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1 Anaerobe Reference Unit, PHLS, 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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The GenBank accession number for the 16S rRNA sequence of CCUG 46090T is AJ507295.
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; Hall et al., 1999, 2001). The use of molecular taxonomic methods [such as 16S rRNA sequencing, amplified 16S rDNA restriction analysis (ARDRA) and protein profiling] have done much to aid the identification of Actinomyces-like organisms and the recognition of novel species (e.g. Collins et al., 2000; Funke et al., 1994, 1997a; Hall et al., 1999, 2001; Lawson et al., 2001; Nikolaitchouk et al., 2000; Pascual et al., 1997; Wüst et al., 1995). However, despite marked improvements in the identification of many Actinomyces-like organisms, especially from human sources, it is evident that many taxonomically problematic organisms remain (Hall et al., 1999, 2001). It is important that atypical or unusual Actinomyces-like organisms are characterized to increase knowledge of their natural habitats and possible clinical associations and prevalence. In the course of an ongoing study exploring the diversity of Actinomyces-like organisms from human sources, we have characterized an organism, which although phenotypically consistent with the genus Actinomyces, does not appear to correspond to any recognized species. Based on the results of a polyphasic taxonomic study, we describe yet another new species of the Actinomyces genus, Actinomyces oricola sp. nov.
The bacterial isolate R5292T was isolated from a dental abscess of a male patient in Bury and was referred to the Anaerobe Reference Unit, PHLS, University Hospital of Wales, 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, incubated anaerobically. The strain was biochemically characterized by using both conventional tests (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 gas–liquid chromatography (Holdeman et al., 1977). PAGE analysis of whole-cell proteins was performed as described by Pot et al. (1994). Long-chain cellular fatty acids were examined using the MIDI system. ARDRAs were performed using HaeIII and HpaII as described previously (Hall et al., 1999). The 16S rDNA of the isolate was amplified by PCR and directly sequenced using a Taq dye-Deoxy terminator cycle sequencing kit (Applied Biosystems) and an automatic DNA sequencer (model 373A; Applied Biosystems). Phylogenetic analyses were performed as described by Collins et al. (2000).
The unidentified organism consisted of Gram-positive, rod-shaped cells, some of which displayed branching, and filaments were observed. Cells were non-acid-fast, non-spore-forming and non-motile. Colonies after 48 h anaerobic incubation on Fastidious Anaerobe Agar with 5 % horse blood were pin-point, breadcrumb-like, white and non-haemolytic. The organism was catalase-negative and facultatively anaerobic, but grew best under anaerobic conditions. The end products of glucose metabolism were acetic acid, lactic acid and succinic acid. Using conventional biochemical testing, the organism produced acid from amygdalin (weak), cellobiose, fructose, glucose, raffinose (weak), salicin, sucrose and trehalose, but not from arabinose or lactose. The organism hydrolysed aesculin, but failed to hydrolyse gelatin or starch. It was lipase-, lecithinase- and urease-negative, and did not produce indole. Employing commercial API biochemical kits the strain was unidentified. Using the API Rapid 32Strep system, the organism was relatively inactive, displaying activity for 
-galactosidase, 
-glucosidase and alanine phenylalanine proline arylamidase (weak reaction). All other tests in this gallery gave negative reactions, producing a numerical code of 220020 00000. Using the API Coryne system, acid was formed from glucose, maltose and sucrose, but not from any of the other carbohydrates. The organism hydrolysed aesculin and displayed activity for alkaline phosphatase, -glucosidase, -galactosidase and pyrazinamidase. All other tests in the API Coryne kit were negative, giving a numerical profile of 2550121. With the API Rapid ID 32A system, the organism produced acid from mannose and raffinose and gave positive reactions for alkaline phosphatase, alanine arylamidase, arginine arylamidase, -galactosidase, -galactosidase, -glucosidase, -glucosidase, glycine arylamidase, proline arylamidase, leucyl glycine arylamidase, leucine arylamidase, phenylalanine arylamidase and tyrosine arylamidase. All other tests were negative, giving an API Rapid ID 32A numerical profile of 4516473700.
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. An examination of the long-chain cellular fatty acids of the unidentified organism revealed the presence of straight-chain saturated and monounsaturated acids: C12 : 0 (0·7 %), C14 : 0 (1·1 %), C16 : 0 (44·0 %), C16 : 1 (2·3 %), C18 : 0 (14 %), C18 : 1 cis9 (37 %) and C20 : 1 cis11 (0·9 %). This fatty acid composition reinforces the relationship of the unknown organism to the genus Actinomyces. To investigate the possible association of the unknown bacterium with Actinomyces species, its whole-cell protein profile was compared with those of known Actinomyces species. Comparative analysis of whole-cell protein profiles showed the unknown organism was distinct from all currently defined species of this genus (data not shown). A dendrogram based on a subset of species depicting comparative protein analysis of the unidentified species and its closest relatives is shown in Fig. 1. Actinomyces howellii (CCUG 32757T, CCUG 35943) displayed the closest similarity to the unidentified clinical organism, joining the latter at about 73 % similarity. Other species displayed much lower levels of similarity (Fig. 1). To further investigate the genetic relatedness of the unidentified organism to Actinomyces species, ARDRA was performed. The unknown organism produced a unique 16S rDNA restriction pattern with HaeIII and HpaII (profile 019/017) and was distinct from the profiles derived from the analysis of over 400 Actinomyces strains (Hall et al., 2001).
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). The observed >5 % 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. In particular, comparative whole-cell protein profiling shows the unidentified organism is distinct from all defined Actinomyces species. Furthermore the unidentified bacterium possesses a very characteristic biochemical profile, which serves to distinguish it from all currently described Actinomyces species. The unidentified organism can be readily distinguished from its nearest phylogenetic relatives using commercial API Rapid ID 32Strep and API Rapid ID 32A kits. In particular, the novel organism differs markedly from Actinomyces bowdenii, Actinomyces catuli, Actinomyces denticolens, Actinomyces israelii, Actinomyces howellii, Actinomyces naeslundii, Actinomyces radicidentis, Actinomyces slackii, Actinomyces viscosus and Actinomyces urogenitalis in producing acid from a far smaller range of sugars. In addition, the unknown organism can be readily distinguished from Actinomyces bovis by producing -galactosidase and -glucosidase. By contrast, Actinomyces bovis gives negative results for these tests.
Therefore, based on the distinct phenotypic characteristics of the unidentified rod-shaped bacterium and molecular phylogenetic evidence, we consider it warrants classification as a new species of the Actinomyces genus, for which the name Actinomyces oricola sp. nov. is proposed. Although only a single strain of Actinomyces oricola is currently known, we consider the formal description of this species together with biochemical criteria to aid its identification, will in the future facilitate its recognition in the routine laboratory, thereby permitting the recovery of additional strains and an evaluation of its distribution, clinical prevalence and possible significance. Tests which are useful in distinguishing Actinomyces oricola from its nearest phylogenetic relatives are shown in Table 1.
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Gram-positive, rod-shaped cells, some of which display branching and filaments may be observed. Cells are non-acid-fast, non-spore-forming and non-motile. Colonies after 48 h anaerobic incubation on Fastidious Anaerobe Agar with 5 % horse blood are pin-point, breadcrumb-like, white and non-haemolytic. Catalase-negative and facultatively anaerobic, but grows best under anaerobic conditions. The end products of glucose metabolism are acetic, lactic and succinic acids. Using conventional tests, acid is produced from amygdalin (weak), cellobiose, fructose, glucose, raffinose (weak), salicin, sucrose and trehalose, but not from arabinose or lactose. Aesculin is hydrolysed, but gelatin and starch are not. Lipase, lecithinase and urease are not detected and indole is not produced. Using commercial API systems, acid is produced from glucose, but not from D-arabitol, L-arabinose, cyclodextrin, glycogen, lactose, mannitol, melibiose, melezitose, methyl--D-glucopyranoside, pullulan, D-ribose, sorbitol, tagatose, trehalose or D-xylose. Acid may or may not be produced from maltose, sucrose and raffinose, depending on the test system used. Aesculin is hydrolysed, but gelatin and hippurate are not. Indole is not produced and nitrate is not reduced. Alanine arylamidase, alanine phenylalanine proline arylamidase, arginine arylamidase, -galactosidase, -glucosidase, -glucosidase, glycine arylamidase, proline arylamidase, pyrazinamidase, leucyl glycine arylamidase, leucine arylamidase, phenyl alanine arylamidase and tyrosine arylamidase are detected. Activity for alkaline phosphatase and -galactosidase may or may not be detected, depending on the test system used. No activity is detected for -arabinosidase, arginine dihydrolase, -fucosidase, -galactosidase-6-phosphate, glutamic acid decarboxylase, glutamyl glutamic acid arylamidase, -glucuronidase, glycyl trypyophane arylamidase, -mannosidase, pyroglutamic acid arylamidase, N-acetyl--glucosaminidase, histidine arylamidase, serine arylamidase or urease. Voges–Proskauer-negative. The major long-chain fatty acids are C16 : 0, C18 : 0 and C18 : 1
9c. Isolated from human dental abscess. Habitat is not known. Type strain is R5292T (=CCUG 46090T=CIP 107639T).
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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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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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