biofilms at some distant time in the past, and may explain the evolution of more virulent strains. Chen T, Hosogi Y, Nishikawa K, Abbey K, Fleischmann RD, Walling J, Duncan MJ. Comparative whole-genome analysis of virulent and avirulent strains of Porphyromonas gingivalis . J Bacteriol 2004: 186: 5473–5479. 29/45

30. Detachment Can be Movement of Individual cells or Biofilm en masse Brading et al have emphasized the importance of physical forces in detachment, stating that the three main processes for detachment are (JADA 1996) erosion or shearing (continuous removal of small portions of the biofilm) sloughing (rapid and massive removal), and abrasion (detachment due to collision of particles from the bulk fluid with the biofilm) 30/45

31. Individual Cell Transfer Erosion - detachment of single cells in a continuous predictable fashion 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. 31/45 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 - formation of the biofilm is not reliant on planktonic cells, which are known to be more susceptible to antimicrobial agents Stoodley 1991 En masse transfer

32. Factors affecting biofilm development and behavior ROLE OF SALIVA Saliva contains – mixture of glycoproteins – mucin. Bacteria – enzymes ( glycosidases ) – split off carb. – utilized as nutrients. Remaining protein – contributes to plaque matrix N euraminidase – separates sialic acid from salivary glycoprotein. Loss of sialic acid - ↓ salivary viscosity - Formation of precipitate – factor in plaque formation 32/45

33. 2. ROLE OF INGESTED NUTRIENTS Most readily utilized nutrients – diffuse easily into plaque – sucrose, glucose, fructose, maltose & less amt. of lactose. Dextran – greater quantity, adhesive properties , relative insolubility & resistance to destruction by bactera . Levan – Used as carbohydrate nutrient by plaque bacteria in absence of exogenous sources. 3. DIET AND PLAQUE FORMATION Consistency affects the rate of plaque formation : Forms rapidly on soft diets, hard chewy food retard it . Dietary supplements of sucrose ↑ plaque formation and affect its bacterial composition. i.e ECM Plaque formation occurs on high protein fat diets and carbohydrate - free diets but in smaller amounts. 33/45

34. IMPORTANCE OF FOOD CHAINS Stimulation of growth of other bacteria ( eg ) stimulation of growth of T.denticola by butyric acid produced by P.gingivalis Increasing the virulence of organisms ( eg ) more virulent strains of P.gingivalis in the presence of S.gordonii . Removal of toxic metabolites ( eg ) protection from hydrogen peroxide by A.neaslundii Utilization of metabolic products for maintaining structural integrity ( eg ) succinic acid produced by T.denticola integrated onto the cell wall of P.gingivalis 34/45

35. MICROBIAL SPECIFICITY 4/42 35/45

36. THEORY DRAWBACK Non-Specific Plaque Hypothesis(NSPH), Individuals with longstanding plaque and gingivitis do not develop periodontitis, while others, with minimal plaque, had lower resistance to disease. The Specific Plaque Hypothesis(SPH), Many of the organisms observed in periodontal health were also observed at diseased sites (Slots, 1977) The Ecological Plaque hypothesis (EPH) Does not address the role of genetic factors of the host that contribute to the composition of dental plaque and to susceptibility to disease The Keystone Pathogen Hypothesis(KPH). P.Gingivalis is one of the easily culturable micro organisms in plaque. Over 700 bacterial speicesare found in dental plaque. So it can be that any one of the uncultured micro-organisms can also create conditions ideal to the growth of periodontopathogens . 36/45

37. MICROBIAL SHIFT/DYSBIOSIS 37/45

38. MICROBIAL SHIFT LEADING TO PERIODONTITIS GRAM +VE AEROBES GRAM -VE ANAEROBES Gradually changes the symbiotic host–microbe relationship to a pathogenic one. Prevotella intermedia Fusobacterium nucleatum P. Gingivalis Tannerella forsythia Treponema denticola 38/45

39. Biofilm and host in conflict O ral microflora has a harmonious and + vely beneficial relationship with the host - microbial homeostasis. 39/45

40. Gingivitis Reduced plaque Increased plaque Reduced inflammation Increased inflammation Low GCF flow High GCF, bleeding, raised pH & temperature, low Eh Gram + ve bacteria Gram - ve anaerobes Gingival health Gingival health Periodontitis Stress Environmental change Ecological shift A schematic representation of the ecological plaque hypothesis in relation to periodontal disease Plaque biofilm accumulation can produce an inflammatory host response; this causes changes in the local environmental conditions and introduces novel host proteins and glycoproteins that favour the growth of proteolytic and anaerobic G – ve bacteria. In order to prevent or control disease, the underlying factors responsible for driving the selection of the putative pathogens must be addressed, otherwise disease will recur.

41. POSSIBLE STRATEGIES TO CONTROL ORAL BIOFILM Inhibiting Adherence with Antagonists Passive Immunization Replacement Therapy Regulating the Levels of Nonpathogenic Bacteria to Influence Virulence Probiotic Approaches Interference with Signaling Mechanisms Targeted Antimicrobial Therapy via a Novel STAMP Technology 41/45

42. 42/45

43. METHODS OF DETECTION OF DENTAL PLAQUE 43/45

44. 1. DIRECT VISION : - Thin plaque – may be translucent & therefore not visible Stained plaque – may be acquired e.g - tobacco stained Thick plaque – tooth may appear dull & dirty 2. USE OF EXPLORER : - Tactile Examination – when calcification has started it appears slightly rough, otherwise it may feel slippery due to coating of soft , slimy plaque Removal Of Plaque – when no plaque is visible , an explorer can be passed over the tooth surface & when plaque is present it will adhere to explorer tip.this technique is used when evaluating plaque index. This can be done by running the explorer or probe along the gingival 3 rd of the tooth 44/45

45. 3. Disclosing Agents: Two tone Erythrosine Bismark Benders Basic Fuschin Disclosing Tablets – Dental Mart, oral B 1 44/45

46. CONCLUSION Dental plaque biofilm cannot be eliminated permanently. However, the pathogenic nature of the dental plaque biofilm can be reduced by reducing the bioburden (total microbial load and different pathogenic isolates within that dental plaque biofilm) and maintaining a normal flora with appropriate oral hygiene methods that include daily brushing,flossing and rinsing with antimicrobial mouthrinses . This can result in the prevention or management of the associated sequelae, including the development of periodontal diseases and possibly the impact of periodontal diseases on specific systemic disorders. 45/45

47. REFERENCES Glickman; Clinical periodontology 4th edition: Saunders Carranza, Newman ;Clinical Periodontology, 8th edition: Saunders Newman, Takei, Klokkevold , Carranza; Clinical Periodontology; 10th Edition: Elseveir Jan Lindhe , Niklaus P. Lang, Thorkildkarring ; Clinical Periodontology And Implant Dentistry: 5th Edition: Blackwell Socransky SS, Haffajee AD. Dental biofilms: difficult therapeutic targets. Peridntol 2000 2002 : 28; 12-55. Marsh PD, Moter A, Devine DA. Dental plaque biofilms: commuinties , conflict and control. Periodontol 2000 2011; 55: 16-35 Max A. Listgarten , The structure of dental plaque , Periodontology 2000, Vol. 5, 1994, 52-65 Interspecies Interactions within Oral Microbial Communities Howard K . et al Microbiology And Molecular Biology Reviews, Dec. 2007, P. 653–670

48. THANK YOU