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Molecular and Cultural Analysis of the Microflora Associated with Endodontic Infections

¹ Department of Microbiology and
² Department of Conservative Dentistry, Guy’s, King’s, and St. Thomas’ Dental Institute, Guy’s Tower, Guy’s Hospital, King’s College London, London SE1 9RT, UK; and
³ Cardiff School of Biosciences, Cardiff University, Cardiff, UK;

* corresponding author, william.wade{at}kcl.ac.uk

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Cultural studies have indicated that a subset of the oral microflora is responsible for endodontic infections. Approximately 50% of oral bacteria are unculturable, so it is likely that currently unknown bacteria are present in such infections. In this study, cultural and molecular analyses were performed on the microflora in aspirate samples collected from 5 infected root canals. 16S rDNA sequences from 261 isolates and 624 clones were identified by comparison with database sequences. Sixty-five taxa were identified, of which 26 were found by the molecular method alone. A mean of 20.2 taxa was found in each sample. A new species of Dialister was the only organism present in all 5 samples. Twenty-seven novel taxa were detected, 18 of which belonged to the phylum Firmicutes and 8 to Bacteroidetes. Culture-independent, molecular analysis has revealed a more diverse microflora associated with endodontic infections than that revealed by cultural methods alone.

KEY WORDS: endodontic • microbiology • 16S rRNA • unculturable • molecular

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Although more than 300 species of bacteria have been isolated from the oral cavity, only a limited number have been consistently isolated from endodontic infections (Sundqvist, 1994). These include species of the genera Streptococcus, Fusobacterium, Prevotella, Porphyromonas, Eubacterium, Peptostreptococcus, Bacteroides, and Lactobacillus (Sundqvist, 1994; Le Goff et al., 1997). However, cultural studies are limited, in that approximately 50% of the oral microflora is unculturable (Socransky et al., 1963). It is therefore probable that unculturable organisms are present in endodontic infections and may play a role in the pathogenesis of the lesions.

Recent advances in molecular techniques based on direct amplification of 16S rDNA genes from DNA extracted from bacteria in clinical samples, followed by cloning and sequencing of the genes, have allowed bacterial communities to be characterized in their entirety, without the biases of culture. The use of these methods to characterize the microflora associated with dento-alveolar abscesses revealed three novel, unculturable, organisms that made up a significant proportion of the organisms present (Dymock et al., 1996; Wade et al., 1997). Studies using a similar approach have demonstrated numerous unculturable taxa in the gingival crevice and in a variety of periodontal infections (Kroes et al., 1999; Paster et al., 2001).

The aim of this study, therefore, was to determine the total diversity of bacteria associated with endodontic infections by means of a combined cultural and molecular approach.

	MATERIALS & METHODS

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Ethical approval for the study was granted by the Guy’s Hospital Research Ethics Committee. Patients participated in the study with their informed consent. Samples were collected from five patients (four females, aged 30-48 yrs, mean 39.2 yrs) from teeth with chronic apical periodontitis with radiological evidence of bone destruction.

The teeth were isolated with a rubber dam and cleaned with hydrogen peroxide. Access cavities were made into the pulp chamber by means of a sterile bur. A 100-µL quantity of sterile saline was introduced to the root canal by means of a needle, and the canal was gently irrigated. The saline was then aspirated, taken immediately to the laboratory and placed inside an anaerobic workstation, and dispersed by passage 5 times through a 25G needle.

Dilutions of the sample in reduced transport medium (RTM) (Bowden and Hardie, 1971) were plated onto fastidious anaerobe agar (FAA), 3 plates per dilution, and blood agar (BA) plates, 6 plates per dilution. FAA plates and one triplicate set of BA plates were incubated in an anaerobic workstation for 7 days, and the remaining set of BA plates was incubated in air + 5% CO₂ for 3 days. Ten isolates were selected at random from the BA plates incubated in air + CO₂. Twenty-five colonies were subcultured at random from both BA and FAA plates incubated anaerobically.

DNA was extracted from the remainder of the sample by the method of Grimont and Grimont (1991). 16S rRNA genes were amplified with use of the primers 27F and 1525R, specific for the domain Bacteria (Lane, 1991). Five replicate amplifications with Ready to Go PCR beads (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) were performed with the use of 1 µL of extract and 1 µL each of the primers at a concentration of 3 µM. Amplifications were carried out on a Biometra UnoII Thermocycler (Whatman Biometra, Glasgow, UK) with 10 cycles of 94°C for 60 sec, 50°C for 30 sec, and 72°C for 2 min followed by a further 20 cycles of 92°C for 30 sec, 50°C for 30 sec, and 72°C for 2.5 min. The PCR products were cloned into the pGEM-T Easy vector (Promega, Southampton, Hampshire, UK), which was then transformed into XL1 Blue MFR' supercompetent cells (Stratagene). One hundred twenty clones from each sample were partially sequenced with the use of the universal sequencing primers 519R (Samples A and B) or 357F (Samples C-E) in an automated DNA sequencer (Beckman CEQ2000, High Wycombe, Buckinghamshire, UK).

We submitted sequences to the Ribosomal Database Project II (Maidak et al., 2001) via the World Wide Web to check for chimeras using Chimera_Check; sequences were provisionally identified by means of the Sequence_Match program. From the phylogenetic position indicated by Sequence_Match, related sequences were selected from sequence databases and aligned by means of Clustal X (Thompson et al., 1997). Further analysis was performed with the use of the PHYLIP suite of programs (Felsenstein, 1993), and phylogenetic trees were viewed by TreeView (Page, 1996).

	RESULTS

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16S rDNA sequence similarity of 98% was used as the cut-off for positive identification of taxa. The nomenclature used was that described by Garrity et al. (2001) for the second edition of Bergey’s Manual of Systematic Bacteriology. Taxa that had less than 98% 16S rDNA sequence similarity with previously described species were identified to genus level, and novel "species" were given an "E" designation. Taxa representing new genera (< 91% sequence similarity) were given an E designation within the relevant family. "Species" within novel genera were given an alphabetical suffix. Some species of streptococci and the subspecies of F. nucleatum could not be definitively identified on the basis of 16S rDNA sequence similarity. For these taxa, the presence of characteristic signature sequences was used for identification, where possible. The two isolates of S. oralis were identified by superoxide dismutase gene (SodA) sequence analysis (Kawamura et al., 1999).

A total of 261 isolates and 624 cloned genes was sequenced and identified in this study. However, 52 clones from samples A and B were identified as Pseudomonas or related genera. These organisms are normally of environmental origin and have been implicated as contaminants of PCR reagents (Tanner et al., 1998). Therefore, for subsequent samples, reagents were UV-irradiated for 15 min to degrade any contaminating DNA. Only 3 clones from samples C-E were identified as belonging to this group. The 52 clones identified as environmental bacteria were therefore excluded from further analysis. In addition, 3 cloned sequences were found to be chimeric and were also excluded from the analysis.

There were no differences in the recovery of individual taxa on BA and FAA incubated anaerobically. Percent recovery rates of taxa on the 2 media were moderately strongly correlated (Spearman correlation coefficient, r = 0.529, p < 0.001).

No aerobes or facultative anaerobes were cultured from samples B and C. All of the aerobic isolates from sample A were Streptococcus mutans and all from sample E were Streptococcus anginosus. From sample D, there were 3 isolates of Micrococcus luteus, 2 of Brachybacterium nesterenkovii, and one each of Acinetobacter lwoffii and a novel Dietzia taxon. In samples A, D, and E, anaerobes outnumbered aerobes and facultative anaerobes by over 200 to 1. Since aerobes and facultative anaerobes were such a minor component of the microflora, the data for the aerobic cultures were not included in the analyses comparing the cultural and molecular methods and estimates of species richness.

The anaerobic culture and clonal analysis data are shown in the Table. Twenty-seven of the 65 taxa could not be positively identified to "species" level, because they did not show 98% or greater similarity to sequences of named species held in GenBank or RDP databases. Seventeen of these novel taxa were detected by the molecular analysis alone. Overall, 19 taxa were detected by culture alone, 20 in both the molecular and cultural analyses, and 26 were found only by the molecular method. A mean of 20.2 taxa was detected in each sample, ranging from 7 in Sample A to 29 in Sample D.

In both analyses, the microflora was dominated by members of the phylum Firmicutes (71.3% of the taxa identified in the molecular analysis, 47.3% by culture, Fig. 1). The phyla Actinobacteria and Bacteroidetes made up around 20% each of the microflora revealed by cultural analysis. In contrast, in the molecular analysis, only 1% of the microflora were found to belong to the Actinobacteria, while 12.1% were Bacteroidetes. Apart from two isolates of Neisseria sicca, Campylobacter gracilis was the sole representative of the phylum Proteobacteria and was more commonly detected in the molecular analysis. The phylum Fusobacteria made up 4.5% of the cultured flora but was not detected in the molecular analysis.

View larger version (16K): [in this window] [in a new window]   Figure 1. Proportion of the microflora, categorized by phylum, recovered by molecular and cultural analyses.

Estimates of the species richness of the microflora infecting dental root canals were made from the data obtained in this study by means of the program EstimateS (Colwell, 1997). From the cultural analysis, total diversity was estimated at 49.1 (Chao 1) or 48.4 (ACE). The molecular analysis gave total taxa figures of 56.9 (Chao 1) and 60.8 (ACE). When the two analyses were combined for each sample, Chao 1 estimated total diversity at 88.1 taxa and ACE 81.4.

Over half of the taxa identified as belonging to the phylum Bacteroidetes were novel taxa, and 3 of these, detected only by the molecular method, represented new "families" within the order Bacteroidales (Fig. 2). Novel taxa within the phylum Firmicutes were widely distributed among this phylum and included novel "species", "genera", and "families" on the basis of their position in the phylogenetic tree (Fig. 3).

View larger version (54K): [in this window] [in a new window]   Figure 2. Phylogenetic tree based on 16S rRNA gene sequence comparisons over 1237 aligned bases showing novel phylotypes within the phylum Bacteroidetes. Tree was constructed by the neighbor-joining method following distance analysis of aligned sequences and was rooted for Lewinella persica. Numbers represent bootstrap values for each branch based on data for 100 trees. Accession numbers for 16S rRNA sequences are given for each strain. Scale bar shows number of nucleotide substitutions per site.

View larger version (57K): [in this window] [in a new window]   Figure 3. Phylogenetic tree based on 16S rRNA gene sequence comparisons over 1131 aligned bases showing novel phylotypes within the phylum Firmicutes. Tree was constructed by the neighbor-joining method following distance analysis of aligned sequences and was rooted for Atopobium rimae. Numbers represent bootstrap values for each branch based on data for 100 trees. Accession numbers for 16S rRNA sequences are given for each strain. Scale bar shows number of nucleotide substitutions per site.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
The results of this study have shown that the microflora associated with endodontic infections is far more diverse than has been shown previously by cultural studies alone. The mean number of taxa recovered from each sample was 20.2, combining both cultural and molecular analyses, and 12.6 by culture alone. Previous studies have recovered from 1 to 12 taxa per sample by cultural methods (Sundqvist, 1992).

The identity of the isolates was consistent with that reported in previous studies, being dominated by anaerobes, particularly Gram-positive taxa of the phylum Firmicutes (Sundqvist, 1992, 1994; Sato et al., 1993). It was interesting, however, that no Porphyromonas species were found. Porphyromonas endodontalis has been linked with endodontic infection in other studies (van Steenbergen et al., 1984; van Winkelhoff et al., 1985), but Haapasalo et al. (1986) reported that P. endodontalis was found only in acute infections. Only chronic lesions were included in this study.

Dialister E1 was the only taxon found in all samples. The isolation of members of this genus is only rarely reported, although D. pneumosintes is common at both periodontally diseased and healthy sites (Moore and Moore, 1994; Contreras et al., 2000). The cells of both D. pneumosintes and Dialister E1 are extremely small, with a diameter of 0.4 µm. It is interesting to speculate that these organisms would be able to penetrate dentinal tubules. It should be possible to test this theory in an in vitro model such as that described by Love et al. (2000) in studies of bacterial penetration of dentinal tubules.

Nineteen taxa were recovered by culture alone. It is probable that primer bias was responsible for the failure of these taxa to be detected in the molecular analysis. Although the primer used is "universal" for the bacterial domain, differential template amplification is a recognized problem with this methodology (Polz and Cavanaugh, 1998).

The molecular analysis revealed numerous novel taxa in the phyla Bacteroidetes and Firmicutes that were not detectable by culture. There are numerous possible reasons for the lack of "culturability". Artificial media may not contain the nutrients required by specific taxa for growth, or the growth of slow-growing organisms may be inhibited by metabolic products from faster-growing organisms. It is also now well-recognized that bacteria in biofilms cooperate to degrade complex substrates. It is possible that some species are completely dependent on others for nutrient supply. Finally, it has been shown that bacteria in biofilms communicate for quorum-sensing purposes (De Kievit et al., 2001) and produce growth factors active across species boundaries (Mukamolova et al., 1998). Signaling molecules have been shown to play a role in the acquisition of hemin and biofilm formation in oral bacteria (Loo et al., 2000; Chung et al., 2001). It is possible that unculturable bacteria are those that are unable to grow when deprived of these signaling networks in vitro.

It has been previously established that of the more than 400 bacterial species found in the mouth, only a subset are found in endodontic infections. This was confirmed in this study, with estimates of species richness giving the total theoretical numbers of species as around 90 or approximately 20% of the total microflora. Presumably those bacteria that are found are those for whom conditions in a necrotic pulp are suitable for their growth. Notwithstanding this, it is interesting that the microflora found in the 5 samples in this study were so different. From this we hypothesize that, within the functional groups of organisms whose metabolism is compatible with the root canal, there is no host restriction via the immune system, because the pulp is dead and the space is inaccessible to inflammatory cells. Thus, although only a quarter of oral bacterial species can colonize the root canal and cause infection, it appears that a variety of permutations of species within that group may be present.

In conclusion, molecular analysis of the microflora associated with endodontic infections has shown it to be far more diverse than demonstrated by culture alone and to include numerous as-yet-uncultivable organisms. The application of molecular analysis and identification by DNA sequence comparison allied with polyphasic taxonomic studies make the description of the oral microflora in its entirety a feasible objective in the short to medium term. The benefit of this as the first stage in the elucidation of bacterial-host relationships in oral health and disease will be enormous.

	ACKNOWLEDGMENTS

 
This study was supported by GKT Dental Institute, King’s College, London.

Received April 1, 2002; Last revision July 25, 2002; Accepted August 7, 2002

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