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1 Anaerobe Reference Laboratory, NPHS Microbiology Cardiff, University Hospital of Wales, Cardiff CF14 4XW, UK
2 School of Food Biosciences, University of Reading, Reading, UK
3 Culture Collection, Department of Clinical Bacteriology, University of Göteborg, Göteborg, Sweden
Correspondence
Val Hall
hallv{at}cardiff.ac.uk
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). In recent years the classification and identification of Actinomyces species has greatly improved (Funke et al., 1997a) and there has been a dramatic expansion in the number of recognized species (e.g. Collins et al., 2000; Funke et al., 1994, 1997b; Hall et al., 2002; Lawson et al., 2001; Nikolaitchouk et al., 2000; Pascual et al., 1997; Wüst et al., 1995). Most of these newly defined species have originated from human sources, and the Actinomyces flora of humans is now much better established. However, despite this increase in the number of recognized species, there is evidence from genetic profiling (amplified 16S rRNA gene sequence restriction analyses) studies of Actinomyces-like organisms recovered from human clinical materials that much diversity remains to be characterized (Hall et al., 2001). In the course of an on-going study to elucidate the taxonomic diversity of the Actinomyces flora of humans, we have characterized an organism that, although phenotypically consistent with the genus Actinomyces, does not appear to correspond to any recognized species. Based on the presented findings, we describe another novel species of the genus Actinomyces, Actinomyces dentalis sp. nov.
The bacterial isolate designated R18165T was isolated in Edinburgh in 2003 from pus of a dental abscess from a 72-year-old female patient with facial swelling. The isolate was referred to the Anaerobe Reference Laboratory, NHPS Microbiology Cardiff, University Hospital of Wales, Cardiff, for identification. No further clinical information is known. For biochemical testing the strain was cultured on Columbia agar (Difco) supplemented with 5 % horse blood at 37 °C and was incubated anaerobically. The strain was biochemically characterized using both conventional tests by the agar plate method of Phillips (1976) and the commercially available API rapid ID32Strep, API rapid ID32A and API Coryne systems according to the manufacturer's instructions (API bioMérieux). Volatile and non-volatile end products of glucose metabolism were detected by GLC (Holdeman et al., 1977). The G+C content of the DNA was determined by HPLC according to the method of Mesbah et al. (1989) except that the methanol content of the chromatographic buffer was decreased to 8 % and the temperature was increased to 37 °C. Amplified 16S rRNA gene sequence restriction analyses were performed using HaeIII and HpaII as described by Hall et al. (1999). The 16S rRNA gene of the isolate was amplified by PCR and sequenced directly using a Taq Dye-Deoxy terminator cycle sequencing kit (Applied Biosystems) and an automatic DNA sequencer (model 373A; Applied Biosystems). The closest known relatives of the new isolate were determined by performing GenBank/EMBL database searches. These sequences and those of other known related strains were retrieved from GenBank and aligned with the newly determined sequence using the program SEQTOOLS (Rasmussen, 2002). The resulting multiple sequence alignment was corrected manually using the program GENEDOC (Nicholas et al., 1997) and a phylogenetic tree was constructed according to the neighbour-joining method with the programs SEQTOOLS and TREEVIEW (Page, 1996); the stability of the groupings was estimated by bootstrap analysis (1000 replications) using the same programs.
The unidentified organism recovered from dental abscess pus consisted of filamentous, beaded, branching, Gram-positive, rod-shaped cells. Cells were non-acid-fast, non-spore-forming and non-motile. After 48 h of anaerobic incubation on fastidious anaerobe agar with 5 % horse blood, colonies were tiny, white and breadcrumb-like and pitted the agar. The organism was catalase-negative and grew under anaerobic conditions; it did not grow in air or in air plus CO2 (5 %). The end products of glucose metabolism were lactic acid (major) and acetic acid (minor); succinic acid was not detected. Using conventional biochemical testing, the organism produced acid from cellobiose, fructose, glucose, lactose (weak), mannose, melibiose, raffinose, sucrose and trehalose but not from arabinose, mannitol or xylose. The organism failed to hydrolyse gelatin or starch. It was urease-negative, did not produce indole and failed to reduce nitrate. Employing commercial API biochemical kits, the strain was unidentified. Using the API Rapid 32Strep system, the organism formed acid from maltose, melibiose, raffinose, sucrose and trehalose but failed to produce acid from D-arabitol, L-arabinose, cyclodextrin, glycogen, lactose, mannitol, melezitose, methyl 
-D-glucopyranoside, pullulan, ribose, sorbitol or tagatose. Activity was detected for 
-galactosidase and -glucosidase; activity for -galactosidase was either weakly positive or negative. All other enzyme tests in the Rapid 32Strep system were negative. The organism failed to hydrolyse hippurate and did not produce acetoin. Using the API Coryne system, acid was formed from glucose, maltose and sucrose but not from glycogen, lactose, mannitol, D-ribose or D-xylose. The organism hydrolysed aesculin but not gelatin, and it failed to reduce nitrate. It displayed activity for -glucosidase and -galactosidase, but all other enzyme tests in this kit were negative. With the API Rapid ID 32A system, the organism produced acid from mannose and raffinose and gave positive reactions for alanine arylamidase, arginine arylamidase, -galactosidase, -galactosidase, -glucosidase, -glucosidase, glycine arylamidase, proline arylamidase, leucine arylamidase, phenylalanine arylamidase and histidine arylamidase (weak). All other tests were negative with this test system.
The cellular morphology and general physiological and biochemical reactions of the organism were consistent with its tentative assignment to the genus Actinomyces, but it did not appear to correspond to any recognized species of this genus. To investigate the possible association of the unknown bacterium with other Actinomyces species, amplified 16S rRNA gene sequence restriction analysis was performed. The unknown organism produced a unique 16S rRNA gene sequence restriction pattern with HaeIII and HpaII (profile 107/099) and was distinct from the profiles derived from analysis of over 800 Actinomyces strains (Hall et al., 2001). To investigate the phylogenetic relationships of the unidentified organism, its almost complete 16S rRNA gene sequence (>1400 nt) was determined. Sequence database searches confirmed that the unknown isolate was most closely related to species of the genus Actinomyces. Treeing analysis demonstrated that the unidentified bacterium was most closely associated with a distinct cluster of species within the genus Actinomyces that includes Actinomyces bovis, the type species (Fig. 1). The nearest sequence to the unknown isolate corresponded to an uncultured rDNA clone from the oral cavity (GenBank accession no. AY278610) whereas the nearest recognized species corresponded to Actinomyces gerencseriae and Actinomyces israelii (Fig. 1).
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). The observed 8 % or more sequence divergence between the unidentified clinical isolate and all currently defined Actinomyces species is therefore consistent with separate species status. Strong support for the separateness of the unknown bacterium also comes from phenotypic evidence. Biochemically the unidentified bacterium can be distinguished from all currently described Actinomyces species. The novel organism can be readily identified using either conventional biochemical criteria or by miniaturized commercial test systems. In particular, the production of API Coryne numerical profile 0450121 serves to distinguish the unknown oral bacterium from all recognized Actinomyces species. Therefore, based on the distinct phenotypic characteristics of the unidentified rod-shaped bacterium and molecular phylogenetic evidence, we consider it warrants classification as a novel species of the genus Actinomyces, for which the name Actinomyces dentalis sp. nov. is proposed. Although only a single strain of A. dentalis is currently known, we consider that the formal description of this species together with biochemical criteria to aid its identification will in the future facilitate its routine recognition in the laboratory, thereby permitting the recovery of additional strains and an evaluation of its distribution, clinical prevalence and possible significance. Tests that are useful in distinguishing A. dentalis from its closest relatives are shown in Tables 1 and 2. Phylogenetically A. dentalis forms a distinct subline within an rRNA subcluster that can be regarded as the genus Actinomyces sensu stricto. It is pertinent to note, however, that current knowledge of the phylogenetic structure of the genus Actinomyces and related taxa is based solely on 16S rRNA gene sequences. Data derived from other independent molecular chronometers are necessary to confirm these phylogenetic inferences.
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Gram-positive, filamentous, beaded, rod-shaped cells. Cells are non-acid-fast, non-spore-forming and non-motile. After 48 h of anaerobic incubation on Fastidious Anaerobe Agar with 5 % horse blood, colonies are tiny, white and breadcrumb-like and pit the agar. Catalase-negative and anaerobic; does not grow in air or in air plus 5 % CO2. The end products of glucose metabolism are acetic and lactic acids. Using conventional tests, acid is formed from cellobiose, fructose, glucose, lactose (weak), mannose, melibiose, raffinose, sucrose and trehalose but not from arabinose, mannitol or xylose. Gelatin, urea and starch are not hydrolysed. Nitrate is not reduced. Using commercial API systems, acid is produced from glucose, maltose, mannose, melibiose, raffinose, sucrose and trehalose but not from D-arabitol, L-arabinose, cyclodextrin, glycogen, lactose, mannitol, melezitose, methyl -D-glucopyranoside, pullulan, ribose, sorbitol, tagatose or D-xylose. Aesculin is hydrolysed but hippurate is not. Indole is not produced. Activity is detected for alanine arylamidase, arginine arylamidase, -galactosidase, -glucosidase, -glucosidase, glycine arylamidase, proline arylamidase, leucine arylamidase, phenylalanine arylamidase and histidine arylamidase (weak); activity for -galactosidase may or may not be detected. Activity is not observed for alanine phenylalanine proline arylamidase, alkaline phosphatase, -arabinosidase, arginine dihydrolase, -fucosidase, glycine arylamidase, -galactosidase-6-phosphate, glutamic acid decarboxylase, glutamyl glutamic acid arylamidase, -glucuronidase, glycyl tryptophan arylamidase, -mannosidase, pyroglutamic acid arylamidase, pyrazinamidase, leucyl glycine arylamidase, N-acetyl--glucosaminidase, tyrosine arylamidase, serine arylamidase or urease. Voges–Proskauer negative. Isolated from human dental abscess. Habitat is not known. The DNA G+C content of the type strain is 62 mol%.
The type strain is R18165T (=CCUG 48064T=CIP 108337T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Funke, G., Stubbs, S., von Graevenitz, A. & Collins, M. D. (1994). Assignment of human-derived CDC group 1 coryneform bacteria and CDC group 1-like coryneform bacteria to the genus Actinomyces as Actinomyces neuii subsp. neuii sp. nov., subsp. nov., and Actinomyces neuii subsp. anitratus subsp. nov. Int J Syst Bacteriol 44, 167–171.
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Hall, V., O'Neill, G. L., Magee, J. T. & Duerden, B. I. (1999). Development of amplified 16S ribosomal DNA restriction analysis for identification of Actinomyces species and comparison with pyrolysis-mass spectrometry and conventional biochemical tests. J Clin Microbiol 37, 2255–2261.
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Wüst, J., Stubbs, S., Weiss, N., Funke, G. & Collins, M. D. (1995). Assignment of Actinomyces pyogenes-like (CDC coryneform group E) bacteria to the genus Actinomyces as Actinomyces radingae sp. nov. and Actinomyces turicensis sp. nov. Lett Appl Microbiol 20, 76–81.[Medline]
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