PLAQUE AS A BIOFILM part 2 Dr Suheti.pptx

Formation and maturation of dental plaque as a biofilm

1. PLAQUE AS A BIOFILM (Part 2) Dr. Suheti Vartak Dept of periodontics and implantology

2. CONTENT 2 - Definitions -History -Development of dental plaque - Role of macroscopic surface characteristics on biofilm -Role of microscopic surface characteristics on biofilm -Development of plaque as a biofilm - Structure of biofilm -Microbial composition -Microbial interactions -Factors affecting biofilm development -Theories on microbial specificity -Conclusion -References

3. DEFINATION Dental plaque is defined clinically as a structured resilient yellow-grayish substance that adheres tenaciously to the intraoral hard surfaces, including removable and fixed restorations. - Carranza defined as a specific but highly variable structural entity resulting from sequential colonization and growth of microorganisms of various strains and species on the surface of the teeth, restorations and other parts of the oral cavity. - WHO(1978) Plaque is defined as a diverse community of micro-organisms found on the tooth surface as a biofilm, embedded in an extracellular matrix of polymers of host and another microbial origin. - Marsh PD (2004) 3

4. BIOFILM Biofilm is described as a relatively undefinable microbial community associated with a tooth surface or any other hard , non shedding material. -According to Wilderer & Charaklis 1989 Biofilms consist of one or more communities of microorganisms, embedded in a shaped matrix or glycocalyx, that are attached to a solid surface. -According to Sigmund S. Socransky & Anne D. Haffajee.2001

5. HISTORY 5 First description of dental plaque biofilm- In the 17th century, Antonie van Leeuwenhoek He reported on diversity and high numbers of animalcules present in scrapings taken from around human teeth. The term ”Biofilm” – was coined by Bill Costerton in 1978. In 2002,Donlan and Costerton –described biofilm as ” A microbially derived sessile community characterized by cells that are irreversibly attached to a substratum or to each other ,embedded in a matrix of extracellular polymeric substances that they have produced and exhibit an altered phenotype with respect to growth rate and gene transcription”

6. The development of dental plaque was studied by Löe et al. (1965) in an experimental gingivitis study on dental students who were restrained from all oral hygiene measures for a period of three weeks. Following observations were made in the study.

7. DEVELOPMENT OF DENTAL PLAQUE 7 Soon after the tooth surface is thoroughly cleaned, it rapidly gets covered with a glycoprotein deposit referred to as "pellicle" which is derived from salivary constituents like albumin, lysozyme, amylase, immunoglobulin A, proline-rich proteins, and mucins, which are selectively adsorbed onto the tooth surface.

8. Further maturation of dental plaque leads to colonization of primarily Gram-negative species. These colonizers are referred to as "tertiary colonizers" which include Porphyromonas gingivalis , Campylobacter rectus, Eikenella corrodens , Aggregatibacter actinomycetemcomitans , and the oral spirochetes (Treponema species). A "corn-cob" like arrangement of microorganisms can be observed microscopically A "hedgehog" like arrangement of bacterial aggregation characterized by a mass of Corynebacterium filaments with Streptococcus at the periphery has been described by Welch et al. (2016). Clusters of Lautropia formed the center of a structure that also contained Streptococcus , Haemophilus / Aggregatibacter ,and Veillonella and was reminiscent of a cauliflower by Welch et al. (2016).

9. The natural history of periodontal disease. The correlation of selected microbiological parameters with disease severity in Sri Lankan tea workers H R Preus , A Anerud , H Boysen, R G Dunford, J J Zambon , H Löe The purpose of this study was to assess the prevalence of A. actinomycetemcomitans , Porphyromonas gingivalis and Prevotella intermedia, and their association with periodontal disease states in a population sample from Sri Lanka. Based on clinical parameters, a total of 536 sites in 268 male Sri Lankan tea workers were categorized as healthy, or showing gingivitis only, moderate or advanced periodontitis. Bacterial samples were obtained from all sites and the three target bacteria identified by indirect immunofluorescence . P. intermedia, P. gingivalis and A. actinomycetemcomitans were found in 76%, 40% and 15% of the subjects , respectively . Of the 536 periodontal sites, 10.5% were categorized with "no disease", 14% "gingivitis only": 59% with moderate and 16% with advanced periodontitis. The prevalence of P. gingivalis and P. intermedia was significantly higher in sites with moderate and advanced periodontitis than in sites with no disease or gingivitis only. A. actinomycetemcomitans was not found in healthy sites, but occurred with equal frequency in sites with gingivitis, moderate and advanced periodontitis. The association between these three bacteria and periodontal diseases in Sri Lankan tea laborers was similar to that described for other non-industrialized and industrialized countries.

10. ROLE OF MACROSCOPIC SURFACE CHARACTERISTICS DURING PLAQUE FORMATION Various macroscopic surface properties that affect bacterial colonization include surface energy, zeta potential, and hydrophobicity. All these properties are interrelated to each other. Surface energy and surface tension Surface energy is defined as the sum of all the intermolecular forces that are present on the surface of a material. It is the sum of the degree of attraction or repulsion forces of a material surface which it exerts on other materials. More is the surface energy; more is the tendency to attract. Surface tension can be defined as the resistance of a fluid to deform or break. The lower is the surface tension, lesser is the resistance to deform or rupture. When the substrate has a high surface energy (high tendency to attract) and the adhesive has a low surface tension, a good wettability is ensured. These are important properties affecting initial colonization of bacteria on the tooth surface

11. Furthermore, microorganisms with high free surface energy tend to adhere to surfaces with high surface free energy and vice versa. It has been demonstrated that most of the oral microorganisms have high surface energy and they tend to attach to the surfaces with high free surface energy, like enamel. The surface energy of enamel surface has been measured to be around 88 mJm2 (225). This finding can provide us another strategy to inhibit plaque formation, i.e. by lowering the surface free energy of the enamel surface. In vitro studies have done a comparative evaluation of plaque formation on enamel (surface free energy ~ 88 mJm³), cellulose-acetate (58 mJm ) and Teflon® (20 mJm ) surfaces. It was demonstrated that fewer microorganisms colonized the Teflon and cellulose-acetate surfaces than enamel.

12. ZETA POTENTIAL Zeta potential is defined as the potential difference existing between the surface of a solid particle immersed in a conducting liquid (e.g. water) and the bulk of the liquid. Bacterial adhesion is facilitated by a low negative charge on the bacterial surface. Various initial colonizers during plaque formation, such as strains of S. mutans, S. sanguis , S. salivarius , Actinomyces viscosus , and Actinomyces odontolyticus show relatively high surface free energies (range, 99-128 mJm ) and carry a negative surface charge which facilitates their adsorption over enamel surface .

13. HYDROPHOBICITY Hydrophobicity of various microorganisms varies considerably. Hydrophobic organisms are attracted towards the solid surfaces to rejection from the aqueous phase. There are several techniques which can be used to determine the degree of hydrophobicity of the bacterial cells. One commonly used technique is BATH (bacterial adherence to hydrocarbons) method, proposed by Rosenberg (1984), which is now more generally known as MATH (microbial adherence to hydrocarbons). Hydrophobicity favors bacterial adhesion. It has been shown that the hydrophobic Streptococci strains adhere better to hydroxyapatite surfaces in vitro as compared to the less hydrophobic strains

14. Role of microscopic surface characteristics during plaque formation The microscopic surface characteristics that affect bacterial adhesion to the tooth surface include surface characteristics of salivary components and microbial adhesins. Salivary components Salivary components can be considered as a major factor during initial bacterial adhesion to the tooth surface . Salivary oligosaccharide-containing glycoproteins have been shown to provide receptors for oral Streptococci in the salivary pellicle . salivary proline-rich protein I and statherin have been shown to provide receptor sites for type 1 fimbriae of Actinomyces viscosus . Microbial adhesins These adhesins are often associated with the surface appendages like fimbria, pilli or high-molecular-weight proteins extending from the microbial surface.

15. Physiochemical process of bacterial adhesion The physiochemical interactions between the bacterial cell and the surface of the substratum have been explained by thermodynamic model and DLVO ( Derjaguin , Landau, Ver wey , Overbeek ) theory. It describes the interaction energies between the interacting surfaces, based on electrostatic interaction and van der Waals forces and their decay with separation distance. According to this theory, the total interactive energy (V₂) of two smooth particles is the sum total of van der Waals forces (attractive energy) (V) and electrostatic energy (V.) (usually repulsive energy). A curve can be plotted to explain total interactive energy with the particle and substratum surface separated by a distance 'D' . In the DLVO graph, the net attraction is described at two values of D. Primary minimum. denotes net attraction at D with a very small value and secondary minimum where D is between 10-20 nm.

16. DENTAL PELLICLE FORMATION REVERSIBLE ADHESION IRREVERSIBLE ADHESION COLONIZATION CO-ADHESION MULTIPLICATION AND BIOFILM MATURATION DETACHMENT AND RECOLONIZATION FORMATION OF PLAQUE AS A BIOFILM

17. 1) Dental pellicle formation Here macromolecules present within the bulk of oral fluid settle onto a substrate through gravitational force or movement of flow. Weak electrostatic forces also play a significant role in this process. 2. Reversible adhesion Involving weak, long-range physicochemical interactions between the bacterial cell surface and the pellicle, which can lead to a stronger adhesin-receptor mediated attachment. Founder microbes attach to the tooth surface primarily through weak van der Waals forces. The reversible adhesion is affected by various factors including pH, temperature, surface energy and electrostatic forces. It has been observed that microbial adhesion strongly depends on the hydrophobic and hydrophilic properties of the interacting surfaces

18. Irreversible adhesion Involves the physical adhesion of bacteria on the tooth surface by overcoming the physical repulsive forces. The bacteria attach irreversibly to the tooth surface using fimbriae or pili that are present on their outer surface. The microorganisms excrete a mixture of exopolysaccharides and DNA, called the extracellular matrix (ECM) which aids in their attachment to the surface and gives them protection from the surrounding environmental conditions. Colonization involves an increase in the bacterial population, which results in the formation of a "proto biofilm" that grows in size by two ways: through the founder microorganisms dividing . microorganisms joining the biofilm from the surrounding environment. The microorganisms that join the biofilm aren't necessarily the same species of bacteria as the founder microorganisms. These organisms join the biofilm by attaching to the sticky extracellular matrix (ECM) of the biofilm.

19. Co-adhesion involves attachment of secondary colonizers to the already attached cells through cell surface interactions. It must be understood that bacterial co adhesion is very specific. Bacterial species attach specifically to other bacterial species. Some bacterial species can attach to many other bacterial species, while others may attach only to a few of them. For example, F. nucleatum can attach to various bacterial species, thus acting as a bridge or nucleus during plaque formation. Multiplication and biofilm maturation Results in a well established biofilm. A mature biofilm has a rough, irregular surface that contains many individual colonies of non-uniform, mushroom-shaped or finger-like columns surrounded by fluid-filled channels in which nutrients, enzymes, and waste products circulate. The water channels permit the passage of nutrients and other agents throughout the biofilm acting as a primitive "circulatory" system.

20. Detachment and re-colonization Involves the formation of new colonies. When the biofilm is large enough, the areas of the ECM are degraded by enzymes which lead to the dispersal of a portion of the biofilm, allowing cells to disperse and establish more biofilms. The bacteria in biofilms are phenotypically distinct from their genomically-identical planktonic counterparts. biofilm dispersal plays an important role in the transmission of bacteria from environmental reservoirs to human hosts, in horizontal and vertical cross-host transmission, and in the exacerbation and spread of infection within a host. Bacteria in biofilms can be up to 1000 times more resistant to antibiotics, and less conspicuous to the immune system because antigens are hidden and key ligands are suppressed.

21. STRUCTURE OF A MATURE BIOFILM 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

22. Composition: Composed of microcolonies of bacterial cells(15-20% by volume) ,distributed in a shaped matrix or glycocalyx (75-80% by volume) Bacteria in biofilm cluster together to from sessile, mushroom shaped colonies. Each microcolony is an independent community with its own living environment. In the lower levels of most biofilms-dense layer of microbes is bound together in a polysaccharide matrix with other organic and inorganic materials Successive layers-loose layer ,often irregular in appearance and may extend into surrounding medium. Fluid layer bordering the biofilm-has stationary and dynamic sub-layers.

23. Voids/Water channels: Voids or water channels present between the microcolonies in the biofilms. Permit the passage of nutrients Act as primitive circulatory systems. Composition of microcolony: Bacteria in centre of microcolony, strict anaerobic environment while those living at the edges of fluid channels maybe living in aerobic environment

24. Exopolysaccharides-the backbone of the biofilm Produced by bacteria present in the biofilm Major component of the biofilm(50-95% of dry weight) Role of EPS Maintain integrity of biofilm Prevent attack by harmful agents Bind essential nutrients such as cations to create favorable environment for specific microorganisms. Act as buffer and assist in retention of extracellular enzymes Many microorganisms can both synthesize and degrade the exopolysaccharides-distinguishing feature of biofilm

25. ATTACHMENT OF BACTERIA Key characteristic of 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 eg.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

26. MICROBIAL COMPOSITION Biofilm micro-organisms form 3 dimensional structured communities with fluid channels for transport of substrate ,waste products and signal molecules. Matrix that holds biofilm together contains-polysaccharides , proteins and DNA secreted by the bacterial cells.

27. MICROBIAL INTERACTIONS The residents in the microbial community display extensive interactions while forming biofilm structures. 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.

28. QUORUM SENSING The term 'quorum-sensing' was first used in a review by Fuqua et al. (1994)", which essentially reflected the minimum threshold level of individual cell mass required to initiate a concerted population response. It has been defined by Miller (2001) as “the regulation of gene expression in response to fluctuations in cell population density”. The signal molecule that is used for communication was called as 'autoinducer', owing to its origin inside the bacterial cell. The desired response can be arrived at by attainment of quorum employing the autoinducer and the process was labeled as 'autoinduction'. Quorum sensing is the regulation of bacterial gene expression in response to fluctuations in local signal concentration. This is done via the production and release of molecular autoinducers that increase in concentration as a function of cell density in a liquid culture; when quorum-sensing cells detect a minimum threshold stimulatory concentration of an autoinducer, gene expression changes *.*. Quorum sensing in bacteria "involves the regulation of the expression of specific genes through the accumulation of signaling compounds that mediate intercellular communication" ". Quorum sensing is dependent upon cell density. With few cells, signaling compounds may be produced at low levels; however, autoinduction leads to increased concentration as cell density increases. Once the signaling compounds reach a threshold level (quorum cell density), gene expression is activated.

29. Signaling is not the only way of transferring information in biofilms. The high density of bacterial cells growing in biofilms facilitates the exchange of genetic information between the cells of the same species and across the species or even genera. For example, genes for antibiotic resistance can be transferred through this efficient process. Other ways of communication between bacteria in a biofilm are, Through small diffusible peptides. Through horizontal gene transfer. Through autoinducer 2, which mediates the communication between Gram-positive and Gram-negative bacteria. QUORUM SENSING has Three types of molecules : Acyl-homoserine lactones (AHLs) - signaling molecules used by many G- ve bacteria, it synthesized by Lux-I family proteins. 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. Several strains of P. intermedia, T. forsythia, F. nucleatum and P. gingivalis were found to produce quorum sensing signal molecules .

30. Quorum sensing provides the biofilm its distinct properties: antibiotic resistance Influence community structure by encouraging the growth of beneficial bacteria and discouraging growth of competitors. Physiological properties of bacteria in community maybe altered

31. ANTIBIOTIC RESISTANCE PROPERTIES IN A BIOFILM Bacteria growing in a biofilm are highly resistant to antibiotics Upto 1,000-1,500 times more resistant than the same bacteria not growing in a biofilm. Slower growth rate of bacteria in the biofilm makes them less susceptible to antibiotics. The exopolymer matrix of biofilm acts as a barrier that may retard diffusion of antibiotics Extracellular matrix enzymes such as lactamase ,formaldehyde lyase and formaldehyde hydrogenase -become trapped in the extracellular matrix,thus inactivating,the typically positively charged ,hydrophilic antibiotics. Recently, the notion of a subpopulation of cells within a biofilm that are "super-resistant" was proposed. Such cells could explain remarkably elevated levels of resistance to certain antibiotics that have been suggested in the literature. Brooun et al. (2000) examined the 109 contribution of multi-drug resistance pumps to antibiotic resistance of microorganisms grown in biofilms. These "pumps" can extrude chemically unrelated antimicrobial agents from the cell.

32. Antibiotic resistance of bacterial biofilms NielsHøibyabThomasBjarnsholtabMichaelGivskovbSørenMolincOanaCiofub International Journal of Antimicrobial Agents April 2010 Abstract A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and DNA. Bacterial biofilms cause chronic infections because they show increased tolerance to antibiotics and disinfectant chemicals as well as resisting phagocytosis and other components of the body's defense system. The persistence of, for example, staphylococcal infections related to foreign bodies is due to biofilm formation. Likewise, chronic Pseudomonas aeruginosa lung infection in cystic fibrosis patients is caused by biofilm-growing mucoid strains. Characteristically, gradients of nutrients and oxygen exist from the top to the bottom of biofilms and these gradients are associated with decreased bacterial metabolic activity and increased doubling times of the bacterial cells; it is these more or less dormant cells that are responsible for some of the tolerance to antibiotics. Biofilm growth is associated with an increased level of mutations as well as with quorum-sensing-regulated mechanisms. Conventional resistance mechanisms such as chromosomal β-lactamase, upregulated efflux pumps and mutations in antibiotic target molecules in bacteria also contribute to the survival of biofilms. Biofilms can be prevented by early aggressive antibiotic prophylaxis or therapy and they can be treated by chronic suppressive therapy. A promising strategy may be the use of enzymes that can dissolve the biofilm matrix (e.g. DNase and alginate lyase) as well as quorum-sensing inhibitors that increase biofilm susceptibility to antibiotics.

33. EXCHANGE OF GENETIC INFORMATION IN BACTERIA Three main methods: Conjugation Transformation Transduction Cells also communicate and interact with one another in biofilms via horizontal gene transfer. 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

34. FACTORS AFFECTING BIOFILM DEVELOPMENT ROLE OF SALIVA Saliva contains – mixture of glycoproteins – mucin. Remaining proteins – contributes to plaque matrix Neuraminidase – separates sialic acid from salivary glycoprotein. Loss of sialic acid - ↓ salivary viscosity Formation of precipitate – factor in plaque formation ROLE OF INGESTED NUTRIENTS - Most readily utilized nutrients( sucrose, glucose, fructose, maltose & less amt. of lactose)- diffuse easily into plaque. Dextran- resistance to destruction by bacteria Levan – Used as carbohydrate nutrient by plaque bacteria in absence of exogenous sources. DIET AND PLAQUE FORMATION Consistency affects the rate of plaque formation : Forms rapidly on soft diets, hard chewy food retard it. Dietary supplements containing sucrose -↑ plaque formation and affect its bacterial composition.

35. Biofilm on dental implants: a review of the literature Abstract Purpose: The aim of this article was to review the current literature with regard to biofilm formation on dental implants and the influence of surface characteristics (chemistry, surface free energy, and roughness) of dental implant and abutment materials and their design features on biofilm formation and its sequelae. Materials and methods: An electronic MEDLINE literature search was conducted of studies published between 1966 and June 2007. The following search terms were used: biofilm and dental implants, biofilm formation/plaque bacterial adhesion and implants, plaque/biofilm and surface characteristics/roughness/surface free energy of titanium dental implants, implant-abutment interface and plaque/biofilm, biofilm and supragingival/subgingival plaque microbiology, biofilm/plaque and implant infection, antibacterial/bacteriostatic titanium, titanium nanocoating/nanopatterning, antimicrobial drug/titanium implant. Both in vitro and in vivo studies were included in this review. Results: Fifty-three articles were identified in this review process. The articles were categorized with respect to their context on biofilm formation on teeth and dental implant surfaces and with regard to the influence of surface characteristics of implant biomaterials (especially titanium) and design features of implant and abutment components on biofilm formation. The current state of literature is more descriptive, rather than providing strong data that could be analyzed through meta-analysis. Basic research articles on surface modification of titanium were also included in the review to analyze the applications of such studies on the fabrication of implant surfaces that could possibly decrease early bacterial colonization and biofilm formation. Conclusions: Increase in surface roughness and surface free energy facilitates biofilm formation on dental implant and abutment surfaces, although this conclusion is derived from largely descriptive literature. Surface chemistry and the design features of the implant-abutment configuration also play a significant role in biofilm formation.

36. CONCLUSION Dental plaque biofilms are complex communities of bacteria . The nature of the biofilm enhances the component bacteria’s resistance to both the host defense system and antimicrobials. If not removed regularly,the biofilm undergoes maturation, and the resulting pathogenic bacterial complex can lead to dental caries, gingivitis and periodontitis. An understanding of the nature and pathophysiology of the dental biofilm is important to implement proper management strategies .Although dental biofilm cannot be eliminated,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 when required. This can result in the prevention or management of the associated sequelae, including the development of periodontal diseases

37. REFRENCES Clinical periodontology- Newman and Carranza (13th edition) Clinical Periodontology and Implant Dentistry-Jan Lindhe (7 th edition) Review article,Dental Plaque:A complex Biofilm,Saini R et al,Pravara Med Rev 2015;7 Subramani K, Jung RE, Molenberg A, Hammerle CH. Biofilm on dental implants: a review of the literature. Int J Oral Maxillofac Implants. 2009 Jul-Aug;24(4):616-26. PMID: 19885401.

38. Thank you 40