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1 Laboratoire de Bactériologie–Virologie, Hôpital de la Timone, CNRS UMR 6020, IFR48, 264 rue Saint-Pierre, 13385 Marseille Cedex 05, France
2 Service de Chirurgie Générale, Clinique Vert-Coteau, 41 rue Brandis, 13005 Marseille, France
Correspondence
Véronique Roux
vroux91{at}hotmail.com
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ABSTRACT |
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to characterize ‘the moderately saccharolytic, predominantly oral Bacteroides species’. Identification at the species level among this group of obligatory anaerobic Gram-negative rods remains problematic due to the lack of characteristic phenotypic and biochemical traits. 16S rRNA gene sequence analysis has improved this situation and the number of recognized Prevotella species has increased over the last few years. Prevotella pallens (Kononen et al., 1998), Prevotella shahii, Prevotella salivae and Prevotella multiformis (Sakamoto et al., 2004, 2005), Prevotella marshii and Prevotella baroniae (Downes et al., 2005) have been described recently. Prevotella species have been isolated from the oral cavity, the upper respiratory tract and the urogenital tract in humans. Some species, such as Prevotella ruminicola and the closely related species Prevotella brevis, Prevotella bryantii and Prevotella albensis, have been recovered from the rumen and hindgut of many mammalian species (Avgustin et al., 1997). Species of the genus Prevotella are considered to be part of the normal flora, but sometimes they can induce disease. Thus, bacteraemia and sepsis caused by representatives of the genus Prevotella have led to liver and spleen abscesses (Brook & Frazier, 1998), appendix abscess (Paneri et al., 2002), cervical abscess, meningitis (Frat et al., 2004) and have also provoked endocarditis in an immunocompromised patient (Dominguez-Castellano et al., 2001). Recently, Prevotella intermedia and Prevotella nigrescens were characterized by specific 16S rRNA gene PCR amplification from artery samples of patients suffering from vascular disease (Fiehn et al., 2005; Iwai et al., 2005). DNA of Prevotella species was PCR amplified and identified after cloning from samples of endodontic infections (Rolph et al., 2001) and from the bacterial microbiota in the human stomach (Bik et al., 2006). Genomic identification of these bacteria will allow a better understanding of their involvement in pathology. In this report, we describe a novel species belonging to the genus Prevotella isolated from a human breast abscess.
A 40-year-old woman underwent breast abscess puncture. Blood sample analysis revealed anaemia (haemoglobin, 109 g l–1) and the erythrocyte sedimentation rate was recorded as 32 mm h–1. Liquid from the punctured abscess was cultured and two different Gram-negative bacteria were isolated. The first isolate was identified as Prevotella disiens using API 20A strips (bioMérieux). However, a doubtful identification was obtained for the second isolate, strain 4401737T. 16S rRNA gene sequence determination was performed. This confirmed that strain 4401737T was a member of the genus Prevotella and was possibly a representative of a novel species. The antimicrobial susceptibility of strain 4401737T was determined according to the National Committee for Clinical Laboratory Standards (NCCLS) criteria. Strain 4401737T showed intermediate susceptibility to penicillin G, but was susceptible to Augmentin, cefotetan, imipenem, metronidazole and vancomycin (30 µg ml–1).
Surface colonies on sheep blood agar (bioMérieux) were circular, white–greyish, smooth, shiny, non-pigmented and 1–2 mm in diameter after 72 h. Growth and haemolytic activity were tested at 37 °C on Columbia agar with 5 % sheep blood. Growth was tested in anaerobic and microaerophilic atmospheres which were created using GENbag anaer and GENbag microaer incubation systems (bioMérieux), respectively. Growth was also tested in the presence of air or 5 % CO2. Growth was tested at different temperatures (25, 30, 37 and 45 °C). Optimum growth of strain 4401737T was obtained at 37 °C, but growth occurred between 25 and 37 °C. Strain 4401737T was strictly anaerobic and non-haemolytic.
The size and ultrastructure of cells of the novel strain were determined by transmission electron microscopy (TEM). Cells were grown in liquid trypticase soy broth (TSB; Becton Dickinson) medium for 48 h, collected by pipetting and stained with 1 % (w/v) phosphotungstic acid. Samples were examined on an electron microscope (Morgagni 268D; Philips) at an operating voltage of 60 kV. The rods were 0.8–1.4 µm long and 0.3–0.5 µm wide and usually occurred singly.
Catalase activity was negative as determined by the ID colour catalase test kit (bioMérieux). Bile resistance was tested by growing the bacteria on peptone/yeast extract/glucose agar plates supplemented with 2 % (w/v) dehydrated gall salt (bile sac powder; MP Biomedicals) equivalent to 20 % (v/v) bile. Antibiotic resistance to 5 µg vancomycin ml–1, 1000 µg kanamycin ml–1 and 10 µg colistin ml–1 was tested in thioglycollate with resazurin broth (bioMérieux). An anaerobic atmosphere was created by the addition of 2 ml paraffin oil. Strain 4401737T was bile sensitive and resistant to vancomycin, colistin and kanamycin.
Commercially available API 20A, rapid API 32A and API 50CH strips (bioMérieux) were used to characterize the biochemical properties of strain 4401737T according to the manufacturer's instructions. Incubation was performed at 37 °C. The results of these tests are given in the species description. The phenotypic characteristics of strain 4401737T were compared with those of Prevotella buccalis ATCC 35310T, P. shahii EHS11T and Prevotella oralis ATCC 33269T which were identified as the most closely related species to strain 4401737T when 16S rRNA gene sequences were analysed. The results are presented in Table 1.
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6,9c/C18 : 0 (16 %; fatty acids could not be separated by the MIDI system). It has been reported previously that the major fatty acids in the genus Prevotella are anteiso-C15 : 0, iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 0 and C18 : 19c (Moore et al., 1994; Sakamoto et al., 2004). The presence of C18 : 26,9c and the absence of iso-C17 : 0 3-OH which were noted for our novel isolate are not usual features for species of the genus Prevotella. However, unexpected fatty acid contents have already been reported for other representatives of the genus Prevotella (Sakamoto et al., 2004; Willems & Collins, 1995).
Bacterial DNA was extracted using the FastDNA Kit (BIO 101; Illkirch) as recommended by the manufacturer. PCR amplification of the 16S rRNA gene was performed using the universal primer pair fD1 and rp2 (Weisburg et al., 1991). PCR products were purified using MultiScreen PCR (Millipore) and sequencing reactions were carried out using a DNA sequencing kit (BigDye Terminator Cycle Sequencing v2.0 Ready Reactions; PE Biosystems) according to the manufacturer's instructions. Sequencing products were purified and electrophoresis was performed with a 3100 Genetic Analyzer (Applied Biosystems). The sequences obtained for strain 4401737T were compared with sequences deposited in the GenBank database by using the BLAST program through the NCBI server. Gene sequences were aligned using the multisequence alignment program CLUSTAL_X (1.8). Phylogenetic relationships with closely related species were determined by using MEGA version 2.1 (Kumar et al., 2001). Distance matrices were determined following the assumptions described by Kimura (1980) and were used to elaborate the dendrogram using the neighbour-joining method (Saitou & Nei, 1987). The maximum-parsimony algorithm was also used to infer phylogenetic relationships. A bootstrap analysis (bootstrap values were obtained for a consensus tree based on 100 randomly generated trees) was performed to investigate the stability of the trees obtained. The tree topology was the same with both methods. Phylogenetic analysis demonstrated that strain 4401737T is a member of the genus Prevotella (Fig. 1). Strain 4401737T was closely related to P. buccalis (94 % 16S rRNA gene sequence similarity), P. salivae (90 %) and P. oris (89.1 %). These percentages of similarity were low (<97 %), confirming that strain 4401737T represents a novel species.
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Description of Prevotella timonensis sp. nov.
Prevotella timonensis (ti.mo.n.en'sis. N.L. fem. adj. timonensis from the name Hôpital de la Timone, the hospital in Marseille, France, from where the type strain was isolated).
Cells are obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative straight rods. Growth occurs on sheep blood agar and in TSB liquid medium. Growth is inhibited by 20 % (w/v) bile. After 72 h growth on blood sheep agar, surface colonies are circular, white–greyish, smooth, shiny and up to 1–2 mm in diameter. Temperature range for growth is 25–37 °C, with an optimum at 37 °C. After 48 h growth in TSB medium, rods are 0.8–1.4 µm in length, 0.3–0.5 µm in diameter and occur singly (as observed by TEM). Catalase-negative. Ferments glucose, lactose and maltose. Tests positive for gelatin hydrolysis using an API 20A strip. With an API 20A strip, tests negative for indole formation, urease activity, aesculin hydrolysis and fermentation of mannitol, sucrose, salicin, xylose, arabinose, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose and trehalose. Using API ID 32A tests, positive for activities of alkaline phosphatase, 
-galactosidase, 
-glucosidase, N-acetyl--glucosaminidase, -fucosidase, arginine arylamidase, leucyl glycine arylamidase and alanine arylamidase. Activities of -galactosidase 6-phosphate and -galactosidase are weakly positive. Negative reaction for indole production, reduction of nitrates and fermentation of mannose and raffinose. No activity is detected for glutamic acid decarboxylase, arginine dihydrolase, -glucosidase, -arabinosidase, -glucuronidase, proline arylamidase, phenylalanine arylamidase, leucine arylamidase, pyroglutamic acid arylamidase, tyrosine arylamidase, glycine arylamidase, histidine arylamidase, glutamyl glutamic acid arylamidase or serine arylamidase. Using an API 50CH strip, only reactions for D-ribose, D-tagatose, potassium 5-ketogluconate and aesculin (iron citrate) are positive after 1 week of cultivation. The fatty acid profile is characterized by the predominance of C14 : 0 (19.5 %), followed by C16 : 0 (15.3 %), iso-C14 : 0 (14 %) and a mixture of C18 : 26,9c and C18 : 0 (16 %).
The type strain, 4401737T (=CIP 108522T=CCUG 50105T), was isolated from a human breast abscess.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bik, E. M., Eckburg, P. B., Gill, S. R., Nelson, K. E., Purdom, E. A., Francois, F., Perez-Perez, G., Blaser, M. J. & Relman, D. A. (2006). Molecular analysis of the bacterial microbiota in the human stomach. Proc Natl Acad Sci U S A 103, 732–737.
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Moore, L. V., Bourne, D. M. & Moore, W. E. (1994). Comparative distribution and taxonomic value of cellular fatty acids in thirty-three genera of anaerobic gram-negative bacilli. Int J Syst Bacteriol 44, 338–347.
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Sakamoto, M., Suzuki, M., Huang, Y., Umeda, M., Ishikawa, I. & Benno, Y. (2004). Prevotella shahii sp. nov. and Prevotella salivae sp. nov., isolated from the human oral cavity. Int J Syst Evol Microbiol 54, 877–883.
Sakamoto, M., Huang, Y., Umeda, M., Ishikawa, I. & Benno, Y. (2005). Prevotella multiformis sp. nov., isolated from human subgingival plaque. Int J Syst Evol Microbiol 55, 815–819.
Shah, H. N. & Collins, D. M. (1990). Prevotella, a new genus to include Bacteroides melaninogenicus and related species formerly classified in the genus Bacteroides. Int J Syst Bacteriol 40, 205–208.
Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 697–703.
Willems, A. & Collins, M. D. (1995). 16S rRNA gene similarities indicate that Hallella seregens (Moore and Moore) and Mitsuokella dentalis (Haapsalo et al.) are genealogically highly related and are members of the genus Prevotella: emended description of the genus Prevotella (Shah and Collins) and description of Prevotella dentalis comb. nov. Int J Syst Bacteriol 45, 832–836.
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