biofilm and its significance in endodontics.pptx

Biofilm significance in endodontics

1. Biofilm and its significance in endodontics- Part 1 1

2. Contents Introduction History Of Biofilm Definition Basic Criteria For A Biofilm Ultra-structure Characteristics Development Of Biofilm Methods Of Observing Biofilms In Endodontics 2

3. Contents Protection Of Biofilm Bacteria From Environmental Threats Nutrient Trapping And Metabolic Cooperativity In A Biofilm Exchange Of Genetic Material Quorum Sensing Resistance Of Microbes In The Biofilm To Antimicrobials Benefits Of Biofilm To Microbes Oral Biofilm 3

4. INTRODUCTION Pulpal and periradicular pathologies - Etiology = microflora . Infection in the oral cavity is caused by a number of organisms from different species found in the human mouth. Oral bacteria - form biofilms on distinct surfaces Fundamental to maintain oral health and prevent dental caries, gingivitis, and periodontitis - control the oral biofilms . 4

5. Biofilm - advantageous for microorganisms - they form 3-D structured communities with fluid channels for transport of substrate, waste products, and signal molecules . Svensäter and Bergenholtz : Biofilm formation in root canals is probably initiated sometime after the first invasion of the pulp chamber by planktonic oral microorganisms after some tissue breakdown.   Svensäter G, Bergenholtz G. Biofilms in endodontic infections.  Endod Topics. 2004;9:27–36. 5

6. Costerton   et al . stated that biofilm consists of single cells and microcolonies , all embedded in a highly hydrated, predominantly anionic exopolymer matrix. Bacteria can form biofilms on any surface that has nutrient-containing fluid Biofilm formation mainly involves the three major components: Bacterial cells, a solid surface, and a fluid medium . Costerton JW, Cheng KJ, Geesey GG, Ladd TI, Nickel JC, Dasgupta M, Marrie TJ Annu Rev Microbiol . 1987; 41():435-64. 6

7. Bacterial biofilms - apical root canals These bacterial endodontic communities are often found adhered to or at least associated with the dentinal canal walls , with bacterial cells encased in an extracellular amorphous matrix . 7

8. In any natural environment , bacteria show the tendency to aggregate in adherent microbic communities. The biofilms forms on any surface that comes in contact with natural liquids. Biofilms - represent a natural scenario for bacterial communication. Pseudomonas aeruginosa Aggregates 8

9. Biofilms grow where there is a combination of : Moisture Nutrient supply Surface Sites include : Natural materials above and below ground Metals & plastics Medical implant materials Plant and body tissue Staphylococcus aureus biofilm on the surface of a catheter 9

10. HISTORY OF BIOFILM Anton Von Leeuwenhoek (17 th century) - saw microbial aggregates (now known to be Biofilms) on scrapings of plaque from his teeth. Bill Costerton (1978) – coined the term ‘Biofilm’ Donlan and Costerton (2002)- offered the most salient description of a biofilm. 10

11. 11 A sessile multi-cellular microbial community characterized by cells that are firmly attached to a surface and enmeshed in a self produced matrix of extracellular polymeric substances.

12. Definition Biofilm is a mode of microbial growth where dynamic communities of interacting sessile cells are irreversibly attached to a solid substratum, as well as each other, and are embedded in a self made matrix of extracellular polymeric substance (EPS). - Ingle 12

13. Biofilm is defined as a community of micro-colonies of microorganisms in an aqueous solution that is surrounded by a matrix of glycocalyx, which also attaches the bacterial cells to a solid substratum. -Grossman 13

14. BASIC CRITERIA FOR A BIOFILM Cadwell et al : Autopoiesis Homeostasis Synergy Communality 14

15. ULTRA STRUCTURE OF BIOFILM Composed primarily of microbial cells and glycocalyx like matrix (Extracellular polymeric substance) Fully developed biofilm is described as heterogeneous arrangement of microbial cells on a solid surface. 15

16. Basic structural unit of a biofilm is the microcolonies or cell cluster formed by the surface adherent bacterial cells. 85% : - Matrix (polysaccharides, proteins, nucleic acid & salts) 15% : - Bacterial cells 16

17. Viable, fully hydrated biofilm appears as ‘ tower ’ or ‘ mushroom ’ shaped structure adherent to the substrate . 17

18. Water channels : establish connection between microcolonies . Facilitates efficient exchange of materials between bacterial cells and bulk fluid Coordinate functions in a biofilm community. 18

19. Matrix A glycocalyx matrix made up of extracellular polymeric substance (EPS) surrounds the microcolonies and anchors the bacterial cell to the substrate. 19

20. Functions of matrix: Maintains the integrity of biofilms Prevents dessication Resists antimicrobial agents Create a nutritionally rich environment Acts as a buffer and retains extracellular enzymes 20

21. Slow bacterial growth = EPS production ↑ EPS is highly hydrated = it prevents lethal desiccation = protect against diurnal variations in humidity. Mayer et al(1999) : EPS helps in mechanical stability of the biofilms - withstand shear forces. 21

22. Different organisms produce differing amounts of EPS Amount of EPS ↑ with age of the biofilm. EPS production is known to be affected by Nutrient status of the growth medium Excess available carbon and limitation of nitrogen, potassium, or phosphate promote EPS synthesis 22

23. Bacterial cells within matrix produce Beta- lactamase against antibiotics Also produce catalases and superoxide dismutase against oxidising ions of phagocytes Elastases and cellulases produced by bacteria are concentrated in the matrix and produce tissue damage. 23

24. CHARACTERISTICS OF BIOFILM Biofilm structure protects the residing bacteria from environmental threats. Structure of biofilm permits trapping of nutrients and metabolic cooperativity between resident cells of the same species and or different species. 24

25. Biofilm structure displays organized internal compartmentalization. Bacterial cells in a biofilm community may communicate and exchange genetic material to acquire new traits. 25

26. Internal Compartmentalization A mature biofilm structure displays gradients in the distribution of nutrition, pH, oxygen, metabolic products and signaling molecules within the biofilm. This would create different microniches that can accommodate diverse bacterial species within a biofilm. 26

27. DEVELOPMENT OF BIOFILM  Bacteria can form biofilm on any surface that is bathed in a nutrition containing fluid. 3 major components involved in biofilm formation Bacterial cells A solid surface A fluid medium 27

28. Stages of biofilm formation According to Kishen : 1 st stage : Formation of conditioning layer 2 nd stage : Planktonic bacterial cell attachment 3 rd stage : Bacterial growth and biofilm expansion 4 th stage : Detachment(seeding dispersal) 28

29. Stage 1 Adsorption of inorganic and organic molecules to the solid surface, forming a thin layer termed as conditioning layer. During dental plaque formation, the tooth surface is conditioned by the saliva pellicle. 29

30. Stage 2 Adhesion of microbial cells to the conditioning layer . • Phase 1 - Transport of microbes to surface. • Phase 2 - Initial non specific microbial-substrate adherence phase. • Phase 3 - Specific microbial-substrate adherence phase. Pioneer organisms : streptococci (major population)  30

31. STAGE 2 : Phase 1 : Transport of microbes to substrate surface Several factors that affect bacterial attachment to a solid substrate. ph Temperature Surface energy of the substrate Flow rate of the fluid passing over the surface Nutrient availability Length of time the bacteria is in contact with the surface Bacterial growth stage Bacterial cell surface charge Surface hydrophobicity .   31

32. STAGE 2 : Phase 1 : Transport of microbes to surface Physicochemical properties such as surface energy and charge density determine the nature of initial bacteria-substrate interaction Microbial adherence to a substrate is also mediated by bacterial surface structures ( fimbriae , flagella, and EPS (glycocalyx) ) Bacterial surface structures form bridges between the bacteria and the conditioning film. 32

33. STAGE 2 : Phase 2 : Initial non specific microbial-substrate adherence phase  Bridges formed are a combination of Electrostatic attraction Covalent and hydrogen bonding Dipole interaction Hydrophobic interaction Bacteria with surface structures: P. gingivalis , S. mitis , Streptococcus salivarius , P. intermedia , P. nigrescens , S. mutans 33

34. STAGE 2 : Phase 3 : Specific microbial-substrate adherence phase Initial bonds : not strong With time these bonds gains in strength, making the bacteria-substrate attachment irreversible. Finally, a specific bacterial adhesion with a substrate is produced via polysaccharide adhesin or ligand formation . 34

35. Adhesin or ligand on the bacterial cell surface will bind to receptors on the substrate . Specific bacterial adhesion is less affected by many environmental factors such as electrolyte, pH, or temperature. 35

36. STAGE 3 Bacterial growth and expansion. 36

37. Co-adhesion Co-Aggregation Two types of microbial interactions occur at the cellular level 37

38. STAGE 4 : Detachment / Dispersion Biofilm transfer particulate constituents from the biofilm to the fluid bathing the biofilm. Detachment plays an important role in shaping the morphological characteristics and structure of mature biofilm. 38

39. Active dispersive mechanism Seeding dispersal 39

40. Because of flow effects, biofilm cells may be dispersed by Shedding of daughter cells from actively growing cells Detachment as a result of nutrient levels Quorum sensing Shearing of biofilm aggregates 40

41. Brading et al have emphasized the importance of physical forces in detachment. 3 main processes for detachment are: Erosion or shearing Sloughing Abrasion 41

42. Methods of Observing Biofilms in Endodontics Light microscopy + histological staining Electron microscopy Scanning electron microscopy (SEM) Atomic force microscopy (AFM) Fluorescent in situ hybridization (FISH) Transmission electron microscopy (TEM) Confocal Laser Scanning Microscopy 42 Mohammadi Z, Palazzi F, Giardino L, Shalavi S. Microbial biofilms in endodontic infections: an update review. Biomedical journal. 2013;36(2):59-70.

43. Methods of Observing Biofilms in Endodontics Microtiter Plate-Based Systems Flow Displacement Biofilm Model Systems Modified Robbins Device Microfluidic Device Fluorescent Microscopic Techniques with Super Resolution 43 Neelakantan P, Romero M, Vera J, Daood U, Khan AU, Yan A, Cheung GS. Biofilms in endodontics—current status and future directions. International journal of molecular sciences. 2017 Aug;18(8):1748.

44. Protection of Biofilm Bacteria from Environmental Threats Bacteria residing in a biofilm experiences certain degree of protection and homeostasis . Bacteria are capable of producing polysaccharides, either as cell surface structures or as extracellular excretion. 44

45. EPS creates microniche . Protects from environmental stresses. Sequestration of metallic cations and toxins. Physically prevents diffusion of certain compounds by acting as ion exchanger.   45

46. Nutrient Trapping And Metabolic Co- operativity In A Biofilm An important characteristics of biofilm growing in a nutrient deprived ecosystem is its ability to concentrate trace elements and nutrients by physical trapping or by electrostatic interaction . 46

47. Highly permeable and interconnected water channels provide an excellent means of material exchange . The complex architecture of biofilm provides opportunity for metabolic cooperation and niches are formed within these spatially well-organized systems. 47

48. EXCHANGE OF GENETIC MATERIAL Bacterial biofilm provides a setting for the residing bacterial cells to communicate with each other. The ability to communicate through quorum-sensing has been shown in some oral streptococci and some periodontal pathogens. -( Loo et al. ) For most oral biofilm micro-organisms Presence and function of signal transduction pathways Quorum-sensing communication 48

49. Quorum Sensing Bacteria in a community may convey their presence to one another by producing, detecting, and responding to small diffusible signal molecules called AUTOINDUCERS . This process of intercellular communication, called QUORUM SENSING Involved in the formation of biofilms and to cope with environmental stresses 49

50. Quorum sensing molecules 50

51. Quorum sensing is mediated by low molecular weight molecules which in sufficient concentration can alter metabolic activity of neighboring cells and coordinates in the function of resident bacterial cells within a biofilm Quorum Sensing 51

52. Dependent on cell density Mediated through signaling compounds. The signals are thought to allow cross-talk between species, causing them to increase their production of exopolysaccharide and the factors that increase their virulence . Quorum sensing 52

53. Quorum sensing gives biofilms their distinct properties. Exchange of genetic material between bacterial species Evolution of microbial communities with different traits 53

54. Resistance of Microbes in the Biofilm to Antimicrobials The nature of biofilm structure and physiological characteristics of resident microorganisms offer an inherent resistance to antimicrobial agents, such as antibiotics, disinfectants, or germicides. Mechanism responsible for resistance: Resistance associated with extracellular polymeric matrix. Resistance associated with growth rate and nutrient availability. 54

55. Resistance associated with EPS : • Diffusion barrier • Direct neutralization of antimicrobials • Inactivation by modified enzymes produced by bacteria. Resistance associated with growth rate and nutrient availability : • Susceptibility towards antimicrobial is directly proportional to growth rate. • Resistance increases as thickness of biofilm increases 55

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57. Benefits Of Biofilm To Microbes Helps the bacteria to survive in unfavorable environmental and nutritional conditions. Resistance to antimicrobial agents. Increase in local concentration of nutrients. Opportunity of genetic material exchange . Ability to communicate between bacterial population of same and or different species. Produce growth factors across species boundaries. 57

58. ORAL BIOFILMS  Oral bacteria have the capacity to form biofilms on distinct surfaces ranging from hard to soft tissues. Oral biofilms are formed in three basic steps : Pellicle formation Bacterial colonization Biofilm maturation Composition : 80% : Water 20% : Inorganic & organic (80% bacteria)   58

59. Organic : Carbohydrates, proteins, and lipids. Carbohydrates : produced by bacteria : glucans , fructans , or levans . - Contribute to the adherence of microorganisms to each other and are the stored form of energy in biofilm bacteria. Proteins ( supragingival biofilm) : derived from saliva Proteins ( subgingival biofilm) : derived from gingival sulcular fluid. Lipid : Endotoxins (LPS) from Gram-negative bacteria. Inorganic : Calcium, phosphorus, magnesium and fluoride.  59

60. Saliva contains proline -rich proteins : aggregate together to form micelle like globules called salivary micelle-like globules (SMGs). SMGs : adsorbed to the clean tooth surface to form acquired enamel pellicle, which acts as a ‘‘ foundation’’ for the future multilayered biofilm . Presence of calcium facilitates the formation of larger globules by bridging 60

61. Acquired Pellicle attracts Gram positive cocci Filamentous bacterium adheres to primary colonizers. Gradually, the filamentous form grows into the cocci layer and replaces many of the cocci. Vibrios and spirochetes appear as the biofilm thickens. 61

62. Coaggregation of F. nucleatum with coccoid bacteria gives rise to “ CORNCOB ” structure, which is unique in plaque biofilms. Presence of these bacteria makes it possible for other non-aggregating bacteria to coexist in the biofilm, by acting as coaggregating bridges . 62

63. Calcified dental biofilm is termed as Calculus . Formed by the precipitation of calcium phosphates within the organic plaque matrix. Factors that regulate the deposition of minerals on dental biofilms are Physicochemical factors : • Plaque pH • Local saturation of Ca, P and fluoride ions Biological factors such as presence of crystallization nucleators /inhibitors. 63

64. Dental biofilm (plaque) formed on the tooth surface is harmless under normal conditions. A shift in microenvironment due to repeated use of ‘‘habit forming’’ substances, diet, and host immune response can lead to biofilm-mediated infections or diseases in the oral cavity. According to the ‘‘ ecological plaque hypothesis ,’’ any environmental change that favours increasing colonization by potential pathogenic bacteria would cause disease 64

65. A decline in the host defense mechanisms caused by disease or immuno -suppressive medicaments may also render generally ‘‘ harmless commensals ’’ to become ‘‘ opportunistic pathogens .’’ Ex. diseases caused by biofilm community: Dental caries Gingivitis Periodontitis Peri-implantitis Apical periodontitis 65

66. Biofilm and its significance in endodontics- Part 2 66

67. Contents - Part 1 Introduction History Of Biofilm Definition Basic Criteria For A Biofilm Ultra-structure Characteristics Development Of Biofilm Methods Of Studying Biofilms In Endodontics Internal Compartmentalization Protection Of Biofilm Bacteria From Environmental Threats Nutrient Trapping And Metabolic Cooperativity In A Biofilm Exchange Of Genetic Material Quorum Sensing Resistance Of Microbes In The Biofilm To Antimicrobials Resistance Of Microbes Benefits Of Biofilm To Microbes Oral Biofilm 67

68. Contents – Part 2 Endodontic Biofilm Microorganisms Involved In Biofilm Formation Endodontic Biofilm Formation Mechanism Types Of Endodontic Biofilm Role Of E. Feacalis  In Biofilm Effects Of Instrumentation On Biofilms Methods To Eradicate Biofilms Conclusion References 68

69. Endodontic biofilm Therapeutically significant One of the basic survival methods employed by bacteria in times of starvation . Responsible for endodontic failures. 69

70. Endodontic biofilms help the bacteria to survive 70

71. Less diverse compared to the oral microbiota Progression of infection alters the nutritional and environmental status within the root canal. Root canal environment : more anaerobic and depleted nutritional levels. 71

72. Microbes : anatomical complexities Shelter the adhering bacteria from cleaning and shaping procedures. Bacterial activities not confined to intracanal spaces, but also beyond the apical foramen. 72

73. The physical conditions available to support the growth of bacteria, pH Ionic concentration Nutrient availability Oxygen supply 73

74. Advantages of Biofilm mode of bacterial growth : Resistance to antimicrobial agents Increase in the local concentration of nutrients Opportunity for genetic material exchange Ability to communicate between bacterial populations of same and/or different species Produce growth factors across species boundaries 74

75. Protection from killing by antimicrobial agents 3 Mechanisms that confer antimicrobial tolerance to cells living in a biofilm 1 st : barrier properties of the EPS matrix Extracellular enzymes such as ß lactamase may become trapped and concentrated in the matrix, thereby inactivating ß lactam antibiotics   75

76. 2 nd : mechanism involves the physiological state of biofilm microorganisms . Bacterial cells residing within a biofilm grow more slowly than planktonic cells, as a result, biofilm cells take up antimicrobial agents more slowly. 3 rd mechanism : microorganisms within the biofilm experience metabolic heterogeneity. 76

77. Interaction between E.faecalis biofilm and root canal dentin substrate By examining the shift in chemical composition of biofilm structure with time. By studying the topography and ultrastructure of the biofilm and dentin substrate. Study Showed : E.faecalis formed biofilm on root canal dentin Bacteria induced dissolution of the mineral fraction from the dentin substrate A precipitated apatite layer was formed in the biofilm structure Kishen A, George S, Kumar R. Enterococcus faecalis ‐mediated biomineralized biofilm formation on root canal dentine in vitro. Journal of Biomedical Materials Research Part A: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials. 2006 May;77(2):406-15. 77

78. 400 different bacterial species - root canal of teeth Endodontic biofilms - 20 species of bacteria(>30) The most frequent bacteria in endodontic biofilms belong to the phyla Firmicutes , Proteobacteria , Spirochaetes , Bacteroidetes , and Actinobacteria Microorganisms involved in biofilm formation 78

79. Gram-positive bacteria Common streptococci Streptococcus intermedius , S. constellatus S. mutans , facultative or microaerophilic streptococci Common enterococci Enterococcus faecalis Gram-positive anaerobes species belonging to the genera Peptostreptococcus , Eubacterium , and Pseudoramibacter Walsh LJ. Novel Approaches to Detect and Treat Biofilms within the Root Canals of Teeth: A Review. Antibiotics. 2020 Mar;9(3):129. 79

80. Gram-negative bacteria Gram-negative anaerobic bacteria species belonging to the genera Fusobacterium , Porphyromonas , Prevotella , and Campylobacter Gram-negative bacteria Tannerella forsythia, Porphyromonas gingivalis , P. endodontalis , Prevotella intermedia , P. nigrescens , Fusobacterium periodonticum , F. nucleatum , and Eikenella corrodens Spirochaetes ( treponemes ) include Treponema denticola , T. socranskii , T. maltophilum , T. lecithinolyticum , T. vincentii , T. pectinovorum , T. amylovorum , and T. medium 80

81. ENDODONTIC BIOFILM FORMATION MECHANISM 81

82. In the breakdown process - steady state is reached where the bacterial mass is held up by host defense mechanisms. Demarcation zone : Inside the root canal near the root canal exit, at the foramen External root surface near the exit of the foramen to the periapical tissue environment. 82

83. Types of endodontic biofilm 83

84. INTRACANAL BIOFILMS Microbial biofilms formed on the root canal dentin of an endodontically infected tooth . Nair et al (1987) Biofilm : Cocci, rods, and filamentous bacteria. Bacterial condensations - palisade structure Monolayer and/or multi-layered bacterial biofilms Morphologically distinct types of bacteria 84

85.  E. faecalis - resist starvation and develop biofilms under different environmental and nutrient conditions Modified according to the prevailing environmental and nutrient conditions. Nutrient-rich environment (aerobic and anaerobic) Surface aggregates of bacterial cells and water channels. Viable bacterial cells were present on the surface of the biofilm. Nutrient-deprived environment (aerobic and anaerobic) Irregular growth of adherent cell clumps were observed. 85

86. Nutrient deprived condition after 1 week Nutrient rich condition after 1 week Nutrient deprived condition after 4 weeks Nutrient rich condition after 4 weeks 86

87. Stages of intracanal bacterial formation Stage I : Adherence and micro colonies Stage II : Dissolution of dentin and Stage III : Mineralization of biofilm ( Carbonated apatite structure ) Siren et al : Ability of E. faecalis to coaggregate with F. nucleatum . Coexist in a microbial community : endodontic infection 87

88. Investigations have also demonstrated biting force-induced retrograde fluid movement into the apical portion of the root canal (apical retrograde fluid movement) Cyclic influx of ion-rich tissue fluid into the apical portion of the root canal can promote persistence of bacteria as biofilms and their mineralization . 88

89. EXTRA-RADICULAR BIOFILM Root surface biofilms Root surface (cementum) adjacent to the root apex of endodontically infected teeth . Reported in Asymptomatic periapical periodontitis Chronic apical abscesses associated with sinus tracts. 89

90. Cocci and short rods, with cocci attached to the tooth substrate. Filamentous and fibrillar forms Riccuci et al - Calcified biofilms  No obvious difference in the biofilm structures formed on the apical root surface of teeth with and without sinus tracts. 90

91. PERIAPICAL MICROBIAL BIOFILMS Isolated biofilms found in the periapical region of an endodontically infected teeth. May or may not be dependent on the root canal. Actinomyces and P. propionicum : Ability to overcome host defense mechanisms Thrive in the inflamed periapical tissue, and subsequently induce a periapical infection. 91

92. Actinomyces species : Grow in microscopic or macroscopic aggregates. ‘‘Sulfur Granules,’’ Microscopically : appearance of rays projecting out from a central mass of filaments. (Ray fungus)   92

93. BIOMATERIAL-CENTERED INFECTION (BCI) Caused when bacteria adheres to an artificial biomaterial surface and forms biofilm structures. Intraradicular Extraradicular Filaments, long rods , and spirochete-shaped bacteria were predominant in the biofilm formed on guttapercha . 93

94. Presence of biomaterials in close proximity to the host immune system can increase the susceptibility to BCI. Major complications associated with prosthesis and/or implant-related infections. Reveals opportunistic invasion by nosocomial organisms 94

95. Coagulase -negative staphyococcus , S.aureus , enterococci , P.aeruginosa and fungi 3 phases of bacterial adhesion to biomaterial surface : phase1 : transport of bacteria to biomaterial surface. phase2 : initial, nonspecific adhesion phase phase3 : specific adhesion phase 95

96. BCI IN ENDODONTICS A study investigated the initial biofilm-forming ability of root canal isolates on gutta-percha points in vitro. E. faecalis and S. sanguinis biofilms were significantly thicker than others. P. gingivalis, and P. intermedia did not form biofilms on gutta-percha. Suggests that Gram-positive facultative anaerobes have the ability to colonize and form extracellular matrices on gutta - percha points Takemura N, Noiri Y, Ehara A, Kawahara T, Noguchi N, Ebisu S. Single species biofilm‐forming ability of root canal isolates on gutta‐percha points. European journal of oral sciences. 2004 Dec;112(6):523-9. 96

97. Extraradicular microbial biofilms formed on tissue or biomaterial surface were related to refractory periapical disease Moine P, Abraham E. Immunomodulation and sepsis: impact of the pathogen. Shock. 2004 Oct 1;22(4):297-308. 97

98. ROLE OF  E. FEACALIS  IN BIOFILM Bacterial isolates from endodontic infections - E. faecalis  - capacity to form biofilms. The persistence of endodontic bacteria via biofilm formation – completely eliminate bacteria during endodontic retreatment and isolate all the existing microorganisms from infected root canals. Complex anatomy of the root canal poses difficulties - biofilms of persistent microorganisms within the root canals may be located on the walls of ramifications and isthmuses. 98

99. E. faecalis  is a gram-positive , facultative anaerobic coccus that is strongly associated with endodontic infections. Opportunistic pathogen - causes nosocomial infections - isolated from the failed root canals undergoing retreatment.   E. faecalis  isolated from infective endocarditis patients were significantly associated with greater biofilm Cause of failure of endodontic therapy - survival of microorganisms in the apical portion of the root-filled tooth. 99

100.   E. faecalis  - suppress the action of lymphocytes leading to endodontic failure. E. faecalis  in dentinal tubules resist ICM of calcium hydroxide for over 10 days by forming a biofilm that helps it resist destruction by enabling the bacteria to become 1000 times more resistant to phagocytosis , antibodies, and antimicrobials than non-biofilm producing organisms. Calcium hydroxide - ineffective to kill  E. faecalis  on its own, if a high pH is not maintained. At a pH of 11.5 or greater,  E. faecalis  is unable to survive. 100

101. EFFECTS OF INSTRUMENTATION ON BIOFILMS By mechanical instrumentation and irrigation with tissue- lytic and microbicidal solutions and antimicrobial medicaments in the root canal, the microbial load is reduced leading to disruption of biofilm. NaOCl in different concentrations is used as a root canal irrigant because of its antimicrobial action and tissue-dissolving property. 101

102. Previous studies have shown that instrumentation and antibacterial irrigation with NaOCl would eliminate bacteria in 50–75% of the infected root canals at the end of the first treatment session, whereas the remaining root canals contain recoverable bacteria. Nair  et al . showed that 88% of root canal of treated mandibular molars showed residual infection of mesial roots after instrumentation, irrigation with NaOCl, and obturation in a one-visit treatment. 102

103. Methods to eradicate biofilms 103

104. Sodium hypocholorite : Most potent disinfectant in endodontics Excellent ability to dissolve vital and necrotic tissues and has antimicrobial activity Used in concentration from 0.5% to 6% Studies show that the antimicrobial activity is not concentration dependent, but tissue dissolution and biofilm disruption are concentration dependent Dunavant et al (2006) concluded that both 1% NaOCl and 6% NaOCl were more efficient in eliminating E.faecalis biofilm than the other solutions tested 104

105. According to Ozdemir et al, the combined application of 17% EDTA and 2.5% NaOCl reduces the amount of intracanal biofilm significantly Effective against biofilms ( p.intermedia , peptostreptococcus micros, streptococcus intermedius , fusobacterium nucleatum and E. Faecalis ) - disrupts oxidative phosphorylation and inhibits DNA synthesis of bacteria . 105

106. The effectiveness of sodium hypochlorite may be improved by warming the solution, use of agitation/activation methods, increasing the volume of the irrigant, and lowering the pH of the irrigant solution 106

107. The endodontic literature is consistent in demonstrating that NaOCl is able to completely disrupt the biofilms within the root canal system. However, Rosen et al., reported a very interesting finding that NaOCl induces a viable but non- culturable state of bacteria in biofilms and that this might contribute to bacterial persistence 107

108. Chlorhexidine digluconate Gram + ve & Gram- ve bacteria Ability to denature the bacterial cell wall while forming pores in the membrane. Lower grade of toxicity compared to sodium hypochlorite and sustained action( substantivity ) Concentration of 2% 108

109. Arias- Moliz et al ., showed that alternating the application of CHX and cetrimide resulted in a higher percentage reduction of E.fecalis compared to the combined use of these 2 agents . Cetrimide facilitates the destruction of EPS matrix allowing CHX to act more directly on E.fecalis thus resulting in a greater bactericidal potential 109

110. Alexidine (ALX) Bisbiguanide Introduced as a root canal irrigant Increased permeability into the bacterial membrane ALX (1%) has been shown to bring about bacterial killing similar to 2% CHX, although both agents do not appear to disrupt the biofilms of E. fecalis 110

111. 111

112. Chelating agent Little or no antimicrobial activity Soares et al , studied the effectiveness of chemomechanical preparation with alternating use of NaOCl and EDTA on an intracanal E. fecalis biofilm and found that the alternating use of these 2 agents promoted the elimination of root canal E. fecalis biofilm Remove the organic and inorganic debris & disrupt microbial biofilms EDTA 112

113. 113

114. Peracetic acid (PAA) 2.25% PAA solution Final irrigant after the use of sodium hypochlorite during instrumentation. Peracetic acid has been shown to be more effective than chlorhexidine against root canal mono-species E. fecalis biofilms . Single irrigant with two purposes demineralizing agent strong antibacterial properties 114

115. Prabhakar and coworkers showed complete inhibition of bacterial growth by MTAD in a 3 week old biofilm Mixture of 3% doxycycline hyclate , 4.25% citric acid 0.5% Tween 80 MTAD 115

116. BioPure MTAD ( Dentsply Tulsa Dental, Johnson City, TN, USA) has been described as a universal irrigating solution. Torabinejad   et al . have shown that MTAD removes the smear layer safely; also, it is effective against  E. faecalis  and it can eliminate bacteria in human root canals that had been infected by whole saliva. 116

117. Tetraclean Pappen FG et al (2010 ) - Tetraclean is more effective than MTAD against E.faecalis biofilm Cetrimide in tetraclean improved the antimicrobial properties of the solutions Ability to eliminate microorganisms and smear layer in dentinal tubules of infected root canals with a final 4-min rinse 117

118. QMiX QMiX consists of EDTA, chlorhexidine and detergent. As effective as NaOCl and superior to CHX against E.fecalis in planktonic and biofilm states It is effective as 6% NaOCl in killing 1-day old E.faecalis Slightly less effective against bacteria in 3 week old biofilm. 118

119. Antibacterial Nanoparticles Antibacterial nanoparticles bind to negatively charged surfaces and have excellent antimicrobial and antifungal activities. Studies have also shown that the treatment of root dentin with Zno nanoparticles , Chitosan -layer- ZnO nanoparticles , or chitosan nanoparticles produces an 80 to 95% reduction in the adherence of E.faecalis to dentin . 119

120. Chitosan Nanoparticles – Antibacterial, antiviral, and antifungal properties Dentin treated with nanoparticles resulted in significantly reduced adherence of Enterococcus faecalis Biofilm bacteria are known to express efflux pumps as a resistance mechanism to antimicrobials Cationic CS-NPs hold significant potential to achieve improved root canal disinfection. Setbacks of CS-NPs The prolonged treatment time required to achieve effective bacterial elimination Effect of tissue inhibitors 120

121. Bioactive Glass: Antibacterial properties BAGs in micro and nano forms have been tested to improve root canal disinfection Silver Nanoparticles : Ag-NPs with significant antibacterial activity could be used for root canal disinfection. use ideally should be limited to medicament rather than as an irrigant. Bioactive glass powder loaded with AgNp demonstrated significant reduction in adhesion of E. fecalis biofilms and this was further exemplified by ultrasonic activation 121

122. Nanoparticles Incorporated Sealers and Restorative Materials : Most of the nanoparticles tested for root canal disinfection depend on contact-mediated and time-dependent antibacterial activity. Thus, the incorporation of various nanoparticles into sealers or root filling materials significantly improved the antibacterial efficacy by inhibition of bacterial biofilm formation on the surface as well as the resin-dentin interface. 122

123. Photoactivated disinfection Combination of photosensitizer solution and low-power laser light . Photodynamic therapy /Light activated therapy destroys an endodontic biofilm when a photosensitizer selectively accumulated in the target cell is activated by a visible light of appropriate wavelength 123

124. The working principle of APDT involves the interaction between a photosensitizer and low-energy laser light in the presence of oxygen, to generate reactive oxygen species (ROS) During APDT treatment in the root canal system, it is possible that the photosensitizers may pass into the periapical tissues through the root apex. This iatrogenic phenomenon may adversely affect the health status of periapical host cells after a photosensitizer is activated by light. Commonly used photosensitizer methylene blue Jiao Y, Tay FR, Niu LN, Chen JH. Advancing antimicrobial strategies for managing oral biofilm infections. International journal of oral science. 2019 Oct 1;11(3):1-1. 124

125. Ultrasonically activated irrigation Passive Ultrasonic Irrigation (PUI ) - defined as the ultrasonic activation of a file inside a pre-shaped root canal space filled with an aqueous irrigant, without touching the canal wall, without any constraint or moving the file about Ultrasonic irrigation has been shown to be significantly more effective than needle-and-syringe irrigation in dislodging debris, smear layer and biofilm 125

126. Bhuva B et al (2010) found that use of ultrasonically activated irrigation using 1% NaOCl, followed by root canal cleaning and shaping improves canal and isthmus cleanliness in terms of necrotic debris/biofilm removal 126

127. EndoVac system Negative apical pressure Automated irrigation mechanism based on aspiration within the root canal system and provides a constant flow and generous supply of disinfectant agent. This disinfectant is transported to the entire working length due the effect of aspiration exerted by this device, and therefore the irrigator solution exerts its effects on this zone and avoids dispersion at periradicular tissues. 127

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129. Endoactivator system Debride into the deep lateral anatomy , Remove the smear layer and Dislodge simulated biofilm clums within the curved canals. 129

130. 130

131. Ozone/ Ozonated water Ozone is a very powerful bactericide that can kill microorganisms effectively. It is an unstable gas, capable of oxidizing any biological entity. It was reported that ozone at low concentration, 0.1 ppm , is sufficient to inactivate bacterial cells including their spores Viera MR et al (1999) reported that Ozone in 0.1 to 0.3 ppm conc is able to completely kill bacteria after 15 or 30 minutes of contact time 131

132. Cardoso evaluated the efficiency of ozonated water as an irrigating agent during endodontic treatment in an attempt to eliminate Candida albicans and Enterococcus faecalis inoculated in root canals . It was possible to see effective antimicrobial action after ten minutes of water ozonization on the microbial suspension. There was no residue found when a second sample was collected seven days later. 132

133. Lasers Induce thermal effect producing an alteration in the bacterial cell wall leading to changes in the osmotic gradients and cell death . Noiri et al found that Er:YAG irradiation reduces the number of viable cells in most of the biofilms ( A.naeslundii , E.faecalis, L.casei , P.acnes , F. Nucleatum, P.gingivalis and P.nigrescenes ) 133

134. Er:YAG laser has been used at very low and short pulses (20 mJ , 50 µs) causing a pure photo-acoustic effect in the canal without thermal effect and vaporization, a protocol called photon-initiated photoacoustic streaming (PIPS) Jaramillo et al. reported that PIPS was effective in eradicating E. faecalis and in inhibiting new bacterial growth Ordinola et al. evaluated the effect of PIPS using 6% NaOCl for the removal of an in vitro biofilm and showed an improved cleaning of the infected dentin on PIPS 134

135. Plasma Dental Probe 135

136. Calcium hydroxide Commonly used ICM Ineffective against biofilms of E. faecalis even after 24 hours of treatment Ineffective in killing E.faecalis on its own, especially when a high pH is not maintained. Combination of calcium hydroxide and camphorated paramonochlorophenol completely eliminates E.faecalis. 136

137. Chlorhexidine and calcium hydroxide when combined together have shown better antimicrobial properties than calcium hydroxide alone. 2% chlorhexidine gel when combined with calcium hydroxide achieves a ph of 12.8 and can completely eliminate E.faecalis within dentinal tubules. 137

138. Triple antibiotic paste Mixture of metronidazole , ciprofloxacin, and minocycline . Used in regenerative endodontic procedure (REP). Effective on infected dentin, intracanal biofilms, and the majority of endodontic pathogens . TAP is significantly better than calcium hydroxide and chlorhexidine in disrupting biofilms of E. faecalis 138

139. COLD ATMOSPHERIC PLASMA (CAP) CAP represents a promising non-antibiotic option for the eradication and control of biofilm infections. Root canals treated with CAP for 40 min showed no detectable re-infection. It has been demonstrated that CAP was effective not only for young biofilms, but also for mature E. faecalis biofilms. After a 10-min of CAP treatment, the regular structure of a 100-μm biofilm was destroyed and replaced with ruptured bacteria Jiao Y, Tay FR, Niu LN, Chen JH. Advancing antimicrobial strategies for managing oral biofilm infections. International journal of oral science. 2019 Oct 1;11(3):1-1. 139

140. Human beta defensins Cationic antimicrobial peptides They bind to the negatively charged molecules on bacterial surface and disrupt bacterial membranes . HBD-3 is strongly inhibitory, whereas HBD-1, -2, and -4 have weak antimicrobial effects on E. faecalis . HBD-1, -2, -3, and -4 are produced in normal and inflamed dental pulp. Synthetic HBD-3 (HBD3-C15) was reported to be effective for disinfecting endodontic biofilm including C. albicans . 140

141. CONCLUSION  The root canal biofilm is a very complex, organized entity and it is difficult, but not impossible to duplicate its characteristics in in vitro experiments. Within root canal systems, the complexity is not only related to the nature of the biofilm, but also the complex anatomy, which houses tissue along with biofilms and removal of such biomasses is as relevant as being able to kill bacteria in biofilms. 141

142. REFERENCES TEXTBOOK OF ENDODONTICS NISHA GARGH Kishen A, George S, Kumar R. Enterococcus faecalis ‐mediated biomineralized biofilm formation on root canal dentine in vitro. Journal of Biomedical Materials Research Part A: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials. 2006 May;77(2):406-15. Jhajharia K, Parolia A, Shetty KV, Mehta LK. Biofilm in endodontics: a review. Journal of International Society of Preventive & Community Dentistry. 2015 Jan;5(1):1. Takemura N, Noiri Y, Ehara A, Kawahara T, Noguchi N, Ebisu S. Single species biofilm‐forming ability of root canal isolates on gutta‐percha points. European journal of oral sciences. 2004 Dec;112(6):523-9. Moine P, Abraham E. Immunomodulation and sepsis: impact of the pathogen. Shock. 2004 Oct 1;22(4):297-308.   Svensäter G, Bergenholtz G. Biofilms in endodontic infections.  Endod Topics. 2004;9:27–36. Chandki R, Banthia P, Banthia R. Biofilms: A microbial home. Journal of Indian Society of Periodontology . 2011 Apr;15(2):111. Swimberghe RC, Coenye T, De Moor RJ, Meire MA. Biofilm model systems for root canal disinfection: a literature review. International endodontic journal. 2019 May;52(5):604-28. 142

143. Neelakantan P, Romero M, Vera J, Daood U, Khan AU, Yan A, Cheung GS. Biofilms in endodontics—current status and future directions. International journal of molecular sciences. 2017 Aug;18(8):1748. Yoo YJ, Perinpanayagam H, Oh S, Kim A, Han SH, Kum KY. Endodontic biofilms: contemporary and future treatment options. Restorative dentistry & endodontics. 2018 Nov 26;44(1). Li YH, Tian X. Quorum sensing and bacterial social interactions in biofilms. Sensors. 2012 Mar;12(3):2519-38. Shrestha A, Kishen A. Antibacterial nanoparticles in endodontics: a review. Journal of endodontics. 2016 Oct 1;42(10):1417-26. Baras BH, Melo MA, Sun J, Oates TW, Weir MD, Xie X, Bai Y, Xu HH. Novel endodontic sealer with dual strategies of dimethylaminohexadecyl methacrylate and nanoparticles of silver to inhibit root canal biofilms. Dental Materials. 2019 Aug 1;35(8):1117-29. Jiao Y, Tay FR, Niu LN, Chen JH. Advancing antimicrobial strategies for managing oral biofilm infections. International journal of oral science. 2019 Oct 1;11(3):1-1. Ruiz‐Linares M, Aguado‐Pérez B, Baca P, Arias‐ Moliz MT, Ferrer‐Luque CM. Efficacy of antimicrobial solutions against polymicrobial root canal biofilm. International endodontic journal. 2017 Jan;50(1):77-83. Walsh LJ. Novel Approaches to Detect and Treat Biofilms within the Root Canals of Teeth: A Review. Antibiotics. 2020 Mar;9(3):129. 143

144. THANK YOU 144