APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2005, p. 87388743 0099-2240/05/$08.000 doi:10.1128/AEM.71.12.87388743.2005 Copyright 2005, American Society for Microbiology.

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Vol. 71, No. 12

Identication and Localization of Extraradicular Biolm-Forming Bacteria Associated with Refractory Endodontic Pathogens
Nobuo Noguchi, Yuichiro Noiri,* Masahiro Narimatsu, and Shigeyuki Ebisu
Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, 1-8, Yamadaoka, Suita, Osaka 565-0871, Japan
Received 11 April 2005/Accepted 3 August 2005

Bacterial biolms have been found to develop on root surfaces outside the apical foramen and be associated with refractory periapical periodontitis. However, it is unknown which bacterial species form extraradicular biolms. The present study aimed to investigate the identity and localization of bacteria in human extraradicular biolms. Twenty extraradicular biolms, used to identify bacteria using a PCR-based 16S rRNA gene assay, and seven root-tips, used to observe immunohistochemical localization of three selected bacterial species, were taken from 27 patients with refractory periapical periodontitis. Bacterial DNA was detected from 14 of the 20 samples, and 113 bacterial species were isolated. Fusobacterium nucleatum (14 of 14), Porphyromonas gingivalis (12 of 14), and Tannellera forsythensis (8 of 14) were frequently detected. Unidentied and uncultured bacterial DNA was also detected in 11 of the 14 samples in which DNA was detected. In the biolms, P. gingivalis was immunohistochemically detected in all parts of the extraradicular biolms. Positive reactions to anti-F. nucleatum and anti-T. forsythensis sera were found at specic portions of the biolm. These ndings suggested that P. gingivalis, T. forsythensis, and F. nucleatum were associated with extraradicular biolm formation and refractory periapical periodontitis. More than 300 different bacterial species are known to inhabit the healthy human mouth (9). This multitude of microbiota can infect the root canal in a nonvital tooth, and the microorganisms and their components might have etiologic roles in refractory or chronic periodontitis. However, only a small numbers of species have been consistently isolated from infected root canals of periapically affected teeth (26). Mixed bacterial biolms in root canals of periapical periodontitisaffected teeth have been investigated microbiologically (3, 5, 10, 17, 25) and morphologically (6, 19). Clinically, we encounter cases in which periapical periodontal disease does not heal despite the debridement of biolm in the root canals. It has been believed that bacteria could not exist in periapical lesions at the chronic phase as a consequence of the local immune response (7). Recently, though, evidence has been found regarding the presence of bacteria within extraradicular areas (29, 30) and periapical lesions (16, 23, 24). In general, an extraradicular area is used clinically as a term that contrasts to a root canal, which ends at the apical foramen of the root apex. On the boundary of the apical foramen, moreover, the root canal surface connects to the tooth surface (cementum) outside the root apex or occasionally to the extruded root canal lling material from a root canal. The solid parts connected with the root canal surface are called the extraradicular area; although the area is located in the periapical lesion, it is expressed as distinguished from the lesion, while the lesions mean the part with the alveolar bone resorption around the root apex in which soft tissue, for example, granulation tissue, exists in many cases.
* Corresponding author. Mailing address: Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, 1-8, Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-2927. Fax: 81-6-6879-2927. E-mail: noiri@dent .osaka-u.ac.jp. 8738

It was thought that viable bacteria could not inhabit the area over the apical foramen without an acute phase of periapical periodontitis. We found morphologically that biolms were formed in the extraradicular area and proposed that extraradicular biolms, developing from the root canal via an apical foramen and consisting of multiple morphotypic bacteria, were attached to the cementum around the root apex (12). It has been reported that gram-positive facultative anaerobes, for example, Enterococcus faecalis, Streptococcus sanguis, and Streptococcus intermedius, have the ability to colonize and form extracellular matrices on the surface of gutta-percha points, while serum plays a crucial role in biolm formation in vitro (27). Gutta-percha points were the root canal lling materials when debridement of bacteria in the root canal was completed. We found that the surface of these points extruded from the root canal could also become a scaffold for biolm formation in vivo (12). As biolms are resistant to antibiotic treatment and immune responses (4), we speculate that microorganisms may also be able to survive at the extraradicular area. Once biolms form in the extraradicular area, it is impossible to remove those on the root surfaces by nonsurgical endodontic treatments. Extraradicular biolms can become refractory periapical pathogens; however, little is known about the bacterial components in extraradicular biolms. Therefore, human extraradicular biolms of refractory periapical periodontitis-affected teeth were used to identify biolm-forming bacteria and to investigate the immunohistochemical localization of detected bacteria.
MATERIALS AND METHODS Subjects and clinical materials. Twenty-seven volunteer patients (eight males and 19 females, 21 to 72 years of age) with refractory periapical periodontitis were enrolled in the study. All selected teeth had previously received endodontic treatment, showed a periapical radiolucent area, and had no root fracture or

VOL. 71, 2005 TABLE 1. Characteristics and clinical features of patients before sample extractiona
Sample no. Patient gender Age (yr) Tooth no. Surgical treatment Spontaneous pain Percussion pain Palpation Fistula

EXTRARADICULAR BIOFILM-FORMING BACTERIA TABLE 2. Prevalence of bacterial species identied in 14 extraradicular samples
Genus or species match No. of samples detected Clone No.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
a

F F F F M F F F M F F F M F M F M F F M M F F F F M F

41 27 55 59 54 21 25 36 40 69 62 29 72 23 62 64 31 49 60 47 47 36 56 48 19 54 25

38 11 25 24 25 46 16 43 46 23 14 17 31 21 22 24 12 12 12 21 21 44 15 24 21 22 25

E AP E E E E AP AP E E AP E E AP AP AP AP AP AP AP AP AP AP AP AP E E

Proportion (% of total)

F, female; M, male; E, extraction; AP, apicoectomy.

periodontal pocket formation at the root apex. Samples were obtained from patients whose periapical lesions could not be healed clinically despite repeated endodontic treatment at the Osaka University Dental Hospital and thus the teeth were clinically judged as having refractory periapical periodontitis. Table 1 shows the characteristics and clinical symptoms of all subjects before extraction. Informed consent was obtained from all patients in accord with the protocol approved by the Ethics Committee of the Osaka University Graduate School of Dentistry. Strains, culture conditions, and preparation of antisera. The sources and culture conditions of Porphyromonas gingivalis 381, Fusobacterium nucleatum 1436, and Tannerella forsythensis ATCC 43037 used in the present study were as previously described (13, 15, 31). P. gingivalis 381 was grown in brain heart infusion broth (Difco Laboratories, Detroit, Mich) containing 5 g/ml hemin and 1 g/ml menadione. F. nucleatum 1436 was grown in Todd-Hewitt broth (Difco) containing 0.5 g/ml L-cysteine. T. forsythensis ATCC 43037 was grown in brain heart infusion broth containing 0.5% yeast exact, 5 g/ml hemin, 0.5 g/ml menadione, 0.001% N-acetylmuramic acid (Sigma Chemical Co., St. Louis, Mo.), and 5% fetal bovine serum (Gibco BRL, Grand Island, N.Y.). Mass cultures were grown anaerobically (90% N2, 5% CO2, and 5% H2) at 37C for 48 h. Cells were also harvested as described (15). The preparation and purication of the antisera against the three bacterial species have been described previously (15, 31). Rabbit antiserum against T. forsythensis was provided by K. Maeda (Kyushu University, Fukuoka, Japan) (31). Sampling procedure, DNA extraction and PCR amplication of the 16S rRNA gene. The 20 samples used were nine extracted teeth and 11 apical fragments obtained from apicoectomies. The teeth and root tips were immediately washed with sterile saline to remove blood and planktonic bacteria. Extraradicular biolm specimens were taken to curettage the root surfaces around the root apex. The method of Rolph et al. (17) was used for DNA extraction. Solution from the Puregene DNA purication kit (Flowgen) was added to samples, which were incubated at 37C for 45 min. Samples were pelleted and resuspended in 100 l of Tris-EDTA buffer and stored at 20C until required. Negative control samples from an intact tooth extracted by orthodontic treatment were processed by the same method. Genomic DNA from P. gingivalis 381 was used as a positive control. PCR was performed by a previously described method (8). The primers used, which target 16S rRNA, were 63f (5-CAGGCCTAACACATGCAAGTC-3) and 1387r (5-GGGCGGWGTGTACAAGGC-3) (8).

Fusobacterium nucleatum Porphyromonas gingivalis Unidentied bacterium Prevotella sp. Tannerella forsythensis Eubacterium sp. Porphyromonas sp. Prevotella intermedia Bacteroides-like sp. Bilophila wadsworthia Corynebacterium matruchotii Desulfobulbus sp. Fusobacterium sp. Porphyromonas gulae Bacteroidales oral clone Dialister sp. Leptotrichia sp. TM7 phylum sp. Actinomyces sp. Campylobacter gracilis Capnocytophaga sp. Capnocytophaga sputigena Chloroexi genomosp. Haemophilus paraphrophilus Lachnospiraceae oral clone Rothia dentocariosa Unidentied bacterium Veillonella sp. Actinomyces gerencseriae Actinomyces naeslundii Atopobium rimae Bacteroides sp. Capnocytophaga granulose Corynebacterium glucuronolyticum Corynebacterium sp. Human oral bacterium Peptostreptococcus sp. Prevotella denticola Providencia stuartii Rahnella sp. Unidentied Prevotella sp. Unidentied Eubacterium

14 12 11 10 8 7 7 7 5 5 5 5 5 5 4 4 4 4 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2

74 340 73 52 136 32 19 27 42 10 5 23 10 40 23 5 4 5 3 3 7 4 12 12 3 6 5 9 7 3 6 12 2 8 3 2 6 2 18 2 22 3

6.13 28.17 6.05 4.31 11.27 2.65 1.57 2.24 3.48 0.83 0.41 1.91 0.83 3.31 1.91 0.41 0.33 0.41 0.25 0.25 0.58 0.33 0.99 0.99 0.25 0.50 0.41 0.75 0.58 0.25 0.50 0.99 0.17 0.66 0.25 0.17 0.50 0.17 1.49 0.17 1.82 0.25

Cloning of mixed 16S rRNA gene products. Mixed PCR products were extracted after agarose gel electrophoresis, using the QIAEX gel extraction kit (QIAGEN, Hilden, Germany) and ligated into the pDrive cloning vector (QIAGEN), followed by transformation into Escherichia coli DH5 cells (TOYOBO, Osaka, Japan). Usually, more than 300 transformants for each PCR library were obtained. Ninety-six colonies were randomly selected from each library and then transferred from the transformation plates to Plusgrow (Nacalai-tesque, Kyoto, Japan) liquid medium containing ampicillin. After overnight incubation at 37C, plasmid DNA was puried with a QIAprep Spin miniprep kit (QIAGEN). Sequencing analysis. Puried plasmid DNA from the cloning procedure was partially sequenced using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) and a Big Dye Terminator cycle sequencing kit (Applied Biosystems). As previously described by Rolph et al. (17), the sequences of each clone were submitted to the NCBI database (http://www.ncbi.nlm.nih.gov /BLAST/) as queries. The BLAST program (version 2.1) (1, 2) was used to determine the highest identity to known sequences in the database, and clone sequences with from 98 to 100% identity with the database were considered to be of the same bacterial species. In addition, sequences with from 90 to 98% identity were considered to be of the same bacterial genus, while an identity of 90% was used as the cutoff for positive identication of taxa. Multiple alignment of

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FIG. 1. Phylogenetic tree of the bacterial genes in an extraradicular biolm based on 16S rRNA genes. The tree was established from an analysis of the sequences examined in samples 1 to 6. The scale bar indicates neighbor-joining distance and 10% differences in nucleotide sequences.

bacterial genes was performed by the CLUSTAL W program (28). Concentration of the unrooted phylogenetic tree was carried out by the neighbor-joining method (18). Evaluation of the topology of the phylogenetic tree and determination of the condence values were carried out by previously described methods (17). Histopathological and immunohistochemical procedures. Five selected teeth and two root tips were carefully xed in 4% paraformaldehyde and 0.1% glutaraldehyde for 12 h at 4C and decalcied for 10 days at 4C with stirring in 10% formic acid-sodium citrate (pH 2.2); 8-m-thick serial frozen sections were prepared. Some of the sections were stained by the Brown and Brenn-modied Gram staining procedure, while others were subjected to the alkaline phosphatase-conjugated streptavidin-biotin method (1315). The results of both staining methods were observed under a light microscope (Optiphot-2; Nikon, Tokyo, Japan).

RESULTS Identication of bacteria in extraradicular biolm. From 14 of the 20 extraradicular biolm samples, 1,207 clones were analyzed and 113 bacterial genera and species were identied.

DNA was not detected in the other six extraradicular samples. Table 2 shows that bacterial DNA was detected in at least two of the 14 positive samples. DNA of F. nucleatum (14 of 14), P. gingivalis (12 of 14), T. forsythensis (8 of 14), and Prevotella intermedia (7 of 14) were frequently detected in all extraradicular samples. Unidentied and uncultured bacterial DNA was also detected in 11 of the 14 samples in which DNA was detected (Table 2). DNA of E. faecalis and Porphyromonas endodontalis was detected in just 1 sample. From the results of the partial 16S rRNA sequences of 35 clones from samples 1 to 6, 23 bacterial strains were identied and 11 uncultured or unidentied bacteria were detected (Fig. 1). Specicity of immunohistochemical labeling. Antisera against P. gingivalis, F. nucleatum, and T. forsythensis were reacted with homogenous bacterial cells. Five gram-positive bacteria (Actinomyces naeslundii ATCC 19246, Streptococcus

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FIG. 2. Histopathological evaluation around the root apex (cross section views). (a) Whole image of a serial section. In the extraradicular area located at the radicular tooth surface outside of the vicinity of the root apex, biolms indicated by arrows are shown. Residual bacteria, indicated by an arrowhead in the root canal, are detected. RC, root canal. Magnication, 40. (b) High-power view of the inset in a. Arrows indicate that gram-negative rods and lamentous microorganisms are located in the extraradicular biolm. Magnication, 625.

mutans MT 8148, Streptococcus sanguis ST-3R, Staphylococcus aureus FDA 209P, and Propionibacterium acnes ATCC 11829), and three other gram-negative bacterial species (Actinobacillus actinomycetemcomitans ATCC 29522, Prevotella intermedia ATCC 33563, and Treponema denticola ATCC 33520) examined for cross-reaction were not labeled with any of the three antisera used. In healthy root apex tissues (negative control), no background or any reaction with the three antisera was observed. Histopathological and immunohistochemical evaluations. In three of the seven root tip samples, extraradicular biolms of 30 to 40 m thickness were found on the tooth surfaces outside of the root apex area (Fig. 2a). Red-stained gramnegative bacteria were observed predominantly in the biolm (Fig. 2b). In the other four samples, microcolonies that formed small numbers of bacterial cells were studded around the cementum surfaces of the root apex. In all samples examined, residual microorganisms were detected in the root canals close to the root apex (Fig. 2a). Positive reactions with anti-P. gingivalis, anti-F. nucleatum, and anti-T. forsythensis sera were found in three of the samples that had observed extraradicular biolm (samples 1, 2, and 3). The photographs are typical images, and their distribution shows a similar tendency for each of the bacterial species in the three positive samples (Fig. 3a to c). Positive reactions with

FIG. 3. Immunohistochemical evaluation in extraradicular biolms. (a, b, and c) Arrowheads indicate the junction between the tooth surface and the biolm. (a and c) Arrows indicate immunohistochemically positive reactions. (a) In the anti-F. nucleatum-positive sample, positive reactions distribute predominantly at the middle within the biolm. Magnication, 400. (b) In the anti-P. gingivalis-positive sample, the reactions are scattered throughout the extraradicular biolm. Magnication, 400. (c) In the anti-T. forsythensis-positive sample, positive reactions are localized at the most supercial layer. Magnication, 400.

anti-P. gingivalis were distributed from the cementum to the supercial layer of the biolm, and were scattered throughout the extraradicular biolm (Fig. 3b). Labeled F. nucleatum and T. forsythensis cells are shown at specic parts of the biolms (Fig. 3a and c). DISCUSSION Initially, we speculated that specic bacteria that could resist host immunocytes might invade in the extraradicular area and form biolm; because it has been considered that the expan-

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sion of periapical lesions results from local immune responses (type III and IV allergy), and that bacteria could not inhabit lesions at the chronic phase (21). Various bacterial species detected are associated with biolm formation at the extraradicular area in many cases of chronic and refractory periapical periodontitis is a novel nding. Infected root canal isolates have been investigated by some researchers using molecular techniques, and F. nucleatum and P. gingivalis were frequently identied (3, 5, 17). However, the method of identifying bacteria using bacterium-specic PCR primers is a technique to detect just the DNA of the target bacterium and is not suitable for qualitative analysis of large numbers of unknown bacterial species. Several studies have reported that obligate anaerobes are not frequently detected in the periapical periodontal disease-affected root canal using conventional culture systems (10, 17, 22, 25). The PCR-based 16S rRNA gene assay is useful for identifying a wide range of anaerobic bacteria that are difcult to grow by standard culture methods. Until recently, it was considered that endodontic treatment failure was in many cases the result of microorganisms persisting in the apical portion of the root canal system. F. nucleatum, P. gingivalis, T. forsythensis, and P. intermedia were detected in the periapical lesion contents of asymptomatic root-lled teeth using DNA and DNA hybridization and uorescence in situ hybridization (23, 24). In their studies, experimental materials were obtained from the periapical lesion contents, and bacteria were detected within the granulation tissues. It was possible that bacteria outside of the lesions in the oral cavity invaded the lesion via the stula and contaminated it. On the other hand, we found extraradicular biolms in both this and previous studies, in 14 of 20 and 9 of 11 (12) samples examined, respectively. Moreover, the bacterial species detected from extraradicular biolms were also detected from the root canal in the same teeth at the high rate of 86.7% (data not shown). These results strongly suggested that bacteria that remained in the root canal but invading from a stula could become a supply source for extraradicular biolm formation and that the bacteria inhabiting not only the root canal but also the extraradicular area were one of the causes of refractory periapical periodontitis. Recently, P. gingivalis cells adhering to the root surface were detected at the bottom of human periodontal pockets (11, 14, 15), and it has been suggested that P. gingivalis plays a role as an early colonizer in biolm formation under most anaerobic conditions. It is proposed that P. gingivalis, observed in close contact with the root surface (Fig. 3b), takes part in the initial adherence as an early colonizer in the extraradicular area, as well as at the bottom of human periodontal pockets. Unidentied and uncultivatable bacteria were detected in extraradicular and root canal biolms in 11 of 20 and ve of six (data not shown) samples examined, respectively, although about half the number of bacterial species inhabiting the human oral cavity were uncultivatable (4). The result of tree view analysis clearly showed that the clone sequences of unidentied and uncultured bacteria represented ones similar to these kinds of genera and species. The correlations between these detected bacteria and clinical symptoms and endodontic pathogenicity remain undetermined. In dental practice, the development of new diagnosis meth-

ods and treatments for extraradicular biolms is needed, since biolms are difcult to remove by routine endodontic therapy, and so their presence encourages and maintains local infections.
ACKNOWLEDGMENTS Katsumasa Maeda is thanked for anti-T. forsythensis antiserum. This study was supported by Grants-in-Aid for Scientic Research (14207080 and 15592019) from the Japan Society for the Promotion of Science and via a 21st Century COE program entitled Origination of Frontier BioDentistry at the Osaka University Graduate School of Dentistry, from the Ministry of Education, Culture, Sports, Science and Technology.
REFERENCES 1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403410. 2. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:33893402. 3. Conrads, G., S. E. Gharbia, K. Gulabivala, F. Lampert, and H. N. Shah. 1997. The use of a 16s rDNA directed PCR for the detection of endodontopathogenic bacteria. J. Endodont. 23:433438. 4. Costerton, J. W., P. S. Stewart, and E. P. Greenberg. 1999. Bacterial biolms: a common cause of persistent infections. Science 284:13181322. 5. Fouad, A. F., J. Barry, M. Caimano, M. Clawson, Q. Zhu, R. Carver, K. Hazlett, and J. D. Radolf. 2002. PCR-based identication of bacteria associated with endodontic infections. J. Clin. Microbiol. 40:32233231. 6. Fukushima, H., K. Yamamoto, K. Hirohata, H. Sagawa, K. P. Leung, and C. B. Walker. 1990. Localization and identication of root canal bacteria in clinically asymptomatic periapical pathosis. J. Endodont. 16:534538. 7. Langeland, K., R. M. Block, and L. I. Grossman. 1977. A histopathologic and histobacteriologic study of 35 periapical endodontic surgical specimens. J. Endodont. 3:823. 8. Marchesi, J. R., T. Sato, A. J. Weightman, T. A. Martin, J. C. Fry, S. J. Hiom, D. Dymock, and W. G. Wade. 1998. Design and evaluation of useful bacterium-specic PCR primers that amplify genes coding for bacterial 16S rRNA. Appl. Environ. Microbiol. 64:795799. 9. Moore, W. E. 1987. Microbiology of periodontal disease. J. Periodont. Res. 22:335341. 10. Munson, M. A., T. Pitt-Ford, B. Chong, A. Weightman, and W. G. Wade. 2002. Molecular and cultural analysis of the microora associated with endodontic infections. J. Dent. Res. 81:761766. 11. Noiri, Y., and S. Ebisu. 2000. Identication of periodontal disease-associated bacteria in the plaque-free zone. J. Periodontol. 71:13191326. 12. Noiri, Y., A. Ehara, T. Kawahara, N. Takemura, and S. Ebisu. 2002. Participation of bacterial biolms in refractory and chronic periapical periodontitis. J. Endodont. 28:679683. 13. Noiri, Y., L. Li, and S. Ebisu. 2001. The localization of periodontal-diseaseassociated bacteria in human periodontal pockets. J. Dent. Res. 80:1930 1934. 14. Noiri, Y., L. Li, F. Yoshimura, and S. Ebisu. 2004. Localization of Porphyromonas gingivalis-carrying mbriae in situ in human periodontal pockets. J. Dent. Res. 83:941945. 15. Noiri, Y., K. Ozaki, H. Nakae, T. Matsuo, and S. Ebisu. 1997. An immunohistochemical study on the localization of Porphyromonas gingivalis, Campylobacter rectus and Actinomyces viscosus in human periodontal pockets. J. Periodont. Res. 32:598607. 16. Ramachandran Nair, P. N. 1987. Light and electron microscopic studies of root canal ora and periapical lesions. J. Endodont. 13:2939. 17. Rolph, H. J., A. Lennon, M. P. Riggio, W. P. Saunders, D. MacKenzie, L. Coldero, and J. Bagg. 2001. Molecular identication of microorganisms from endodontic infections. J. Clin. Microbiol. 39:32823289. 18. Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406425. 19. Sen, B. H., B. Piskin, and T. Demirci. 1995. Observation of bacteria and fungi in infected root canals and dentinal tubules by SEM. Endodont. Dent. Traumatol. 11:69. 20. Socransky, S. S., R. J. Gibbons, A. C. Dale, L. Bortnick, E. Rosenthal, J. B. Macdonald. 1963. The microbiota of the gingival crevice area of man. I. Total microscopic and viable counts and specic organisms. Arch. Oral Biol. 8:275280. 21. Stashenko, P. 1998. Etiology and pathogeneses of pulpitis and apical periodontitis, p. 4267. In D. rstavic and T. R. Pitt Ford (ed.), Essential endodontology, vol. 3. Blackwell Science, Oxford, United Kingdom. 22. Sunde, P. T., I. Olsen, G. J. Debelian, and L. Tronstad. 2002. Microbiota of periapical lesions refractory to endodontic therapy. J. Endodont. 28:304310. 23. Sunde, P. T., I. Olsen, U. B. Gobel, D. Theegarten, S. Winter, G. J. Debelian,

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L. Tronstad, and A. Moter. 2003. Fluorescence in situ hybridization (FISH) for direct visualization of bacteria in periapical lesions of asymptomatic root-lled teeth. Microbiology 149:10951102. Sunde, P. T., L. Tronstad, E. R. Eribe, P. O. Lind, and I. Olsen. 2000. Assessment of periradicular microbiota by DNA-DNA hybridization. Endodont. Dent. Traumatol. 16:191196. Sundqvist, G. 1992. Associations between microbial species in dental root canal infections. Oral Microbiol. Immunol. 7:257262. Sundqvist, G. 1994. Taxonomy, ecology, and pathogenicity of the root canal ora. Oral Surg. Oral Med. Oral Pathol. 78:522530. Takemura, N., Y. Noiri, A. Ehara, T. Kawahara, N. Noguchi, and S. Ebisu. 2004. Single species biolm-forming ability of root canal isolates on guttapercha points. Eur. J. Oral Sci. 112:523529.

EXTRARADICULAR BIOFILM-FORMING BACTERIA

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24. 25. 26. 27.

28. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specic gap penalties and weight matrix choice. Nucleic Acids Res. 22:46734680. 29. Tronstad, L., F. Barnett, and F. Cervone. 1990. Periapical bacterial plaque in teeth refractory to endodontic treatment. Endodont. Dent. Traumatol. 6:73 77. 30. Tronstad, L., F. Barnett, K. Riso, and J. Slots. 1987. Extraradicular endodontic infections. Endodont. Dent. Traumatol. 3:8690. 31. Yoneda, M., T. Hirofuji, N. Motooka, K. Nozoe, K. Shigenaga, H. Anan, M. Miura, H. Kabashima, A. Matsumoto, and K. Maeda. 2003. Humoral immune responses to S-layer-like proteins of Bacteroides forsythus. Clin. Diagn. Lab. Immunol. 10:383387.