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1 Université Montpellier 1, EA 3755, Faculté de Pharmacie, Laboratoire de Bactériologie-Virologie, 15, Avenue Charles Flahault, BP 14491, 34093 Montpellier Cedex 5, France
2 Institut Pasteur, Centre National de Référence des Bactéries Anaérobies et du Botulisme, 25 rue du Dr Roux, 75724 Paris Cedex 15, France
3 Centre Hospitalier Universitaire de Montpellier, Hôpital Arnaud de Villeneuve, Laboratoire de Bactériologie, 371 Avenue du Doyen Gaston Giraud, 34295 Montpellier Cedex 5, France
4 R. M. Alden Research Laboratory, Santa Monica Blvd, Santa Monica, CA 90404, USA
5 National Microbiology Laboratory – Public Health Agency of Canada, 1015 Arlington St., Suite H5040, Winnipeg, MB R3E 3R2, Canada
Correspondence
Hélène Marchandin
h-marchandin{at}chu-montpellier.fr
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Jonquetella anthropi ADV 126T is EF436500.
A supplementary figure showing the PFGE analysis of strains of Jonquetella anthropi gen. nov., sp. nov. is available with the online version of this paper.
This work was presented in part at the 8th Congress of the Anaerobe Society of the Americas, Boise, Idaho, USA, July 2006.
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). However, this name is illegitimate due to its prior use for a genus and we therefore suggest the name ‘Synergistetes’ for this candidate phylum. A comprehensive phylogenetic analysis demonstrated that this candidate phylum corresponds to a robust deep-branched clade that groups bacteria currently classified in diverse taxa by Bergey's Manual of Systematic Bacteriology (Garrity & Holt, 2001): the family Syntrophomonadaceae in the phylum Firmicutes and Synergistes jonesii in the phylum Deferribacteres (A. Damay, H. Marchandin, L. Roudière, C. Teyssier, H. Dechaud, H. Jean-Pierre, & E. Jumas-Bilak, unpublished results). To date, bacteria belonging to the phylum ‘Synergistetes’ have been found in a large range of ecosystems, including the guts of mammals (Godon et al., 2005). Culture-independent studies have demonstrated their presence in man during endodontic or periodontic infections, in dental caries (Munson et al., 2002, 2004; Siqueira & Rôças, 2005, 2007; Hutter et al., 2003), in subgingival plaque (Paster et al., 2001), in faeces (Horz et al., 2006) and in vaginal flora (A. Damay, H. Marchandin, L. Roudière, C. Teyssier, H. Dechaud, H. Jean-Pierre, E. Jumas-Bilak, unpublished results). However, only eight human isolates have been reported to date (Munson et al., 2002; Warren et al., 2005; Horz et al., 2006).
In this study, we characterized six strains of unknown, anaerobic Gram-negative rods with fastidious growth, including one isolate initially studied by Warren et al. (2005). The five strains labelled ‘ADV’ were isolated between February 2002 and November 2006 from different patients hospitalized in the University Hospital of Montpellier, France. Strain ADV 116 was recovered in pure culture from a peritoneal fluid sample (abdominal lymphocele due to wound slacking after stomach liposuction), whereas strains ADV 256, ADV 126T, ADV 62 and ADV 808 were recovered in mixed cultures from wounds, cysts and abscesses. Strain RMA 10849 was obtained from the collection of the R. M. Alden Research Laboratory, Santa Monica, CA, USA. This isolate originated from a sacral wound and was previously described as an unnamed organism by Warren et al. (2005) and further described as belonging to ‘Synergistes’ group organisms by Horz et al. (2006). Colony morphology and the responses of the strains to special potency disks (Jousimies-Somer et al., 2002) were observed on Columbia sheep blood agar plates. For further characterization, the novel strains were maintained anaerobically in trypticase/glucose/yeast extract (TGY) medium under anaerobic conditions at 37 °C, as described previously (Carlier et al., 2004). Biochemical reactions were performed according to the procedures of the VPI Anaerobe Laboratory Manual (Holdeman et al., 1977) by using trypticase/yeast extract (TY) medium supplemented with 1 % (w/v) of each sterilized substrate. Colonies appeared on Columbia sheep blood agar plates after 3–5 days at 37 °C in anaerobic conditions generated by the Anaerogen System (Oxoid Unipath). Colonies were very small (0.5 mm), non-haemolytic, circular, shiny and grey. Cells were non-motile. Gram stain showed polymorphic, Gram-negative rods arranged in pairs or short chains. Spores were not observed. Catalase, cytochrome oxidase and urease activities were not detected. Nitrate reductase activity was detected in only two of the six strains (ADV 126T and RMA 10849). No gas was produced in deep TGY agar cultures. Indole production was not detected, gelatin was not liquefied and milk was not coagulated. Aesculin was not hydrolysed. Acid was not produced from arabinose, cellobiose, aesculin, fructose, galactose, glucose, glycerol, inositol, lactose, maltose, mannose, mannitol, melezitose, melibiose, raffinose, rhamnose, ribose, salicin, sorbitol, sucrose, starch, trehalose or xylose. The strains displayed susceptibility to a bile disk (1 mg) and to kanamycin (1 mg) and showed resistance to vancomycin (5 µg; Rosco). Apart from strain ADV 116, resistance was observed to the 10 µg colistin disk (Rosco). The enzymic profile determined by the Rapid ID 32 A system (bioMérieux) according to the manufacturer's recommendations, showed leucyl glycine and glycine arylamidase activities (API code 0000040400) for all strains. The combination of these phenotypic data did not result in the identification of the novel isolates.
DNAs were extracted and purified using the Aquapure DNA isolation kit (Bio-Rad) in accordance with the supplier's protocol. 16S rRNA genes were selectively amplified by PCR using primers 27f and 1492r as previously described (Carlier et al., 2002). The PCR products were directly sequenced on an Applied Biosystems Automatic Sequencer (Genome Express) by using forward and reverse primers. The sequences were compared with sequences in the GenBank and EMBL databases using the BLAST program (Altschul et al., 1997) and LALIGN software (www.expasy.org) and with sequences deposited in the RDPII database by using the SEQMATCH program (Cole et al., 2007). The six novel isolates displayed at least 99 % 16S rRNA gene sequence similarity with each other and were most closely related to species of the genus Dethiosulfovibrio, but with no more than 88.7 % gene sequence similarity. The 16S rRNA gene sequences selected from the GenBank database were aligned by CLUSTAL_X, version 1.83 (Thompson et al., 1997). The alignment was checked and corrected manually before reconstruction of phylogenetic trees. Phylogenetic trees were inferred by using the distance, parsimony and maximum-likelihood methods. The F84 substitution model (Felsenstein, 1993) was used to reconstruct neighbour-joining and maximum-likelihood (ML) evolutionary trees by using PHYLIP (Felsenstein, 1993) and PHYML (Guindon & Gascuel, 2003), respectively. The robustness of the trees was evaluated by bootstrap analysis of 100 resamplings. Distance and ML trees gave congruent results. The strains formed a homogeneous group together with the Flexistipes sp. E3_33 E1 oral isolate that was remote from species of the genus Dethiosulfovibrio and was supported by a high bootstrap value (Fig. 1). From 16S rRNA gene sequence analysis and phylogeny, the six strains studied can be considered to represent a new genus and a novel species. The Flexistipes sp. E3_33 E1 oral isolate should be considered to represent a seventh strain of the novel species. The novel strains clearly belonged to the phylum ‘Synergistetes’ but were distant from other bacteria classified in the family Syntrophomonadaceae (Fig. 1).
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).
For electron microscopy, cells were prepared as described previously for negative staining (Marchandin et al., 2003a; Jumas-Bilak et al., 2005) and were observed with a Hitachi H7100 electron microscope. The morphology of the novel strains was not similar to that of species of the genus Dethiosulfovibrio (Table 1) as the cells were straight rods of 0.8–0.9x1.4–1.7 µm with an irregular surface (Fig. 2).
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). The major metabolic end product was acetic acid (8.5–14.1 mmol l–1). Smaller volumes of propionic (0.9–2.2 mmol l–1), isovaleric (1.5–3.4 mmol l–1), lactic (0–4.1 mmol l–1) and succinic (0–2.5 mmol l–1) acids and trace amounts (below 0.8 mmol l–1) of isobutyric and phenylacetic acids were also detected. The production of 3-methylbutyric acid (isovaleric acid) was detected. However, under the analytical conditions used in this study, it could not be separated from anteisovaleric acid (2-methylbutyric acid) and thus, the latter may be produced. Consequently, this profile appeared relatively similar to that of D. peptidovorans DSM 11002T consisting of acetate, isobutyrate, isovalerate and 2-methylbutyrate production (Table 1
).
The cellular fatty acid (CFA) composition of the five French isolates and of the type strain of D. peptidovorans was analysed by GC according to Veys et al. (1989) after growth on Mueller–Hinton agar plates for 5 days. The CFA profiles obtained for the five novel strains were roughly similar, with the major CFAs being iso-C15 : 0 and C16 : 0, whereas D. peptidovorans DSM 11002T showed different CFA ratios and lacked anteiso-C15 : 0 (Table 2).
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without NaCl. Growth of the novel strains in this medium was extremely poor and was enhanced by NaCl (Table 1). The optimum growth temperature for strains ADV 126T, ADV 62, ADV 256 and ADV 808 was 37 °C. No growth was observed at 42 °C (Table 1). The addition of thiosulfate did not enhance growth. In contrast, strain ADV 116 was similar to D. peptidovorans DSM 11002T since its optimum growth temperature was 42 °C and thiosulfate was reduced to hydrogen sulfide. However, this strain reduced sulfite and thus differs from species of the genus Dethiosulfovibrio (Table 1). None of the novel strains contained desulfoviridin. Based on leucyl glycine and glycine arylamidase activities observed with the Rapid ID 32 A kit, the effect of glycine and leucine on growth was investigated in TGY medium supplemented with 10 mM of each amino acid. In addition, the novel strains were tested in an undefined medium containing (l–1, deionized H2O): 1 g trypticase (BBL), 1 g yeast extract (Difco), 500 mg cysteine HCl, 5 mg haemin, 2 mg resazurin, 5 µg vitamin B12, 500 µg menadione, l mg thiamine HCl, 1 mg nicotinic acid, 500 µg riboflavin, 100 µg para-aminobenzoic acid, 50 µg biotin, 1 mg calcium pantothenate, 0.5 µg pyridoxamine and 500 µg folic acid. The pH of the medium was 6.8–7. The medium was prepared in an anaerobic chamber and dispensed as 10 ml aliquots into 30x60 mm bottles (Supelco). Bottles were closed with butyl rubber stoppers and fastened with crimp seals. The growth of the novel strains was not significantly enhanced in the presence of leucine plus glycine or with vitamins. The maximal OD600 was similar to that of controls in TGY broth and varied between 0.4–0.6 after 5 days of incubation.
Due to the very limited growth of the strains in broth media, it was not possible to obtain sufficient biomass for efficient DNA–DNA hybridization experiments. Therefore, another genomic approach was performed using PFGE, as described previously (Marchandin et al., 2001, 2003b). Large-scale chromosome structure has previously been described as a sensitive indicator of phylogenetic relationships between bacteria (Liu et al., 1999; Marchandin et al., 2003a; Jumas-Bilak et al., 2005). Both chromosomal size and rrn skeleton clearly distinguished the novel strains from D. peptidovorans DSM 11002T. Three of the novel strains displayed a mean genomic size estimated at 1.940±0.11 Mb with 3 rrn operon copies, whereas the type strain of D. peptidovorans showed a clearly distinct rrn skeleton with 5 rrn copies and a genome size of 3.48 Mb (see Supplementary Fig. S1 in IJSEM Online).
The DNA G+C content, as determined by HPLC at the Identification Service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany) for strain ADV 126T was 59.4 mol%, a value distinct from that of members of the genus Dethiosulfovibrio (Table 1).
On the basis of 16S rRNA gene sequence divergence greater than 11 % from the genus Dethiosulfovibrio, phylogenetic and genomic structure data, morphology, absence of motility, inability to reduce thiosulfate and metabolic end products, the novel strains should be classified within a new genus and novel species, for which the name Jonquetella anthropi gen. nov., sp. nov., is proposed. J. anthropi sp. nov. is the first characterized species of the candidate phylum ‘Synergistetes’ that includes human isolates.
Description of Jonquetella gen. nov.
Jonquetella (Jon.que'te.lla. N.L. fem. dim. n. Jonquetella named in honour of Professor Jonquet, the clinician who first diagnosed infection involving this novel genus).
Cells are Gram-negative, non-motile, non-spore-forming, straight rods of 0.8–0.9x1.4–1.7 µm in size. Strictly anaerobic. Colonies are very small (0.5 mm), non-haemolytic, circular, shiny and grey after 3–5 days at 37 °C on Columbia sheep blood agar. Oxidase- and catalase-negative. Carbohydrates are not fermented. Positive for leucyl glycine and glycine arylamidase activities. NaCl is not required for growth, but enhances growth on DP medium (Magot et al., 1997). Major cellular fatty acids are iso-C15 : 0 and C16 : 0. The G+C content of the DNA is 59.4 mol%. Members of the genus can be distinguished from other genera of the phylum ‘Synergistetes’ by 16S rRNA gene sequencing and DNA G+C content. Members of the genus can be differentiated from the closest genus, Dethiosulfovibrio, by morphology, absence of motility, inability to reduce thiosulfate at 37 °C, metabolic end products and genomic structure. The type species is Jonquetella anthropi.
Description of Jonquetella anthropi sp. nov.
Jonquetella anthropi (an.thro'pi. Gr. n. anthropos a human being; N.L. gen. n. anthropi of a human being, since virtually all strains thus far recovered are from human clinical specimens and since it represents the first characterized species including human clinical isolates in the phylum ‘Synergistetes’).
Displays the following characteristics in addition to those given in the genus description. Optimum temperature for growth is 37 or 42 °C. Gas is not produced. Unreactive in most of the conventional biochemical tests. Some strains can reduce thiosulfate and sulfite to sulfide. The metabolic end products in TGY broth are acetic acid, propionic acid and isovaleric acid. Lactic acid and succinic acid may be produced. Trace amounts (
0.8 mmol l–1) of isobutyric acid and phenylacetic acid are produced. Mean genomic size is 1.940±0.11 Mb with 3 rrn operons. The DNA G+C content of the type strain is 59.4 mol%.
The type strain, ADV 126T (=AIP 136.05T=CIP 109408T=CCUG 53819T), was isolated from human clinical samples.
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ACKNOWLEDGEMENTS |
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) end of the carbon chain; cis isomer is indicated by the suffix c.