Biofilms

A biofilm is a highly organized structure consisting of bacterial cells enclosed in a self-produced extracellular polymeric matrix attached on a surface. Root canal infections are biofilm mediated. The complexity and variability of the root canal system, together with the multi-species nature of biofilms, make disinfection of this system extremely challenging.

1. BIOFILM S Dr. K. PALANI SELVI MDS

2. • Introduction • Definition • Basic criteria • Characteristics Ultrastructure of a biofilm • Formation and factors affecting its formation • Endodontic biofilms • Role of E .feacalis • Microbial diversity in endodontic biofilm Current therapeutic options in endodontic biofilm • Eradication of biofilm • Biofilm assessment methods Conclusion

3. From an ecological perspective, the root canal can be considered a highly controlled environment with a limited number of niches. T he main limiting factors in root canal niches that influence bacterial colonization are, for instance, oxygen and nutrient availability . INTRODUCTION JOE - Redefining the Persistent Infection in Root Canals: Possible Role of Biofilm Communities - Luis Chávez 2014

4. Biofilm is defined as a sessile multi cellular microbial community characterized by cells that are firmly attached to a surface and embedded in a self produced matrix of extracellular polymeric substances Biofilms are formed whenever there is free flow of fluid , microorganisms and a solid surface. It is one of the basic survival strategies employed by bacteria DEFINITION • Costerton JW, Lewandowski Z, DeBeer D, Caldwell D, Korber D, James G. Biofilms, the customized microniche . J Bacteriol . 1994;176:2137-42.

5. BASIC CRITERIAS Caldwell DE, Atuku E, Wilkie DC, Wivcharuk KP, Karthikeyan S, Korber DR, et al. Germ theory vs. community theory in understanding and controlling the proliferation of biofilms. Adv Dent Res. 1997;11:4-13 . Biofilms should possess 1. AUTOPOIESIS- ability to self organize 2. HOMEOSTASIS-resist environmental pertubations 3.SYNERGY- effective in association than in isolation 4.COMMUNITY- respond to environmental changes as a unit rather than single individual

6. ULTRASTRUCTURE OF A BIOFILM

7. Basic structure - heterogenous arrangement of microbial cells on a solid surface Glycocalyx matrix - E xtrapolymeric substance surrounds the microcolonies and anchors the bacterial cell to the substrate 85% - matrix and 15% - cells M ushroom shape/ tower shape Water channels are primitive circulatory system in biofilms ULTRASTRUCTURE OF A BIOFILM 85% MATRIX 15% CELLS

8. HOW BIOFILM FORMS First stage - a conditioning film forms- t ransport of microbe to the substrate surface Second stage – initial non-specific microbial-substrate adherence phase Third stage - B acterial growth and biofilm expansion Fourth stage - D etachment of biofilm

9. STAGES OF BIOFILM FORMATION

10. FACTORS INFLUENCING BIOFILM FORMATION PH, temperature, surface energy of substrate, flow rate of fluid, nutrient availability, bacterial growth stage, surface hydrophobicity

11. GROWTH OF BIOFILM

12. CHARACTERISTICS

13. CHARACTERISTICS

14. Detachment of biofilm- seeding dispersal Erosion- continous detachment of single cells and small portions of biofilm Sloughing- rapid massive loss of biofilm

15. QUORUM SENSING

16. QUORUM SENSING

17. Intracanal Biofilm Forms on root canal dentin of an infected tooth Identified by Nair 1987 Cocci, rods , filaments and spirochetes are seen Morphologically distinct t ype of bacteria are seen Eg - E . faecalis

18. Extraradicular biofilms Root surface biofilms - adjacent to root apex of endodontically treated teeth Seen in teeth with asymptomatic periapical periodontitis and chronic apical abscess with sinus tract Multispecies in nature- F. nucleatum , Po. gingivalis , and Tannerella forsythensis Dominated by cocci and short rods with cocci attached to tooth substrate

19. Periapical biofilms Isolated biofilms in the periapical area of endodontically involved teeth Eg - A cti n omyc es , P. propionicum

20. Biomaterial centered biofilm Bacteria adheres to artificial biomaterial surface and forms biofilms O pportunis tic infection- nosocomial organisms Eg- coagulase negative staphylococcus, s. aureus, enterococci, streptococci, p.aeruginosa Serum plays a significant role in biofilm formation GP coated with biofilm

21. E. FEACALIS & BIOFILM • E. faecalis is a gram-positive, facultative anaerobic cocci that is strongly associated with endodontic infections. • It causes nosocomial infections • Frequently isolated from the failed root canals undergoing retreatment . Duggan JM, Sedgley CM. Biofilm formation of oral and endodontic Enterococcus faecalis. J Endod . 2007;33:815-8. Al-Ahmad A, Muller N, Wiedmann -Al-Ahmad M, Sava I, Hubner J, Follo M, et al. Endodontic and salivary isolates of Enterococcus faecalis integrate into biofilm from human salivary bacteria cultivated in vitro. J Endod . 2009;35:986-91. They can grow in extremely Alk aline pH, salt concentrated environment - survive a temperature of 60°C for 30 min. • E. faecalis is able to suppress the action of lymphocytes, potentially contributing to endodontic failure. • E.faecalis has the ability to form biofilm that can resist calcium hydroxide dressing by maintaining pH homeostasis.

22. The development of E. faecalis biofilm on the root canal dentin involves three stages as follows: Stage 1: Microcolonies are formed as E. faecalis cells adhere on the root canal dentin surface Stage 2: Bacterial-mediated dissolution of the mineral fraction from the dentin substrate leads to localized increase in the calcium and phosphate ions causing mineralization (or calcification) of the E. faecalis biofilm Stage 3: Due to this interaction of bacteria and their metabolic products on dentin, E. faecalis biofilm is mineralized.

23. CURRENT THREUPATIC OPTIONS FOR ENDODONTIC BIOFILM

24. CURRENT THREUPATIC OPTIONS FOR ENDODONTIC BIOFILM

25. O Z ONE U L T RASONIC IRRIGATION RECENT METHODS - ENDODONTIC BIOFILM ERADICATION

26. Biofilm Detection Atomic force microscopy – The forces of interaction among bacterial cells and between bacterial cells and substrates has been studied by AFM. Micromanipulators have been used to sample individual cells or biofilm compartments. Laser-based optical tweezers are noninvasive and non- contact tools that can probe the interaction between microscopic objects such as bacteria and collagen. Fourier transform infrared (FTIR) spectroscopy is used to characterize the chemical composition of mature biofilm structures qualitatively and quantitatively. Solid-state nuclear magnetic resonance (NMR) is a powerful analytical tool to study the constituents of bacterial biofilm, as well as to obtain metabolic information

27. Micromanipulator-assisted analysis Green fluorescent protein (GFP) tagging Confocal laser scanning microscopy (CLSM) Flow cytometry Fluorescence in situ hybridization (FISH) - have made biofilm characterization very comprehensive . Biofilm Detection RECENT ADVANCES

28. It is foremost to understand that how the biofilm formed by root canal bacteria resists endodontic treatment measures. Elimination or significant reduction of endodontic bacterial biofilms and prevention of recontamination of the root canal after treatment are the essential elements for successful outcomes of endodontic treatment. CONCLUSION