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Applied and Environmental Microbiology, December 2005, p. 8738-8743, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8738-8743.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification and Localization of Extraradicular Biofilm-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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial biofilms 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 biofilms. The present study aimed to investigate the identity and localization of bacteria in human extraradicular biofilms. Twenty extraradicular biofilms, 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. Unidentified and uncultured bacterial DNA was also detected in 11 of the 14 samples in which DNA was detected. In the biofilms, P. gingivalis was immunohistochemically detected in all parts of the extraradicular biofilms. Positive reactions to anti-F. nucleatum and anti-T. forsythensis sera were found at specific portions of the biofilm. These findings suggested that P. gingivalis, T. forsythensis, and F. nucleatum were associated with extraradicular biofilm formation and refractory periapical periodontitis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 biofilms in root canals of periapical periodontitis-affected 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 biofilm 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 filling 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.

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 biofilms were formed in the extraradicular area and proposed that extraradicular biofilms, 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 biofilm formation in vitro (27). Gutta-percha points were the root canal filling 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 biofilm formation in vivo (12).

As biofilms are resistant to antibiotic treatment and immune responses (4), we speculate that microorganisms may also be able to survive at the extraradicular area. Once biofilms form in the extraradicular area, it is impossible to remove those on the root surfaces by nonsurgical endodontic treatments. Extraradicular biofilms can become refractory periapical pathogens; however, little is known about the bacterial components in extraradicular biofilms. Therefore, human extraradicular biofilms of refractory periapical periodontitis-affected teeth were used to identify biofilm-forming bacteria and to investigate the immunohistochemical localization of detected bacteria.

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 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.

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TABLE 1. Characteristics and clinical features of patients before sample extractiona

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 µg/ml hemin and 1 µg/ml menadione. F. nucleatum 1436 was grown in Todd-Hewitt broth (Difco) containing 0.5 µg/ml L-cysteine. T. forsythensis ATCC 43037 was grown in brain heart infusion broth containing 0.5% yeast exact, 5 µg/ml hemin, 0.5 µg/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 37°C for 48 h. Cells were also harvested as described (15).

The preparation and purification 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 amplification 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 biofilm 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 purification kit (Flowgen) was added to samples, which were incubated at 37°C for 45 min. Samples were pelleted and resuspended in 100 µl of Tris-EDTA buffer and stored at –20°C 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).

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 37°C, plasmid DNA was purified with a QIAprep Spin miniprep kit (QIAGEN).

Sequencing analysis.
Purified 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 identification of taxa. Multiple alignment of 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 confidence values were carried out by previously described methods (17).

Histopathological and immunohistochemical procedures.
Five selected teeth and two root tips were carefully fixed in 4% paraformaldehyde and 0.1% glutaraldehyde for 12 h at 4°C and decalcified for 10 days at 4°C with stirring in 10% formic acid-sodium citrate (pH 2.2); 8-µm-thick serial frozen sections were prepared. Some of the sections were stained by the Brown and Brenn-modified Gram staining procedure, while others were subjected to the alkaline phosphatase-conjugated streptavidin-biotin method (13-15). The results of both staining methods were observed under a light microscope (Optiphot-2; Nikon, Tokyo, Japan).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of bacteria in extraradicular biofilm.
From 14 of the 20 extraradicular biofilm samples, 1,207 clones were analyzed and 113 bacterial genera and species were identified. 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. Unidentified 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 identified and 11 uncultured or unidentified bacteria were detected (Fig. 1).

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TABLE 2. Prevalence of bacterial species identified in 14 extraradicular samples

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FIG. 1. Phylogenetic tree of the bacterial genes in an extraradicular biofilm 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.

Specificity 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 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 biofilms of 30 to 40 µm thickness were found on the tooth surfaces outside of the root apex area (Fig. 2a). Red-stained gram-negative bacteria were observed predominantly in the biofilm (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).

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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, biofilms indicated by arrows are shown. Residual bacteria, indicated by an arrowhead in the root canal, are detected. RC, root canal. Magnification, x40. (b) High-power view of the inset in a. Arrows indicate that gram-negative rods and filamentous microorganisms are located in the extraradicular biofilm. Magnification, x625.

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 biofilm (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 anti-P. gingivalis were distributed from the cementum to the superficial layer of the biofilm, and were scattered throughout the extraradicular biofilm (Fig. 3b). Labeled F. nucleatum and T. forsythensis cells are shown at specific parts of the biofilms (Fig. 3a and c).

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FIG. 3. Immunohistochemical evaluation in extraradicular biofilms. (a, b, and c) Arrowheads indicate the junction between the tooth surface and the biofilm. (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 biofilm. Magnification, x400. (b) In the anti-P. gingivalis-positive sample, the reactions are scattered throughout the extraradicular biofilm. Magnification, x400. (c) In the anti-T. forsythensis-positive sample, positive reactions are localized at the most superficial layer. Magnification, x400.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Initially, we speculated that specific bacteria that could resist host immunocytes might invade in the extraradicular area and form biofilm; because it has been considered that the expansion 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 biofilm formation at the extraradicular area in many cases of chronic and refractory periapical periodontitis is a novel finding.

Infected root canal isolates have been investigated by some researchers using molecular techniques, and F. nucleatum and P. gingivalis were frequently identified (3, 5, 17). However, the method of identifying bacteria using bacterium-specific 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 difficult 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-filled teeth using DNA and DNA hybridization and fluorescence 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 fistula and contaminated it. On the other hand, we found extraradicular biofilms 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 biofilms 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 fistula could become a supply source for extraradicular biofilm 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 biofilm 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.

Unidentified and uncultivatable bacteria were detected in extraradicular and root canal biofilms in 11 of 20 and five 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 unidentified 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 methods and treatments for extraradicular biofilms is needed, since biofilms are difficult to remove by routine endodontic therapy, and so their presence encourages and maintains local infections.

 
Katsumasa Maeda is thanked for anti-T. forsythensis antiserum.

This study was supported by Grants-in-Aid for Scientific 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.

 
* 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{at}dent.osaka-u.ac.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, December 2005, p. 8738-8743, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8738-8743.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Noguchi, N. Articles by Ebisu, S. Search for Related Content PubMed PubMed Citation Articles by Noguchi, N. Articles by Ebisu, S. Agricola Articles by Noguchi, N. Articles by Ebisu, S.