Prevotella histicola sp. nov., isolated from the human oral cavity

Prevotella species constitute part of the commensal oral microbiota in humans and are also associated with various oral diseases and infections at other body sites. The oral mucosa is heavily colonized with bacteria, and bacteria are known to invade the epithelial cells lining the cheeks (Rudney et al., 2005). In a recent study on the bacteria present in healthy oral tissue and in tumours of the oral mucosa, three Prevotella strains were identified that could not be assigned to any species with validly published names (Hooper et al., 2006). The aim of this study was to perform a thorough phenotypic and genotypic characterization of these strains.

Strain T05-04^T was isolated from oral squamous cell carcinoma tissue, while strains N12-20 and N19-30 were isolated from non-tumorous oral mucosal tissue, as described previously (Hooper et al., 2006). Prevotella melaninogenica ATCC 25845^T was obtained from the ATCC and Prevotella veroralis CCUG 15422^T from the CCUG. P. melaninogenica strains W1528, W2198 and W7559 and P. veroralis strains W1627, W1660 and W1700 were obtained from the departmental collection and their identities were confirmed using 16S rRNA gene sequence analysis.

Strains were grown at 37 °C on fastidious anaerobe agar (FAA; LabM) supplemented with 5 % horse blood under anaerobic conditions (80 % N₂, 10 % H₂, 10 % CO₂) in an anaerobic workstation (Don Whitley Scientific). Colonial morphologies were determined after 3 days incubation, using a dissecting microscope. Cellular morphology was recorded after Gram-staining of smears prepared from 2-day-old FAA plate cultures. Cellular motility was investigated by using the hanging-drop method and phase-contrast microscopy with preparations taken from cultures grown in peptone/yeast extract/glucose broth (Holdeman et al., 1977) for 18 h.

Biochemical and physiological tests were performed using standard methods (Holdeman et al., 1977; Jousimies-Somer et al., 2002). Fermentation tests were performed using pre-reduced, anaerobically sterilized sugars prepared in an anaerobic workstation (Holdeman et al., 1977). Susceptibility to special-potency antibiotic discs containing vancomycin (5 μg), kanamycin (1 mg) or colistin (10 μg) was determined on FAA (Jousimies-Somer et al., 2002). Bacterial strains were grown in peptone/yeast extract broth with and without glucose, and short-chain volatile and non-volatile fatty acids produced as metabolic end products were extracted by using standard methods and analysed by gas chromatography (Holdeman et al., 1977). Enzyme profiles were obtained using the Rapid ID 32A anaerobe identification kit (bioMérieux) according to the manufacturer's instructions, using bacteria harvested from Columbia agar plates (LabM) supplemented with 5 % horse blood; the tests were performed in duplicate.

The G+C content of the DNA of all three strains was determined by using an HPLC method described previously (Wade et al., 1999). A thermal denaturation method was used to estimate the extent of DNA–DNA relatedness between strains (Huß et al., 1983).

The 16S rRNA genes of the three strains were sequenced as described previously (Downes et al., 2005). Sequences were assembled using the BioEdit program (Hall, 2004) and their closest relatives were identified by using blast interrogation of the GenBank database (Altschul et al., 1990). Sequences were aligned using clustal w within BioEdit. Phylogenetic trees were constructed using mega 3.1 (Kumar et al., 2004), using the neighbour-joining method, from distance matrices prepared using the Jukes–Cantor correction (Jukes & Cantor, 1969).

The results of the phenotypic tests can be viewed in table format as supplementary data (Supplementary Table S1) in IJSEM Online. Strains T05-04^T, N12-20 and N19-30 were obligately anaerobic, non-motile, variably pigmenting, Gram-negative bacilli that were 0.7 μm wide by 0.8–3.0 μm long (and occasionally up to 5 μm long). After 3 days incubation on FAA plates, colonies were 1.5–2.0 mm in diameter, circular, entire, convex, cream and opaque with a solid non-translucent internal appearance when viewed under a plate microscope (Fig. 1a⇓). Upon further incubation, strain N19-30 developed a deep reddish-brown pigment in the centre of the colony, forming a bull's-eye appearance (Fig. 1b⇓). Strains T05-04^T and N12-20 remained unpigmented even after 2 weeks incubation on FAA with 5 % rabbit blood. However, in the presence of a metronidazole disc (5 μg; Oxoid), strains T05-04^T and N12-20 formed some black-pigmented colonies around the edge of the zone of growth inhibition on FAA (Fig. 1c⇓). When these pigmented colonies were subcultured they retained their black pigmentation even in the absence of metronidazole (Fig. 1d⇓).

Strains were resistant to the special-potency discs containing vancomycin and kanamycin but were sensitive to colistin. Growth of all strains in peptone/yeast extract broth consisted of moderately turbid suspensions (3+ on a scale of 0 to 4+). Growth was markedly enhanced by the addition of 1 % fermentable carbohydrates (4+). Biochemical characteristics of the strains are summarized in the species description, Table 1⇓ and Supplementary Table S1. The DNA G+C content of all three strains was 43 mol%.

In the Rapid ID 32A panel of tests, all three strains gave positive reactions for α-galactosidase, β-galactosidase, β-galactosidase 6-phosphate, α-glucosidase, N-acetyl-β-glucosaminidase, α-fucosidase, mannose fermentation, raffinose fermentation, alkaline phosphatase, arginine arylamidase, leucyl glycine arylamidase, alanine arylamidase and glutamyl glutamic acid arylamidase. The reactions for leucine arylamidase were either negative or weakly positive but were scored as negative, and negative reactions were obtained for the remaining 15 enzymes, resulting in a profile of 4707 4502 22. The type strain and three other strains of P. melaninogenica displayed a profile that was almost identical to that for the novel strains (the only difference being that the type strain of P. melaninogenica was either negative or weakly positive for histidine arylamidase). The profile for the type strain and three other strains of P. veroralis was 4707 4402 22, being negative for arginine arylamidase.

Phylogenetic analysis of the 16S rRNA gene sequence of strain T05-04^T revealed this organism to belong to the genus Prevotella (Fig. 2⇓). An extended phylogenetic tree is available as Supplementary Fig. S1 in IJSEM Online. The complete sequence of the 16S rRNA gene was also obtained for strains N12-20 and N19-30, and both showed ≥99.8 % sequence identity with respect to strain T05-04^T over 1453 unambiguously aligned bases. A blast interrogation of the GenBank database revealed the strains to be closely related to a sequence obtained from Prevotella species oral clone BE073, with a sequence identity of 99.8 %. The most closely related species with validly published names were P. veroralis and P. melaninogenica, both of which showed 97.8 % sequence identity with respect to strain T05-04^T.

The level of DNA–DNA relatedness between strains T05-04^T and N12-20 and between T05-04^T and N19-30 was 96 %. The levels of DNA–DNA relatedness between strain T05-04^T and P. melaninogenica ATCC 25845^T and P. veroralis CCUG 15422^T were 34 and 25 %, respectively. Strain N12-20 showed DNA–DNA relatedness values of 34 % with respect to P. melaninogenica ATCC 25845^T and 27 % with respect to P. veroralis CCUG 15422^T.

Strains T05-04^T, N12-20 and N19-30 constitute a homogeneous group and are clearly distinct from any species with validly published names (see Table 1⇑ for characteristics that serve to distinguish them from recognized species of the genus Prevotella). Therefore strains T05-04^T, N12-20 and N19-30 represent a novel species of the genus Prevotella, for which the name Prevotella histicola sp. nov. is proposed.