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1 Université Nancy I, UMR CNRS 7565, UFR de Médecine, Laboratoire de Bactériologie, Vandoeuvre-les-Nancy, France
2 Centre Hospitalier et Universitaire de Nancy, Hôpital Central, Laboratoire de Bactériologie, Nancy, France
3 Institut Pasteur, Centre National de Référence des Bactéries Anaérobies et du Botulisme, Paris, France
4 Centre Hospitalier et Universitaire de Montpellier, Hôpital Arnaud de Villeneuve, Laboratoire de Bactériologie, Montpellier, France
5 Université Montpellier 1, UFR de Pharmacie, Laboratoire de Bactériologie-Virologie, EA 3755, Montpellier, France
Correspondence
A. Lozniewski
a.lozniewski{at}chu-nancy.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and rpoB gene sequences determined in this study are respectively AY957555 and EF405531 for P. nanceiensis LBN 293T, EF405529 and EF405532 for P. nanceiensis LBN 297 and EF405530 and EF405533 for P. nanceiensis LBN 298. Those for the rpoB gene sequences of P. marshii E9.34T and P. shahii JCM 12083T are respectively EF405534 and EF405535.
Transmission electron micrographs of cells of strain LBN 293T and a comparison of its fatty acid profile with those of related type strains are available as supplementary material with the online version of this paper.
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). These bacteria, which are part of the human oral, intestinal and urogenital flora, may be involved in various infections of the head and neck, lower respiratory tract, central nervous system, abdominal and female genital tract and in bacteraemia (Jousimies-Somer et al., 2002). The use of molecular methods, such as 16S rRNA gene sequencing, for the identification of anaerobic bacteria has revealed that a significant proportion of strains did not correspond to species with validly published names (Jousimies-Somer, 1997; Jousimies-Somer et al., 2003) and has led to a great expansion of the genus Prevotella, with the description of ten novel species within the last 3 years. In this study, three strains of anaerobic Gram-negative rods, isolated from different human clinical samples and affiliated to the genus Prevotella by presumptive identification tests, were subjected to a comprehensive range of phenotypic, genotypic, genomic and phylogenetic tests.
Strains LBN 293T and LBN 297 were both recovered in pure culture. Strain LBN 293T was isolated in 2003 from blood cultures from a 78-year-old man (Mory et al., 2005) and strain LBN 297 was isolated in 2004 from lung abscess pus from a 67-year-old man. Strain LBN 298 was isolated in 2005 from a broncho-alveolar lavage fluid from a 66-year-old woman. The broncho-alveolar lavage fluid also contained Pseudomonas aeruginosa and a coagulase-negative Staphylococcus sp. Strains were grown at 36 °C on Brucella agar supplemented with 5 % sheep blood, haemin and vitamin K1 (BBA) in an anaerobic chamber (Concept 1000; Ruskinn).
DNA was extracted by using the QIAamp DNA Mini kit (Qiagen). The 16S rRNA gene was amplified by PCR and sequenced as described previously (Carlier et al., 2004). A 374 bp fragment of the gene rpoB was amplified using the primer pair Prev3250F (5'-AACCCGTTGGGTGTGCC-3') and Prev3623R (5'-AGIGCCCAAACCTCCATCTCTCC-3') (Berger et al., 2005). Nucleotide sequences were analysed by using SeqScape software (version 2.5; Applied Biosystems). The sequences were compared to those deposited in the GenBank and Ribosomal Database Project II databases using the BLAST program (Altschul et al., 1997) and Seqmatch program (Cole et al., 2007), respectively. The isolates displayed the highest 16S rRNA gene sequence similarity to members of the genus Prevotella. Maximum similarity (99.4 %) was observed with the sequence of Prevotella sp. oral clone BI027, obtained from human subgingival plaque (Paster et al., 2001). The 16S rRNA gene sequences were aligned against sequences of all known Prevotella type strains and that of Prevotella oral clone BI027 using the DIALIGN program (Morgenstern, 2002). The distance matrix constructed using the Similarity table program of the PHYLIP package (Felsenstein, 1993) showed that strains LBN 293T, LBN 297 and LBN 298 shared more than 99.6 % of their 16S rRNA gene nucleotide positions. The best sequence matches with strains of species with validly published names were obtained with Prevotella marshii E9.34T and Prevotella shahii JCM 12083T, but the similarity levels (89.8 and 89.0 %, respectively) were relatively low. The three isolates displayed 100 % identity in rpoB gene sequences. Maximum similarity was again observed with the sequences of P. marshii E9.34T and P. shahii JCM 12083T (83.1 and 82.8 %, respectively). Altogether, the sequencing results suggested that the three strains belonged to a new taxon. This prompted us to investigate the taxonomic position of these strains by a polyphasic approach.
Evolutionary trees were reconstructed using the PHYLIP suite of programs (Felsenstein, 1993) by maximum-parsimony (Kluge & Farris, 1969) and by neighbour-joining (algorithm F84 for substitution model) (Saitou & Nei, 1987; Kishino & Hasegawa, 1989). The robustness of the nodes was evaluated by 1000 bootstrap replications using SEQBOOT and CONSENSE programs (Felsenstein, 1993). The maximum-likelihood (ML) tree was reconstructed using phyML software with GTR (gamma distribution and invariable sites) as the substitution model and 100 bootstrap replications (Guindon & Gascuel, 2003). The 16S rRNA gene-based ML phylogenetic tree is shown in Fig. 1. Nodes labelled by asterisks were found by all three phylogenetic methods, but other branches moved according to the phylogenetic method. Strains LBN 293T, LBN 297 and LBN 298 formed a robust homogeneous group distinct from other species of the genus Prevotella.
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; Avgu
tin et al., 1997) and was clearly distinct from the DNA G+C contents determined for the most closely related species, P. marshii (51.0 mol%; Downes et al., 2005) and P. shahii (44.3 mol%; Sakamoto et al., 2004).
Large-scale chromosome structure analysis was previously described as a sensitive indicator of phylogenetic relationships between bacteria (Liu et al., 1999; Marchandin et al., 2003a; Jumas-Bilak et al., 2005). Chromosome size and rrn skeletons of the three isolates were studied in comparison with P. marshii DSM 16973T and P. shahii DSM 15611T using PFGE, as described previously (Jumas-Bilak et al., 1998; Marchandin et al., 2003a, b). All three strains studied possess a unique and circular chromosome (data not shown). The chromosome of the novel strains was 3.02 to 3.09 Mb in size, similar to the P. marshii chromosome (2.95 Mb) but clearly distinct from the P. shahii chromosome (4.01 Mb). I-CeuI profiles showed that all the strains tested possess four rrn operons (Fig. 2). The rrn skeleton distinguished the novel strains from their most closely related phylogenetic neighbours, P. marshii and P. shahii, each species exhibiting a specific I-CeuI profile (Fig. 2).
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-haemolysis zone. For examination of the cell-wall ultrastructure of strain LBN 293T, cells were prepared as described previously (Carlier et al., 2004) and electron photomicrographs were taken with a JEOL 1010 transmission electron microscope operating at 80 kV. Rods, 0.4–0.5x0.5–1.0 µm in size, were observed. Transmission electron microscope examination of ultrathin sections of strain LBN 293T showed the presence of a typical Gram-negative cell wall composed of a thin peptidoglycan layer surrounded by an outer membrane (see Supplementary Fig. S1 in IJSEM Online). All strains were susceptible to bile (1 mg tablet) and resistant to kanamycin (1 mg disc), vancomycin (5 µg tablet) and colistin (10 µg tablet).
Biochemical tests were performed in triplicate using the API 20A anaerobe identification kit (bioMérieux) as recommended by the manufacturer. Xylan fermentation was determined in trypticase/yeast extract broth as recommended by Holdeman et al. (1977). Metabolic end products were assayed by quantitative gas chromatography as described previously (Carlier, 1985). Enzyme profiles were generated with the Rapid ID 32A anaerobe identification kit (bioMérieux), according to the manufacturer's instructions, and performed in triplicate. The results of these tests are given in the species description.
Characteristics summarized in Table 1 for the novel strains and related Prevotella species showed that the novel strains were phenotypically very similar to Prevotella buccalis and Prevotella veroralis. However, the novel strains could be distinguished from P. buccalis by the absence of arginine arylamidase production and from P. veroralis by the absence of fermentation of xylan.
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9c are differential characteristics between strain LBN 293T and closely related species. On the basis of the above-mentioned findings, we propose that strains LBN 293T, LBN 297 and LBN 298 should be classified within a novel species of the genus Prevotella, Prevotella nanceiensis sp. nov.
Description of Prevotella nanceiensis sp. nov.
Prevotella nanceiensis (nan.ce.i.en'sis. N.L. fem. adj. nanceiensis pertaining to Nanceium, the old name of Nancy, the French city where the strains supporting the description of the species were isolated).
Cells are obligately anaerobic, non-spore-forming, non-motile, Gram-negative coccoid and short rods (0.4–0.5x0.5–1.0 µm). After 4 days incubation on BBA, colonies are 0.5–1 mm in diameter, circular, entire, slightly convex and smooth, white, non-pigmented and surrounded by a -haemolysis zone. Growth is inhibited in the presence of bile. Strains are saccharolytic and ferment glucose, lactose, maltose, mannose, raffinose and sucrose. Fermentation of cellobiose is variable among strains. Acid is not produced from arabinose, glycerol, mannitol, melezitose, rhamnose, salicin, sorbitol, trehalose, xylan or xylose. The major metabolic end products are acetic, lactic and succinic acids. Aesculin is hydrolysed. Gelatin and urea are not hydrolysed. Indole and catalase are not produced and nitrate is not reduced. All strains are positive for 
-galactosidase, -galactosidase, -galactosidase-6-phosphate, -glucosidase, N-acetyl--glucosaminidase, alkaline phosphatase, leucyl glycine arylamidase, alanine arylamidase, -fucosidase and glutamyl glutamic acid arylamidase in the Rapid ID 32A identification panel, while reactions for mannose and raffinose fermentation and for -glucosidase are variable among strains. All strains are negative for the remaining 16 tests, resulting in a Rapid ID 32A profile of 470/1 1/5/7 440222. The fatty acid profile predominantly comprises iso-C15 : 0, anteiso-C15 : 0, C16 : 0, C16 : 0 3-OH, C18 : 29,12c and summed feature 11 (iso-C17 : 0 3-OH and/or C18 : 2 dimethylacetal). Chromosomal genomic size ranges from 3.02 to 3.09 Mb, with four rrn operon copies. The DNA G+C content of the type strain is 39.4 mol%.
The type strain is LBN 293T (=AIP 261.03T =CIP 108993T =CCUG 54409T), isolated from a blood culture. Two other strains were recovered from clinical respiratory samples.
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ACKNOWLEDGEMENTS |
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