In Vitro Biofilm Formation from Root Canal Isolates: A Pilot Study
Biofilms in infected root canals are known as multispecies biofilms, which are composed of restricted groups of bacteria. Even though evidence of biofilm in infected root canals has been reported, the knowledge of biofilm formation in root canals is still limited. The aim of this study was to generate biofilm from root canal isolates under different nutrient conditions. Five species of bacteria isolated from an infected root canal were cultured on bovine root canal dentin and on cover slips in rich and in diluted media for one and for four weeks. The biofilm formation was examined by scanning electron microscopy. The viability of bacterial cells in the biofilm was analyzed by viability stain and confocal laser scanning microscopy. A greater amount of mixed-species bacterial aggregation was found on bovine root canal dentin than on cover slips. Cocci were predominantly observed in the one-week biofilms, with very few rods, whereas more rods co-aggregated with cocci were found in the four-week biofilms. Furthermore, biofilm formation in diluted medium was greater than that in rich medium. More live bacteria than dead cells were found in both media. It was concluded that the root canal isolates could form biofilm in vitro on bovine root canal dentin in diluted medium and that the co-aggregation between cocci and rods was found in the four-week biofilms.
1. Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol 1965; 20: 340-349.
2. Moller AJ, Fabricius L, Dahlen G, Ohman AE, Heyden G. Influence on periapical tissues of indigenous oral bacteria and necrotic pulp tissue in monkeys. Scand J Dent Res 1981; 89: 475-484.
3. Fabricius L, Dahlen G, Holm SE, Moller AJ. Influence of combination of oral bacteria on periapical tissues of monkeys. Scand J Dent Res 1982; 90: 200-206.
4. Fabricius L, Dahlen G, Ohman AE, Moller AJ. Predominant indigenous oral bacteria isolated from infected root canals after varied times of closure. Scand J Dent Res 1982; 90: 134-144.
5. Jefferson KK. What drives bacteria to produce a biofilm? FEMS Microbiol Lett 2004; 236: 163-173.
6. Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 2002 Apr; 15: 167-193.
7. Nair PNR. Light and electron microscopic studies of root canal flora and periapical lesions. J Endod 1987; 13: 29-39.
8. Molven O, Olsen I, Kerekes K. Scanning electron microscopy of bacteria in the apical part of root canals in permanent teeth with periapical lesions. Endod Dent Traumatol 1991; 7: 226-229.
9. Siqueira JF, Jr., Lopes HP. Bacteria on the apical root surfaces of untreated teeth with periradicular lesions: a scanning electron microscopy study. Int Endod J 2001 Apr; 34: 216-220.
10. Sen BH, Piskin B, Demirci T. Observation of bacteria and fungi in infected root canals and dentinal tubules by SEM. Endod Dent Traumatol 1995 Feb; 11: 6-9.
11. Leonardo MR, Rossi M, Silva L, Ito I, Bonifacio K. EM evaluation of bacterial biofilm and microorganisms on the apical external root surface of human teeth. J Endod 2002; 28: 815-818.
12. Noiri Y, Ehara A, Kawahara T, Takemura N, Ebisu S. Participation of bacterial biofilms in refractory and chronic periapical periodontitis. J Endod 2002; 28: 679-683.
13. Richardson N, Mordan NJ, Figueiredo JA, Ng YL, Gulabivala K. Microflora in teeth associated with apical periodontitis: a methodological observational study comparing two protocols and three microscopy techniques. Int Endod J 2009; 42: 908-921.
14. Wilson M. Susceptibility of oral bacterial biofilms to antimicrobial agents. J Med Microbiol 1996; 44: 79-87.
15. Larsen T. Susceptibility of Porphyromonas gingivalis in biofilms to amoxicillin, doxycycline and metronidazole. Oral Microbiol Immunol 2002;17: 267-271.
16. Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet 2001; 358: 135-138.
17. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM. Microbial biofilms. Annu Rev Microbiol 1995; 49: 711-745.
18. Spratt DA, Pratten J, Wilson M, Gulabivala K. An in vitro evaluation of the antimicrobial efficacy of irrigants on biofilms of root canal isolates. Int Endod J 2001; 34: 300-307.
19. Distel JW, Hatton JF, Gillespie MJ. Biofilm formation in medicated root canals. J Endod 2002; 28: 689-693.
20. Takemura N, Noiri Y, Ehara A, Kawahara T, Noguchi N, Ebisu S. Single species biofilmforming ability of root canal isolates on guttapercha points. Eur J Oral Sci 2004; 112: 523- 529.
21. George S, Kishen A, Song KP. The role of environmental changes on monospecies bio-film formation on root canal wall by Enterococcus faecalis. J Endod 2005; 31: 867-872.
22. Dunavant TR, Regan JD, Glickman GN, Solomon ES, Honeyman AL. Comparative evaluation of endodontic irrigants against Enterococcus faecalis biofilms. J Endod 2006; 32: 527-531.
23. Sena NT, Gomes BP, Vianna ME, Berber VB, Zaia AA, Ferraz CC, et al. In vitro antimicrobial activity of sodium hypochlorite and chlorhexidine against selected single-species biofilms. Int Endod J 2006; 39: 878-885.
24. Chavez de Paz LE, Bergenholtz G, Dahlen G, Svensater G. Response to alkaline stress by root canal bacteria in biofilms. Int Endod J 2007; 40: 344-355.
25. Chavez de Paz LE, Bergenholtz G, Svensater G. The effects of antimicrobials on endodontic biofilm bacteria. J Endod 2010; 36: 70-77.
26. Liu H, Wei X, Ling J, Wang W, Huang X. Biofilm formation capability of Enterococcus faecalis cells in starvation phase and its susceptibility to sodium hypochlorite. J Endod; 36: 630-635.
27. Li J, Helmerhorst EJ, Leone CW, Troxler RF, Yaskell T, Haffajee AD, et al. Identification of early microbial colonizers in human dental biofilm. J Appl Microbiol 2004; 97: 1311-1318.
28. Love RM. Invasion of dentinal tubules by root canal bacteria. Endod Topics 2004; 9: 52-65.
29. Switalski LM, Butcher WG, Caufield PC, Lantz MS. Collagen mediates adhesion of Streptococcus mutans to human dentin. Infect Immun 1993; 61: 4119-4125.
30. Love RM, McMillan MD, Jenkinson HF. Invasion of dentinal tubules by oral streptococci is associated with collagen recognition mediated by the antigen I/II family of polypeptides. Infect Immun 1997; 65: 5157- 5164.
31. Tronstad L, Sunde PT. The evolving new understanding of endodontic infections. Endod Topics 2003; 6: 57-77.
32. Camargo CH, Siviero M, Camargo SE, de Oliveira SH, Carvalho CA, Valera MC. Topographical, diametral, and quantitative analysis of dentin tubules in the root canals of human and bovine teeth. J Endod 2007; 33: 422-426.
33. Bond PL, Smriga SP, Banfield JF. Phylogeny of microorganisms populating a thick, subaerial, predominantly lithotrophic biofilm at an extreme acid mine drainage site. Appl Environ Microbiol 2000; 66: 3842-3849.
34. Brake SS, Hasiotis ST. Eukaryote-dominated biofilms in extreme environments: overlooked sources of information in the geologic record. Palaios 2008; 23: 121-123.
35. Lapaglia C, Hartzell PL. Stress-Induced Production of Biofilm in the Hyperthermophile Archaeoglobus fulgidus. Appl Environ Microbiol 1997; 63: 3158-3163.
36. Dupraz C, Visscher PT. Microbial lithification in marine stromatolites and hypersaline mats. Trends Microbiol 2005; 13: 429-438.
37. Overman PR. Biofilm: A new view of plaque. J Contemp Dent Pract 2000; 1: 1-8.
38. Stepanovic S, Cirkovic I, Ranin L, SvabicViaholic M. Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface. Appl Microbiol 2004; 38: 428-432.
39. Dewanti R, Wong AC. Influence of culture conditions on biofilm formation by Escherichia coli O157:H7. Inter J Food Microbiol 1995; 26: 147-164.
40. Stepanovic S, Davic I, Opavski N, Jezek P, Ranin L. Influence of the growth medium composition on biofilm formation by Staphylococcus sciuri. Ann Microbiol 2003; 53: 63-74.
41. Hood SK, Zottola EA. Adherence to stainless steel by foodborne microorganisms during growth in model food systems. Inter J Food Microbiol 1997; 37: 145-153.
42. Khemaleelakul S, Baumgartner JC, Pruksakorn S. Autoaggregation and coaggregation of bacteria associated with acute endodontic infections. J Endod 2006; 32: 312-318.