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1 Pfizer Inc., Veterinary Medicine Research and Development, 7000 Portage Road, Kalamazoo, MI 49001, USA
2 Western Michigan University, Department of Biological Sciences, 3441 Wood Hall, Kalamazoo, MI 49008, USA
3 University at Buffalo, School of Dental Medicine, Department of Oral Biology, 219 Foster Hall, Buffalo, NY 14214, USA
Correspondence
John M. Hardham
john.m.hardham{at}pfizer.com
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Odoribacter denticanis strains B106T, B107, B113, B126, B129, B135, B140, B150, B151, B152, B155, B163, B171, B172, B174, B183 and B264 are AY560020, AY560021, AY560022, AY560023, AY560024, AY560025, AY560026, AY560027, AY560028, AY560029, AY560030, AY560031, AY560032, AY560033, AY560034, AY560035 and AY560036, respectively. The GenBank/EMBL/DDBJ accession number for the fimA gene of strain B106T is AY573801.
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). Historically, the genera Porphyromonas and Bacteroides have been greatly intertwined, due, in part, to the original classification of many Porphyromonas isolates as belonging to the genus Bacteroides (Shah & Collins, 1988). Porphyromonas species and Bacteroides species both generally appear pigmented on blood agar after extended incubation. Members of these two genera are Gram-negative, non-spore-forming, non-motile rods (Holt et al., 1994). At the time of writing, the genera Porphyromonas and Bacteroides comprised approximately 16 and 77 recognized species, respectively (List of Prokaryotic names with Standing in Nomenclature; http://www.bacterio.cict.fr).
We previously conducted an extensive field study that resulted in the collection of a large number of gingival crevicular fluid samples from companion animals with periodontitis. Analysis of these samples permitted the types and frequencies of pigmented anaerobic bacteria present in diseased crevicular spaces to be determined (Hardham et al., 2003, 2005a). Briefly, paper-point samples were taken from the crevicular spaces of dogs and cats with periodontitis. Samples were only taken from teeth with pocket depths of greater than 2 mm (cats) or 3 mm (dogs). The samples were diluted, plated on Brucella blood agar (Anaerobe Systems) and incubated at 37 °C in an anaerobic environment (5 % H2/5 % CO2/90 % N2). Individual, well-isolated colonies were restreaked on Brucella blood agar in order to obtain pure cultures. A region of the 16S rRNA gene was PCR amplified directly from individual colonies using the universal primers D56 (5'-GGATTAGATACCCTGGTAGTC-3') (Rumpf et al., 1999) and D57 (5'-CCCGGGAACGTATTCACCG-3') (Slots et al., 1995) (Invitrogen). PCR fragments were purified and directly sequenced. The DNA sequences obtained were analysed for relatedness to DNA sequences in the available databases at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/) using the BLASTN software (Altschul et al., 1990). Nearest matches were used to presumptively classify the bacterial genus and species. We identified numerous isolates whose 16S rRNA gene sequences did not have highly similar matches in the available databases, indicating that they may represent novel isolates. One group of these isolates (Table 1) appeared to represent a novel species originally referred to as ‘Porphyromonas denticanis’ (Hardham et al., 2003, 2005a, b). Herein, we propose that this novel species should be placed into a new genus with the name Odoribacter gen. nov.
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). The sample was processed as described above. The purified cells were subjected to biochemical analysis using a RapID ANA II clinical test kit (Remel) and the Vitek Anaerobe Identification (ANI) card system (bioMérieux). Briefly, confluent bacterial cells on Brucella blood agar plates were resuspended to McFarland no. 3 equivalency. The suspension was added to the RapID ANA II test wells or the Vitek ANI card and incubated for 4 h at 37 °C. At the end of the incubation period, the results of the biochemical tests were recorded.
Table 2 shows the results of RapID ANA II testing for strain B106T as well as for six related bacteria. Of the 18 tests performed by using the RapID ANA II kit, only two (leucyl-glycine and indole) were positive for strain B106T. In comparison, Porphyromonas gingivalis ATCC 33277T, Prevotella intermedia ATCC 25611T, Tannerella forsythensis ATCC 43037T, Bacteroides thetaiotaomicron ATCC 29148T, Bacteroides fragilis ATCC 25285T and Bacteroides splanchnicus ATCC 29572T yielded 5, 5, 10, 9 (plus 1 variable), 11 and 7 (plus 1 variable) positive tests, respectively.
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). Strain B106T yielded only two positive tests (indole and triphenyl tetrazolium), whereas Porphyromonas gingivalis ATCC 53977 yielded five positive tests (indole, p-nitrophenyl phosphate, p-nitrophenyl N-acetyl β-D-glucosaminidase, N-benzoyl-DL-arginine p-nitroanilide and triphenyl tetrazolium). Of the 12 tests that were in common between the RapID ANA II and the Vitek ANI card, all yielded similar results. Using the database supplied by Vitek, strain B106T was identified incorrectly as F. nucleatum with a confidence level of 63 % (data not shown). The misidentification may be due to a lack of veterinary pigmented anaerobic bacterial isolates in the Vitek database.
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) (Invitrogen). The PCR products were pooled, purified and desalted, and subjected to direct DNA sequence analysis. BLASTN (Altschul et al., 1990) searches of the non-redundant nucleotide database at the NCBI using the 16S rRNA gene sequence of strain B106T indicated that strain B106T was related to members of the genus Bacteroides. The 16S rRNA gene sequence of strain B106T was most closely related to the 16S rRNA gene sequence of Bacteroidetes sp. oral clone FX069 (GenBank accession number AY134906), with 96.3 % identity over 1465 bp. Bacteroidetes sp. oral clone FX069 was isolated from a human patient with necrotizing ulcerative periodontitis during a study to define the bacterial species associated with necrotizing ulcerative periodontitis in HIV-positive patients (Paster et al., 1994).
Phylogenetic analysis based on 16S rRNA gene sequences was performed using CLUSTAL_X version 1.81 software (Thompson et al., 1997). Phylogenetic trees were generated using the neighbour-joining method (Saitou & Nei, 1987). Bootstrap values were obtained using 1000 replicates. Fig. 1 shows the results of phylogenetic analysis for strain B106T. The placement of the major genera (Porphyromonas, Bacteroides, Prevotella, Tannerella, etc.) is in agreement with previously published phylogenetic trees for the phylum Bacteroidetes (Paster et al., 1994; Sakamoto et al., 2002; Shah et al., 1998).
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), several oral isolates and numerous uncultured strains. An 88.9 % bootstrap confidence value at the branch point of O. denticanis and B. splanchnicus indicates a moderately high degree of certainty that these two organisms are related. However, historically, there has been discussion about the placement of B. splanchnicus within the genus Bacteroides (Paster et al., 1994; Shah et al., 1998). Paster et al. (1994) suggested that B. splanchnicus may represent a new genus. Shah et al. (1998) indicated that B. splanchnicus shares many chemotaxonomic properties with members of the genus Porphyromonas. Our data are in agreement with this hypothesis, as the B. splanchnicus/O. denticanis B106T group branches closer to the genus Porphyromonas than to the genus Bacteroides (Fig. 1
).
An approximately 580 bp region of the 16S rRNA gene from 15 other canine clinical isolates of O. denticanis (Table 1) was PCR amplified (in triplicate), using the primers D56 and D57 described above. The PCR products were purified, desalted and pooled. The DNA sequence of the PCR products was then determined. The partial 16S rRNA gene sequences from the 16 O. denticanis isolates were found to all cluster together into six groups of identical sequences (I: B106T, B107, B126, B129, B135, B140, B163, B174; II: B150, B151, B152; III: B171, B183; IV: B113; V: B155; and VI: B172). The largest divergence between these six groups was between groups II and VI, which showed 97 % identity over the 516 bp analysed. Each of these partial 16S rRNA gene sequences was found to be highly related to that of strain B106T (Fig. 1). Recently, Elliot and others (unpublished sequence submission) have deposited in the NCBI nucleotide databases six partial 16S rRNA gene sequences from bacteria found in canine dental plaque that have homology to O. denticanis 16S rRNA gene sequences. Additional sequences deposited recently by Baldwin and others (unpublished sequence submission) and Eckburg et al. (2005) from bacteria isolated from human periodontal pockets and human intestine, respectively, also have homology to the O. denticanis 16S rRNA gene sequences. Based on these observations, all of these isolates appear to be varying strains of the same species.
In order to analyse further the relationship between strain B106T and the genus Porphyromonas, the fimA gene was PCR amplified (in triplicate) using the degenerate PCR primers D122 (5'-TGGCTAARYTGACYGTAATGGTYTA-3') and D123 (5'-AGTTYACYAATACAGGRTAATAGGT-3') (Invitrogen). The PCR products were purified, desalted and pooled, and subjected to direct DNA sequence analysis. BLASTP searches (Altschul et al., 1990) of the non-redundant polypeptide database at the NCBI using the FimA amino acid sequence indicated that the FimA protein of strain B106T was related to the FimA protein from members of the genus Porphyromonas. Fig. 2 shows the phylogenetic relationships of the various FimA protein sequences. A very strong relationship existed (bootstrap value of 100 %) between the protein of strain B106T and several of the type I fimbrillin proteins of Porphyromonas species.
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) and the FimA phylogenetic data (Fig. 2), we propose that the subgroup of bacteria containing B. splanchnicus should be reclassified in a new genus, Odoribacter gen. nov., in the family ‘Porphyromonadaceae’ (Fig. 1). Additionally, we propose that B. splanchnicus should be reclassified as Odoribacter splanchnicus comb. nov.
Structural analysis
Strain B106T and Porphyromonas gulae B 243 (Hardham et al., 2003, 2005a) were subjected to scanning electron microscopy to determine the cellular morphology. Briefly, 36-h liquid cultures in modified phytone/yeast/glucose medium (Hardham et al., 2003) were centrifuged at 1800 g. The medium was replaced with 3 % electron microscopy-grade glutaraldehyde (Polysciences) in a 0.1 M phosphate buffer (pH 7.3) and incubated at 4 °C for 1 h. The cells were then washed twice in 0.1 M phosphate buffer (pH 7.3) and suspended in buffered 1 % osmium tetroxide (Polysciences) for 1 h at room temperature. The samples were dehydrated in a graded ethanol series with final dehydration done by immersion in hexamethyldisilazane for 15 min, followed by air-drying for 1 h. Samples were mounted on aluminium stubs and coated with 200 Å gold in a Polaron SEM autocoating unit (Polaron Instruments). Each sample was examined by using a DS-130 scanning electron microscope (ISI). Images were captured using a Noran 4485 digital beam control interface (Noran Instruments). Fig. 3 shows scanning electron micrographs of Porphyromonas gulae B 243 and strain B106T. The short rod-shaped morphology (mean length of 1.25 µm) of Porphyromonas gulae B 243 (Fig. 3a) is typical of members of the genus Porphyromonas. In contrast, strain B106T appeared fusiform with tapered ends, with a mean cell length of 5.9 µm (Fig. 3b). The morphology of strain B106T is analogous to that of T. forsythensis under the same growth conditions (Tanner et al., 1986). The morphological difference between Porphyromonas gulae B 243 and strain B106T further supports the placement of strain B106T outside of the genus Porphyromonas.
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). As the O. denticanis isolates described herein were isolated from the crevicular space of dogs with periodontal disease, we sought to determine whether these isolates could cause disease in an experimental model of periodontal disease. We tested the isolates in an oral mouse model of periodontal disease (Baker et al., 1994). In this model, mice that had been pre-treated with antibiotics to reduce their oral flora were orally challenged with the bacteria in question. After a 42-day infection period, the maxillary buccal surfaces of each mouse were scored for alveolar bone loss. The distance from the cemento–enamel junction (CEJ) to the alveolar bone crest (ABC) is indicative of the amount of bone loss.
Fig. 4 shows the results of alveolar bone height measurements. The negative control animals (sham infected) were used to determine the base-line CEJ–ABC distance. Porphyromonas gingivalis ATCC 53977 was used as a positive control. The alveolar bone loss observed following Porphyromonas gingivalis ATCC 53977 infection was consistent with previously published results for this isolate (Baker et al., 2000; Evans et al., 1992a, b). Strain B106T was capable of eliciting alveolar bone loss as shown by the increased CEJ–ABC distances in infected animals. The bone loss elicited by strain B106T was equal to or greater than that generated by infection with Porphyromonas gingivalis ATCC 53977. It can be concluded that strain B106T is capable of inducing periodontitis in infected animals.
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, 2005a, b) and others (Fournier et al., 2001) has indicated that the periodontal pathogens in companion animals may be slightly different from those of humans. For example, Porphyromonas gulae has been identified in companion animals (Fournier et al., 2001; Hardham et al., 2003, 2005a), but not in humans. Since vaccines for periodontitis in humans (Page, 2000) and companion animals (Hardham et al., 2003, 2005b) would aid in periodontal disease prevention programmes, O. denticanis should be considered as an important constituent in such vaccines. We have presented data that demonstrate that a bacterium isolated from the periodontal pockets of dogs represents a previously unidentified species. Furthermore, we propose that the subgroup of bacteria in the genus Bacteroides containing B. splanchnicus should be reclassified as belonging to the family ‘Porphyromonadaceae’ in the genus Odoribacter gen. nov. We also propose that B. splanchnicus should be reclassified as Odoribacter splanchnicus comb. nov. and that the novel group of clinical isolates described herein should be classified as representing Odoribacter denticanis sp. nov.
All procedures in this study were approved by the Institutional Animal Care and Use Committee and were conducted in compliance with the Guide for Care and Use of Laboratory Animals, as well as with all internal company policies and guidelines.
Description of Odoribacter gen. nov.
Odoribacter [O.do.ri.bac'ter. L. n. odor smell; N.L. masc. n. bacter rod; N.L. masc. n. Odoribacter rod of (bad) smell].
Cells are anaerobic, Gram-negative, non-spore-forming, non-motile, fusiform in shape and catalase-positive. Colonies demonstrate pigmentation after >7 days of anaerobic growth at 37 °C on medium containing blood. The type species is Odoribacter splanchnicus.
Description of Odoribacter splanchnicus comb. nov.
Basonym: Bacteroides splanchnicus Werner et al. 1975 (Approved Lists).
The description is that given by Werner et al. (1975).
The type strain is ATCC 29572T (=CCUG 21054T=CIP 104287T=DSM 20712T=LMG 8202T=NCTC 10825T).
Description of Odoribacter denticanis sp. nov.
Odoribacter denticanis (den.ti.ca'nis. L. n. dens -tis tooth; L. gen. n. canis of a dog; N.L. gen. n. denticanis of the tooth of a dog).
Exhibits the following properties in addition to those given in the genus description. Colonies are punctiform and begin to show pigmentation (tan–black) after 10–12 days of incubation at 37 °C in an anaerobic environment on medium containing blood. Colonies are haemolytic on Brucella blood agar, and are resistant to kanamycin, vancomycin and colistin (antibiotic discs from Anaerobe Systems). Colonies on egg yolk agar (Anaerobe Systems) have neither lecithinase nor lipase activities. There is no evidence of bacterial swarming since distinct colonies appear on numerous media types.
The type strain is strain B106T (=ATCC PTA-3625T=CNCM I-3225T). Strain B106T is the subject of the US patent application US20030228328 and the worldwide patent application WO2003054755. The strain is now available to the public.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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-arabinosidase, and hydrolysis of leucyl-glycine, glycine and proline generated the same results for the bacteria indicated. +, Positive; –, negative; V, variable.
, >950; 
, >850; 
, >700; 
, >500; no designation, <500). Bar, 0.02 substitutions per nucleotide position.
