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Review Article
Deepak Viswanath*,1, Prabhuji MLV2,

1Dr. Deepak Viswanath Preventive Dentistry, Krishnadevaraya College Of Dental Sciences, Bangalore, Karnataka, India.

2Professor and Head, Department Of Pedodontics and Preventive Dentistry, Krishnadevaraya College Of Dental Sciences, Bangalore, Karnataka, India.

*Corresponding Author:

Dr. Deepak Viswanath Preventive Dentistry, Krishnadevaraya College Of Dental Sciences, Bangalore, Karnataka, India., Email:
Received Date: 2016-05-10,
Accepted Date: 2016-06-15,
Published Date: 2016-07-31
Year: 2016, Volume: 8, Issue: 2, Page no. 33-40, DOI: --
Views: 590, Downloads: 9
Licensing Information:
CC BY NC 4.0 ICON
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0.
Abstract

Biofilms are highly structured, hydrated microbial communities containing sessile cells embedded in a self-produced extracellular polymeric matrix (containing polysaccharides, DNA and other components). The formation of biofilms might facilitate certain survival and virulence characteristics under some situations. Several mechanisms have been postulated in the biofilm antimicrobial resistance, which includes: slow penetration of the antimicrobial agent into the biofilm, changes in the chemical micro-environment within the biofilm leading to zones of slow or no growth, adaptive stress responses and presence of a small population of extremely resistant “persister” cells. Biofilm biology has become an expanding field of research and the knowledge accumulated suggests that organisms growing in biofilms develop properties different to those dwelling in the planktonic stage. This review article covers the concept of biofilms and its role in endodontic infections.

<p>Biofilms are highly structured, hydrated microbial communities containing sessile cells embedded in a self-produced extracellular polymeric matrix (containing polysaccharides, DNA and other components). The formation of biofilms might facilitate certain survival and virulence characteristics under some situations. Several mechanisms have been postulated in the biofilm antimicrobial resistance, which includes: slow penetration of the antimicrobial agent into the biofilm, changes in the chemical micro-environment within the biofilm leading to zones of slow or no growth, adaptive stress responses and presence of a small population of extremely resistant &ldquo;persister&rdquo; cells. Biofilm biology has become an expanding field of research and the knowledge accumulated suggests that organisms growing in biofilms develop properties different to those dwelling in the planktonic stage. This review article covers the concept of biofilms and its role in endodontic infections.</p>
Keywords
Biofilm, endodontic biofilm, bacterial adaptation, microbial ecology, pathogens
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INTRODUCTION

           Microorganisms are essential in the development of periradicular diseases and are the major causative factors associated with endodontic treatment failures. “Bacteriaassociated endodontic failures together with pulpperiapical infections refractory to conventional treatment represent the unresolved bacteri ological problems in endodontics”.1 It is evident that an infected root canal system is a unique niche for a range of species of microorganisms. The composition of root canal microflora has been the focus of considerable research and interest over the years. Results of studies have clearly defined the microbial differences between primary endodontic treatment and also retreatment.2 Apical periodontitis persisting after root canal treatment presents a more complex etiological and therapeutic solution.3 Another important factor is that the microbes in the root canals grow not only as planktonic cells, but also form biofilms consisting of a complex network of different microorganisms.

The term 'biofilm' was introduced to designate the thin layered condensation of microbes (e.g. bacteria, fungi, protozoa) that may occur on various surface structures in nature. Free-flowing bacteria existing in an aqueous environment, so-called planktonic microorganisms, are a prerequisite for biofilm formation. Biofilms are highly organised structures consisting of mushroom-shaped clumps of bacteria bound together by a carbohydrate matrix that contains water channels to deliver nutrients and remove wastes.4 Bacteria sequestered in biofilms are shielded and are often harder to kill than their planktonic counterparts. Biofilm bacteria are 1000 times more resistant to phagocytosis, antibodies and antibiotics.4 The dominant mechanisms of biofilm resistance are due to:

  • Delayed penetration of antimicrobial agents through the exo-polysaccharide complex
  • Modified nutrient environments and suppression of growth rate within the biofilm, thus affording protection from antimicrobial killing
  • A subpopulation of microorganisms in a biofilm can develop into a spore state that is highly protected: a phenotypic state known as a “persister”

           Most antimicrobial agents may be effective on the superficial layer of microorganisms in a biofilm, as the matrix layer may prevent direct contact of the agents with the microorganisms.5 In the endodontic field, biofilms did not receive wide attention until it was reported by Sen et al.6 The genera most frequently implicated as persistent are streptococci, enterococci, staphylococci, fusobacteria, peptostreptococci, and lactobacilli.

BIOFILMS – AN OVERVIEW

  • Structure: The basic structural units of a biofilm are the colonies or cell clusters formed by the surface adherent bacterial cells. The bacterial cells are distributed in a spatial manner within a biofilm. A glycocalyx matrix made up of extracellular polymeric substances surrounds the microcolonies and anchors the bacterial cell to the substrate. The biofilm structure by volume is made up by 85% with matrix material and the rest with cells. The structure and composition of a biofilm modifies according to the environmental conditions. The structural feature of a biofilm that has the highest impact in chronic bacterial infection is the tendency of microcolonies to detach from the biofilm community; and during this process of detachment, there is transfer of particulate matter from the biofilm to the fluid bathing the biofilm. Detachment occurs by two types; erosion where there is continual detachment of single cells and small portions of the biofilm and by sloughing, which is rapid and massive loss of biofilm. Detachment plays an important role in shaping the morphological characteristics of and also the structure of a mature biofilm
  • Composition: Biofilms are composed of carbohydrates, proteins and lipids which make up the organic portion; and calcium, phosphorus, magnesium and fluoride which make up the inorganic portion.7
  • Characteristics8, 9: Bacteria in a biofilm show distinct capacity to survive tough growth and environmental conditions; this is due to the following features:

            1.  Biofilm structure protects the residing bacteria from environmental threats

            2.  Biofilm structure permits trapping of nutrients and metabolic cooperativity between resident cells of same species and/or different species

            3.  Biofilm structure allows bacterial species with different growth requirements to survive

            4.  Bacteria in biofilms may communicate and exchange genetic materials to acquire new traits

ENDODONTIC PATHOGENS

           According to the results of studies, primary root canal infection is a dynamic process and bacterial species differ during various stages. Steeg and van der Hoeven10 showed that the most important factors are availability of nutrition, oxygen level (redox potential) and the local pH within the root canal. Facultative anaerobic bacteria grow well in anaerobic conditions, their primary source of energy being carbohydrates. Obviously they flourish when there is decreased availability of carbohydrates in the root canals. Endogenous proteins and glycoproteins are the main nutrients in the root canal system of primary endodontic cases. The main source of proteins in the root canal is a process of degradation of the small volume of pulpal tissue and influx of exudates from periapical tissues into the canal due to inflammatory process. Bacterial metabolism of the serum-like fluid also causes reduction of the redox potential and a rise in the pH within the root canal.11

           Currently, there is no substantial evidence indicating that certain microorganisms are more virulent than others. Sundqvist and Figdor2 stated that a proper definition for endodontic pathogens should include every organism capable of inducing the tissue destruction in apical periodontitis. In reality, however, the majority of endodonticmicrobiology studies refer to the endodontic pathogens as the bacteria isolated from a symptomassociated root canal that grows in the laboratory in a specific media. By this approach, the most frequently recovered species will assume the role of major endodontic pathogen.

           With the exception of Actinomyces, other species commonly associated with persistent intraradicular infection such as candida and enterococci are opportunistic pathogens. For microbes to maintain apical periodontitis and continue to cause disease, they must not only survive in the root-filled canal, but also possess the pathogenic properties necessary to perpetuate inflammation external to the root canal system. In general, microorganisms involved in persistent infections implement one of the three strategies to evade immune response- sequestration, cellular or humoral evasion12. Sequestration involves a physical barrier between the microbe and the host; cellular evasion means that microorganisms avoid leukocyte dependant antibacterial mechanisms and humoral evasion means that extracellular bacteria avoid the host's antibodies and complement.

           The frequent occurrence of E. faecalis in the potential colonization and overgrowth in endodontic infections as the dominant organism in post-treated apical periodontitis has often been isolated from root canals; and its pathogenicity is well documented.2 E. faecalis is an opportunistic pathogen and one of the leading causes of nosocomial infections. The ability of E. faecalis to form biofilms may confer an ecological advantage in certain situations. In endodontic infections, E. faecalis first adheres to the tissue surfaces by a physical association; in a second step there is permanent bonding by specific bacterial adhesins to complementary receptors on the host surfaces. Once the bacterial cell is bound, it is able to use available nutrients and a biofilm structure is necessary to contend with host defense mechanisms and for resistance to antibacterial treatments. For these reasons, experimental data suggest that viable E. faecalis cells can be recovered from root canals after an effective chemo-mechanical instrumentation treatment.13,14When compared to detection of E. faecalis by culturing (24-70%), E. faecalis has been found at higher percentages (67-77%) when a PCR detection method is used.15

           E. faecalis possesses certain virulence factors including lytic enzymes, cytolysin, aggregation substance, pheromones and lipoteichoic acid.15 It has been shown to adhere to host cells, express proteins and alter host responses.15,16 E. faecalis suppresses the action of lymphocytes, potentially contributing to endodontic failure.17 The potential survival and virulence factors of E. faecalis can be summarised as:

  • It endures prolonged periods of nutritional deprivation
  • Binds to dentin, proficiently invading the dentin tubules
  • Alters the host responses Ÿ Suppresses the action of lymphocytes
  • Utilises serum as nutritional source
  • Forms a biofilm

           Prevotella species such as P. intermedia and P. nigrescens were more often found in infected root canals; these two species have been cultured from 26-40% of root canals of teeth with apical periodontitis18. Further, P.nigrescens was more common than P.intermedia19. Some species of microorganisms are strongly associated with primary endodontic cases. These are Fusobacterium nucleatum, Veillonella parvula, Eubacterium and other species. In root canals, some of them are associated with other species; and numerous studies have shown the importance of food chain in which the metabolism of one species supplies nutrients for the growth of others.20 One example of synergistic association between microbial species could be the strong association of F. nucleatum with P. micros, P. endodontalis and Campylobacterrectus.20 Strong associations were also detected between Pr. intermedia and P. micros and also between P. anaerobius and the Eubacteria and Peptostreptococcus anaerobius.20

CONDITION FOR PERSISTENT INFECTION

           Persistent endodontic treatment disease involves multiple microbial and location factors. Microorganisms must possess an ability to survive the antimicrobial treatment and require 'persistence' characteristics such as a capacity for starvation survival and an ability to utilize serumlike periapical transudate as a source of nutrition. The location of the microbes within the root canals is crucial for access to nutrients; they must be situated near the apical foramen and also have an open communication for the free exchange of fluid, molecules and to inflame the periapical tissue. Together, these microbial characteristics and the opportunities of location determine whether microorganisms that survive treatment are able to maintain apical periodontitis following such treatment

ENDODONTIC BIOFILMS

           Though surface-associated microbial communities are the main form of colonization and retention by oral bacteria, biofilms also form in the root canals having the same properties as the parent communities colonising the enamel and cementum. Biofilms form when planktonic bacteria in a natural liquid phase are deposited on a surface containing an organic conditioning polymeric matrix or “conditioning film”. According to Svensater and Bergenholtz8, the biofilms in root canals are initiated at some time after the first invasion of the pulp chamber by planktonic oral organisms. There is inflammation which moves towards the apex providing the fluid vehicle for the invading planktonic organisms so that they multiply and attach to the root canals. The bacteria in the infected root canals is a restricted group compared to oral flora and largely comprised of facultative bacteria and strict anaerobes.

The progression of infection alters the nutritional and environment of the root canals; the initial polymicrobial environment of the infected root canal becomes more anaerobic thus depleting the nutritional level. These changes will offer a tough ecological niche for the surviving microorganisms. The endodontic bacterial biofilms can be categorised as:

  • Intracanal biofilms
  • Extraradicular biofilms
  • Periapical biofilms and
  • Biomaterial centered infections

           During the various stages of biofilm development, cells are in different physiological states. The cells that are at the base of the biofilm, may be dead where as those at the top may be actively growing. The majority of the time cells even with extremes of diversity, are in a state equivalent to cells in the stationery phase of growth.21,22 From the perspective of the persisting root canal flora, the “stationery-phase” cells might maintain a low but sufficient metabolic activity to provoke periapical inflammation.

           The characteristic features in cell-cell and microbe-substrate interactions were explained based on the phenomena of microbial adherence.23, 24 Many studies have shown the ability of E.faecalis to resist starvation and also develop biofilms under different environmental and nutrient conditions; but they modified according to the prevailing conditions. E. faecalis produced typical biofilm structures with bacterial cells and water channels under nutrient-rich environment. In vitro experiments have revealed three distinct stages in the development of E. faecalis biofilm:

Stage 1: Formation of microcolonies on the root canal

Stage 2: There is bacterial-mediated dissolution of the mineral fraction from the dentin substrate thus leading to increase in calcium and phosphate ions; finally promoting the mineralization of the biofilm.

Stage 3: The mature biofilm structure formed after 6 weeks carbonated-apatite structure as compared to natural dentin which had carbonated fluorapatite structure.

ANTI-MICROBIAL AGENTS AND BIOFILMS

           Anti-microbial agents have been developed and optimised for their activity against fast growing, dispersed populations containing a single organism. Antibiofilm substances can inhibit biofilm formation (preventive effect) or alternatively act on biofilms already formed (therapeutic effect). The mechanism of action against established biofilms may be through disruption of biofilm biomass and/or direct killing of the biofilm bacteria. It is very important for an endodontic irrigant or medicament to act primarily on established biofilms attached to the root canal walls so as to promote their elimination.

           Some of the newer antibiofilm agents like Farnesol, Xylitol, Lactoferrin and also Salicylic acid have removed the biofilm. Farnesol has a unique property of both inhibiting the biofilm formation and also disrupting the already formed biofilms.25- 27Farnesol, when applied topically reduces the biofilm matrix content26 and also kills biofilm bacteria.28 Xylitol only minimally reduces bacterial viability in biofilms29; but can synergistically act with Farnesol inhibiting the growth of Staphylococcus aureus.30, 31Lactoferrin has great potential to act synergistically with xylitol to disrupt biofilm structure and reduce bacterial viability.29,32 Specifically, xylitol disrupts biofilm integrity whereas Lactoferrinpermeabilizes bacterial membranes.29 Salicylic acid prevents bacterial attachment to medical devices, inhibits biofilm formation preferentially affects certain species.33- 35

CONCLUSION

           It is evident that in primary endodontic cases, root canal environment provides nutritional supply rich with peptides and amino acids for bacterial inhabitants of root canal system favouring the growth of anaerobic proteolytic species. The formation of biofilms carries particular clinical significance for defence mechanisms, and therapeutic benefits including chemical and mechanical antimicrobial treatment measures. As far as endodontic infections are concerned, the biofilm concept has gained very little attention. Further research is required to explore the conditions that may affect the efficacy of antimicrobials so that their clinical effects can be better predicted.

Supporting File
References
  1. Orstavik D. Antibacterial properties of endodontic materials. IntEndod J. 1988; 21: 161-9.
  2. Sundqvist G, Figdor D. Life as an endodontic pathogen. Etiologic differences between untreated and root-filled root canals. Endod Top. 2003; 6: 3- 28.
  3. Nair PRN, Sjogren U, Kahnberg KE, Krey G, Sundqvist G. Intraradicular bacteria and fungi in rootfilled, asymptomatic human teeth with therapy-resistant periapical lesions: a longterm light and electron microscopic study. J Endod. 1990; 16: 580-8.
  4. Costerton JW, Lewandowski Z, Debeer D, Caldwell D, Korber D, James G. Biofilms, the customised microniche. J Bacteriol. 1994; 176: 2137-42.
  5. Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant micro organisms. ClinMicrobiol Rev. 2002; 15: 167-93
  6. Sen BH, Safavi KE, Spangberg LSW. Antifungal effects of sodium hypochlorite and chlorhexidine in root canals. J Endod. 1999; 25: 235-8.
  7. Tronstad L, Sunde PT. The evolving new understanding of endodontic infections. Endo Topics. 2003; 6: 57-77.
  8. Svensater G, Bergenholtz G. Biofilms in endodontic infections. Endo Topics. 2004; 9: 27- 36.
  9. Lewis K. Riddle of biofilm resistance. Antimicrob Agents Chemother. 2001; 45: 999-1007. 
  10. TerSteeg PF, van der Hoeven JS. Development of periodontal microflora on human serum. Microb Ecol Health Dis. 1989; 2: 1-10.
  11. Marsh PD. Are dental diseases examples of ecological catastrophes? Microbiology. 2003; 149: 279-94.
  12. Nataro JP, Blaser MJ, Cunningham-Rundles S. Persistent bacterial infections: commensalism gone awry or adaptive niche? In: Nataro JP, Blaser MJ, Cunningham-Rundles S, eds. Persistent bacterial infections. Washington, DC: ASM Press, 2000: 3-10.
  13. Bystrom A, Sundqvist G. The antibacterial action of sodium hypochlorite and EDTA in 60 cases of endodontic therapy. IntEndod J . 1985; 18: 35-40.
  14. Jenkinson HF, Lamont RJ. Streptococcal adhesion and colonization. Crit Rev Oral BiolMed. 1997; 8: 175-200.
  15. Rocas IN, Siquera JF, Santos KRN. Association of Enterococcus faecalis with different forms of periradicular disease. J Endod. 2004; 30: 315-20.
  16. Love RM. Enterococcus faecalis: a mechanism for its role in endodontic failure. IntEndod J. 2001; 34: 399-405.
  17. Lee W, Lim S, Son H, Bae K. Sonicated extract of Enterococcus faecalisinduces irreversible cell cycle arrest in phytohemagglutin-activated human lymphocytes. JEndod. 2004; 30: 209-212.
  18. Wasfy MO, McMahon KT, Minah GE, Falkler WA Jr. Microbiological evaluation of periapical infections in Egypt. Oral MicrobiolImmunol .1992; 7: 100-5.
  19. Baumgartner JC, Bae KS, Xia T, Whitt J, David LL. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and polymerase chain reaction for differentiation of Prevotella intermedia and Prevotellanigrescens. J Endod. 1999; 25: 324-28.
  20. Sundqvist G. Associations between microbial species in dental root canal infections. Oral MicrobiolImmunol. 1992; 7: 257-62.
  21. Nystrom T. Not quite dead cells: on bacterial life, culturability, senescence, and death. Arch Microbiol. 2001; 176: 159-64.
  22. Nystrom T. Conditional senescence in bacteria: death of the immortals. MolMicrobiol. 2003; 48: 17-23.
  23. Seet AN, Buschner JH, van der Mei HC. Initial microbial adhesion events: mechanisms and implications 1998. In: 2011 Allison DG, Gilbert P, Lappin-Scott HM eds., Wilson M 64: 1736-42. 
  24. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science 1999; 284: 1318-22
  25. Jabra-Rizk MA, Meiller TF, James CE, Shirtliff ME. Effect of Farnesol on staphylococcus aureus biofilm formation and antimicrobial susceptibility. Antimicrob Agents Chemother 2006; 50(4): 1463- 9.
  26. Jeon JG, Pandit S, Xiao J, Gregoire S, Falsetta ML, Klein MI, et al. influences of trans-trans Farnesol, a membrane-targeting sesquiterpenoid, on Streptococcus mutans physiology and survival within mixed-species oral biofilms. Int J Oral Sci 2011; 3(2): 98-106
  27. Koo H, Schobel B, Scott-Anne K, Watson G, Bowen WH, Cury JA, et al. Apigenin and ttfarnesol with fluoride effects on S. mutans biofilms and dental caries. J Dent Res 2005; 84(11): 1016- 20.
  28. Gomes F, Teixeira P, Cerca N, Azeredo J, Oliveira R. Effect of Farnesol on structure and composition of staphylococcus epidermidis biofilm matrix. CurrMicrobiol 2011; 63(4): 354-9.
  29. Ammons MC, Ward LS, Fisher ST, Wolcott RD, James GA. In vitro susceptibility of established biofilms composed of a clinical wound isolate of Pseudomonas aeruginosa treated with Lactoferrin and xylitol. Int J Antimicrob Agents 2009; 33(3): 230-6.
  30. Katsuyama M, Itchikawa H, Ogawa S, Ikezawa Z. A novel method to control the balance of skin microflora. Part 1. Attack on biofilm of Staphylococcus aureus without antibiotics. J DermatolSci 2005; 38(3): 197-205. 
  31. Katsuyama M, Kobayashi Y, Itchikawa H, Mizuno A, Miyachi Y, Matsunaga K, et al. A novel method to control the balance of skin microflora. Part 2. A study to assess the effect of a cream containing Farnesol and xylitol on atopic dry skin. J DermatolSci 2005; 38(3): 207-13.
  32. Ammons MC, Ward LS, James GA. Antibiofilm efficacy of a Lactoferrin/xylitol wound hydrogel used in combination with silver wound dressings. Int Wound J 2011; 8(3): 268-73.
  33. Arciola CR, Montanaro L, Caramazza R, Sassoli V, Cavedagana D. Inhibition of bacterial adherence to a high-water-content polymer by a watersoluble, nonsteroidal, anti-inflammatory drug. J Biomed Mater Res 1998; 42(1): 1-5.
  34. Prithviraj B, Bais HP, Weir T, Suresh B, Najarro EH, Dayakar BV, et al. Down regulation of virulence factors of Pseudomonas aeruginosa by salicylic acid attenuates its virulence on Arabidopsis thaliana and Caenorhabditis elegans. Infect Immun 2005; 73(9): 5319-28.
  35. Muller E, Al-Attar J, Wolff AG, Farber BF. Mechanism of salicylate-mediated inhibition of biofilm in Staphylococcus epidermidis. J Infect Dis 1998; 177(2): 501-3.
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