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1 Institute of Dental Research, Westmead Centre for Oral Health and Westmead Millennium Institute, Westmead Hospital, Wentworthville, NSW 2145, Australia
2 Centre National de Référence des Bactéries Anaérobies et du Botulisme, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France
3 Centre Hospitalier Universitaire de Montpellier et EA 3755, Laboratoire de Bactériologie, Hôpital Arnaud de Villeneuve, 371 avenue du Doyen Gaston Giraud, 34295 Montpellier Cedex 5, France
Correspondence
Roy Byun
roy_byun{at}wmi.usyd.edu.au
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8), which is consistent with other members of the genus Veillonella. Based on these observations, strains RBV81 and RBV106T represent a novel species, for which the name Veillonella denticariosi sp. nov. is proposed, with the type strain RBV106T (=CIP 109448T =CCUG 54362T =DSM 19009T).
A matrix of DNA–DNA hybridization results and 16S rRNA gene sequence similarities is available as supplementary material with the online version of this paper.
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). Presently, the genus Veillonella belongs to the family ‘Acidaminococcaceae’ of the phylum Firmicutes, and is represented by eight species, Veillonella parvula, V. dispar, V. atypica, V. caviae, V. rodentium, V. ratti, V. criceti (Mays et al., 1982; Rogosa, 1965), V. montpellierensis (Jumas-Bilak et al., 2004) and the recently identified ‘V. rogosae’ (Arif et al., 2008). Of these, only V. parvula, V. dispar, V. atypica, V. montpellierensis and ‘V. rogosae’ have been isolated from humans, and they are seldom associated with disease.
With the exception of V. criceti, Veillonella species are unable to ferment carbohydrates or amino acids and rely on the fermentation of lactate, pyruvate, malate, fumarate and/or oxaloacetate as a source of carbon and energy (Foubert & Douglas, 1948; Rogosa, 1984). Veillonella species are frequently isolated from the oral cavity and, in the polymicrobial community of dental plaque, are commonly associated with bacteria that are capable of fermenting carbohydrates to lactic acid, such as strains of Actinomyces, Streptococcus and Lactobacillus. It is believed that veillonellae play an important role in modulating the acid flux (Hoshino et al., 1981), where the metabolic conversion of lactate into the weaker acetic and propionic acids (Ng & Hamilton, 1971) is considered to be anti-cariogenic, as the weaker acids are less capable of decalcifying enamel (Mikx et al., 1972; van der Hoeven et al., 1978). Likewise, in the anaerobic environment of deep dentinal caries, the conversion of a lactate-dominant to a less acidic environment would favour the growth and proliferation of various anaerobic bacterial species that are commonly found in the deeper regions of carious dentine (Chhour et al., 2005; Edwardsson, 1974; Hoshino, 1985). In this study, we describe the isolation and characterization of two strains of a novel species of Veillonella isolated from human carious dentine.
The bacterial isolates RBV81 and RBV106T were selectively isolated from carious lesions of two independently extracted teeth of patients serviced at the Westmead Centre for Oral Health (Sydney, Australia) in 2005. Carious dentine was extracted as described previously (Martin et al., 2002) and plated on Veillonella agar (Rogosa, 1956; Rogosa et al., 1958) supplemented with vancomycin (final concentration 7.5 µg ml–1). Plates were incubated at 37 °C in an anaerobic atmosphere (90 % N2, 5 % H2, 5 % CO2 by vol.) for 48 h in an anaerobic workstation (Don Whitley Scientific). Isolates were presumptively identified by PCR-RFLP analysis of 16S rRNA genes (Sato et al., 1997). Strains RBV81 and RBV106T were identified as having an identical restriction profile to that of V. rodentium ATCC 17743T.
Colonies of strains RBV106T and RBV81 cultured on Veillonella agar in an anaerobic atmosphere for 48 h at 37 °C were circular, smooth, beige coloured, opaque, convex and approximately 2 mm in diameter. Both strains had Gram-negative, coccus-shaped cells arranged singularly or in pairs. Cells were non-spore-forming and non-motile.
For negative staining, bacteria were grown in Veillonella broth at 37 °C in an anaerobic atmosphere for 48 h. A drop of the bacterial suspension was held on a 200-mesh copper grid with a carbon-coated pioloform/Formvar support film for 1 min and removed. The grids were then negatively stained with phosphotungstic acid for 30 s and removed. After allowing the grids to dry for 30 min, they were observed under a Philips CM10 electron microscope. The cells were spherical in shape with a convoluted surface and varied in size from 0.3 to 0.5 µm, with a mean size of 0.4 µm. This is consistent with previous observations of other members of the genus Veillonella (Bladen & Mergenhagen, 1964; Jumas-Bilak et al., 2004).
For electron microscopy of ultrathin sections, cells were fixed in Karnovsky's fixative for 1 h. The bacteria were then washed in phosphate buffer and dehydrated using graded ethanol and embedded in fresh Spurr's resin. Ultrathin sections were cut using a Reichert Ultracut E microtome and picked up on 400-mesh copper grids. The sections were stained with uranyl acetate and lead citrate and observed under a Philips CM10 electron microscope at high magnification to distinguish cellular structures (Fig. 1). The ultrathin sections allowed visualization of structural components (outer membrane, thin peptidoglycan layer and cytoplasmic membrane) that are characteristic of a Gram-negative cell wall and consistent with previous reports for other members of the genus Veillonella (Bladen & Mergenhagen, 1964; Jumas-Bilak et al., 2004).
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; Holdeman et al., 1977). For the Rapid ID 32A system (bioMérieux), cells were grown for 24 h on Veillonella agar at 37 °C in an anaerobic atmosphere and then collected and resuspended in sterile distilled water and analysis was performed according to the manufacturer's instructions. Cellular fatty acid composition was analysed by GC according to Veys et al. (1989). Briefly, strains were grown anaerobically in 10 ml trypticase-glucose-yeast extract (TGY) medium at 37 °C for 48 h in an anaerobic jar containing 5 % H2, 5 % CO2 and 90 % N2 (by vol.). Methyl esters were chromatographed on a fused-silica capillary column (25 mx0.25 mm i.d.) coated with 5 % methyl phenyl silicone (film thickness 0.25 µm).
Strains RBV81 and RBV106T were strictly anaerobic, catalase-negative, indole-negative and reduced nitrate to nitrite. The strains were resistant to vancomycin (5 µg) but susceptible to kanamycin (1 mg), colistin (10 µg), metronidazole (4 µg) and bile identification discs, which is characteristic of other Veillonella species, except V. montpellierensis and V. ratti (Jumas-Bilak et al., 2004). Interestingly, gas was not produced in TGY deep agar, which is atypical for the genus Veillonella. Biochemical analysis showed that neither strain was able to ferment carbohydrates (glucose, fructose, lactose, maltose, mannose and sucrose) but could metabolize lactate to acetate and propionate (5.5 and 5.4 mM, respectively, for strain RBV106T; 5.8 and 6.4 mM, respectively, for strain RBV81). This was supported by the use of the Rapid ID 32A system, which showed that both strains were only positive for the reduction of nitrates (NIT) and for the hydrolysis of arginine (ARG). Cellular fatty acid profiles for RBV81 and RBV106T were similar to each other and consistent with those of other Veillonella species (Table 1), with the dominant fatty acids C13 : 0 and C17 : 18 being synthesized (Jumas-Bilak et al., 2004). Minor fatty acids included C12 : 0, iso-C14 : 0, C14 : 0, anteiso-C15 : 0, C15 : 0, C16 : 19c, C16 : 0, C18 : 19c and C18 : 0. Fatty acids C11 : 0, iso-C16 : 0, C17 : 0
, C17 : 0, C16 : 0 2-OH, C18 : 19t and C20 : 0 were found in trace amounts. Strains RBV81 and RBV106T were found to differ from other Veillonella species in the production of significant amounts of iso-C14 : 0 as well as three unknown compounds.
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; Paster et al., 2001). Sequences were aligned using the program CLUSTAL_X (version 1.83) and phylogenetic analysis was performed using MEGA (version 3.1). Distance matrices were constructed using Kimura's two-parameter method and phylogenetic trees were constructed by the neighbour-joining method (Figs 2 and 3). Bootstrap analysis was based on 1000 replicates and support is represented as percentages.
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). The 16S rRNA gene sequences of strains RBV106T and RBV81 differed at only a single site (99.9 %) and were phylogenetically most closely related to V. rodentium ATCC 17743T, with 99.1 and 99.0 % sequence identity, respectively (Supplementary Table S1, available in IJSEM Online).
Because of the high level of sequence conservation in the 16S rRNA gene sequence between several Veillonella species and the relatively high level of intrachromosomal heterogeneity in some Veillonella isolates (Jumas-Bilak et al., 2004; Marchandin et al., 2003), the 70 kDa heat-shock protein gene (dnaK) was also partially sequenced as an alternate genetic marker. Analysis of partial dnaK sequences (640 bp) of strains RBV106T and RBV81 showed that they share 99.5 % identity. Consistent with observations in the 16S rRNA gene, both strains were phylogenetically most closely related to V. rodentium ATCC 17743T (Fig. 3
), with 95.2 and 95.7 % sequence identity, respectively. This is comparable to the level of sequence identity observed between the dnaK sequences of the type strains of the closely related species V. parvula and V. dispar (Jumas-Bilak et al., 2004), supporting the claim that strains RBV106T and RBV81 represent a novel species. The phylogenetic tree for dnaK is congruent with that of the 16S rRNA gene, which shows that strains RBV106T and RBV81 are distinct from other Veillonella species and most closely related to V. rodentium.
DNA–DNA hybridization to determine genetic relatedness was performed in NucleoLink micro-well strips (Nalge Nunc International) as described previously (Christensen et al., 2000), except that hybridization was performed at 42 °C for 18 h with 50 ng photobiotin acetate-labelled DNA per well in 100 µl 2x SSC buffer, 5x Denhardt's solution, 3 % (w/v) dextran sulphate, 50 % (v/v) formamide and 50 µg salmon sperm DNA ml–1 (Sigma). The control wells contained purified genomic DNA from Escherichia coli XL1-Blue. Strains RBV81 and RBV106T showed a high level of DNA relatedness (87 %; Supplementary Table S1) and are most closely related to V. rodentium ATCC 17743T (49 and 48 %, respectively).
Description of Veillonella denticariosi sp. nov.
Veillonella denticariosi (den.ti.ca.ri.o'si. L. n. dens, dentis tooth; L. adj. cariosus rotten, decayed; N.L. gen. n. denticariosi of a decayed tooth).
Cells are non-motile, non-spore-forming cocci, arranged singly or in pairs, with a mean diameter of 0.4 µm. Irregular masses of cells are also observed in TGY broth. Colonies on Veillonella agar are circular, smooth, beige coloured, opaque and convex and are approximately 2 mm in diameter after 48 h at 37 °C in an anaerobic atmosphere. Cells are strict anaerobes, Gram-negative, with a convoluted surface. Cells are catalase-negative, reduction of nitrates is positive, arginine dihydrolase is present and gas is not produced. Lactate is fermented, with the major metabolic end products being acetate and propionate. Major cellular fatty acids produced are C13 : 0 and C17 : 18, consistent with other Veillonella species, with unique fatty acids iso-C14 : 0 and three unknown compounds being produced. Can also be differentiated from other Veillonella species by 16S rRNA gene and dnaK sequencing.
The type strain RBV106T (=CIP 109448T =CCUG 54362T =DSM 19009T) and a reference strain RBV81 (=CIP 109449 =CCUG 54361 =DSM 19010) were isolated from human carious dentine.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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