|
|
||||||||
1 Laboratoire de BactériologieVirologie, Faculté de Pharmacie, 15 Avenue Charles Flahault, BP 14491, 34060 Montpellier Cedex 5, France
2 Centre National de Référence des Bactéries Anaérobies et du Botulisme, Institut Pasteur, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France
3 Laboratoire de Bactériologie, Hôpital Arnaud de Villeneuve, 371 Avenue du Doyen Gaston Giraud, 34295 Montpellier Cedex 5, France
Correspondence
Hélène Marchandin
h-marchandin{at}chu-montpellier.fr
| ABSTRACT |
|---|
|
|
|---|
The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence of Veillonella montpellierensis ADV 281.99T (=CIP 107992T=CCUG 48299T) is AF473836.
| MAIN TEXT |
|---|
|
|
|---|
During a survey of anaerobic, Gram-negative cocci in human clinical samples, we isolated three nitrate-reducing strains that showed a resistant phenotype when tested with a 10 µg colistin disc (Rosco). The aim of the present study was to determine the taxonomic status of these isolates by using a polyphasic approach.
Bacterial strains and growth conditions
Three strains (designated ADV 281.99T, ADV 2216.03 and ADV 3198.03) were isolated from clinical samples of three different patients. Strain ADV 281.99T was isolated in 1999 from gastric fluid of a newborn. It was recovered together with Gardnerella vaginalis, Escherichia coli and serotype III group B Streptococcus. Strains ADV 2216.03 and ADV 3198.03 were isolated in 2003 from amniotic fluid samples from two women (28 and 21 years old, respectively) who were hospitalized in the obstetric surgical unit of Montpellier University Hospital. Isolate ADV 2216.03 was recovered together with coagulase-negative Staphylococcus, Lactobacillus,
-haemolytic Streptococcus and Ureaplasma urealyticum. Isolate ADV 3198.03 was recovered together with serotype III group B Streptococcus. Cultures were performed on Columbia sheep blood agar, incubated for 4 days in an anaerobic jar with the AnaeroGen system (Oxoid Unipath). The three isolates were anaerobic, Gram-negative cocci that were identified presumptively as Veillonella sp. on the basis of their capacity to reduce nitrate.
Strains ADV 281.99T, ADV 2216.03 and ADV 3198.03 were studied further in comparison with strains of each of the seven species currently described within the genus Veillonella. V. atypica ATCC 17744T, V. dispar ATCC 17748T, V. parvula ATCC 17745, V. ratti ATCC 17746T and V. rodentium ATCC 17743T were purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA. V. caviae DSM 20738T and V. criceti DSM 20734T were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DMSZ), Braunschweig, Germany, and V. parvula CIP 60.1 from the Collection de l'Institut Pasteur (CIP), Paris, France.
16S rDNA analysis and phylogeny
16S rDNA was amplified from genomic DNA as described previously (Carlier et al., 2002
). The 16S rDNA PCR product was sequenced directly on an Applied Biosystems automatic sequencer (Genome Express). A partial 16S rDNA sequence of 1409 nt was determined previously for strain ADV 281.99T (GenBank accession no. AF473836; Marchandin et al., 2003a
) and partial 16S rDNA sequences of 1388 and 1374 nt were determined in this study for isolates ADV 2216.03 and ADV 3198.03, respectively. As 16S rDNA sequences of V. rodentium and V. caviae were not available, we determined almost-complete 16S rDNA sequences for the type strains of these two species. GenBank accession numbers for 16S rDNA sequences determined in this work are as follows: V. caviae DSM 20738T, AY355140; V. rodentium ATCC 17743T, AY514996; strain ADV 2216.03, AY355141; and strain ADV 3198.03, AY355142.
Alignment of the 16S rDNA sequences by using LALIGN software (www.expasy.ch) showed >99·5 % similarity between the sequences of strains ADV 281.99T, ADV 2216.03 and ADV 3198.03. This indicated that the three isolates were closely related and could belong to the same species. BLAST analysis showed that the tested organisms were related most closely to the currently uncharacterized Veillonella sp. strain 2001-112662 (GenBank accession no. AY244769), with 99·399·6 % sequence similarity. This suggested that this strain might represent the fourth member of the novel taxon. Highest similarity was found with members of the genus Veillonella, from 92·5 % with V. criceti DSM 20734T to 94·7 % with V. atypica ATCC 17744T. The 16S rDNA sequences of strains ADV 281.99T, ADV 2216.03 and ADV 3198.03 were aligned against sequences of representative strains and clones retrieved from GenBank by using the DIALIGN program (Morgenstern, 2002
). The resulting 1309 nt alignment was checked manually and used for construction of phylogenetic trees. Evolutionary trees were inferred by using the maximum-likelihood (ML) (Olsen et al., 1994
), maximum-parsimony (Kluge & Farris, 1969
) and neighbour-joining (NJ) (Saitou & Nei, 1987
) methods from the PHYLIP suite of programs (Felsenstein, 1993
). The F84 algorithm (Kishino & Hasegawa, 1989
) was used to generate evolutionary distance matrices for the NJ method structure; the evolutionary distance tree obtained for members of the genus Veillonella is shown in Fig. 1
. Robustness of trees was evaluated by bootstrap analysis of 1000 resamplings by using the SEQBOOT and CONSENSE programs from the PHYLIP package (Felsenstein, 1993
). It is evident from the 16S rDNA-based phylogeny that strains ADV 281.99T, ADV 2216.03 and ADV 3198.03, together with Veillonella sp. strain 2001-112662, formed a distinct, monophyletic unit. All three treeing algorithms supported the taxonomic integrity of this clade. The monophyly of this group was supported strongly by the high bootstrap value of 100 % of the ML analysis, as well as by parsimony and NJ analyses (96 and 100 %, respectively) (Fig. 1
). A previously published phylogenetic analysis placed strain ADV 281.99T in an intermediate position among members of the genus Veillonella (Marchandin et al., 2003a
). In the present study, the clade that contains strain ADV 281.99T is more deeply branched. This discordance could be explained by the sequences sampled; particularly, the sequences of V. caviae and V. rodentium and those of strains ADV 2216.03, ADV 3198.03 and Veillonella sp. 2001-112662 have been included in the present trees. Nevertheless, the branching of strains ADV 281.99T, ADV 2216.03, ADV 3198.03 and Veillonella sp. 2001-112662, relative to other Veillonella strains, is supported by a good bootstrap value of 83 % in the NJ analysis, which is better than that obtained in the previous tree (67 %), and by the congruence observed between the three phylogenetic methods.
|
dnaK sequence analysis and phylogeny
The 70 kDa heat-shock protein gene (dnaK) was generally present as one copy in bacterial genomes and showed higher interspecies variability than the 16S rRNA gene. These two characteristics should avoid the pitfalls of the 16S rDNA-based taxonomy of the genus Veillonella. A 700 bp dnaK fragment was amplified and sequenced as described previously (Marchandin et al., 2003b
) for strains ADV 281.99T, ADV 2216.03, ADV 3198.03, V. caviae DSM 20738T, V. criceti DSM 20734T, V. parvula ATCC 17745, V. ratti ATCC 17746T and V. rodentium ATCC 17743T. Sequences were deposited in GenBank under accession numbers AY353717, AY353715, AY353716, AY353714, AY353713, AY514999, AY514998 and AY514997, respectively. Sequence data obtained were aligned with those of V. atypica ATCC 17744T, V. dispar ATCC 17748T and V. parvula CIP 60.1 (previously deposited in databases) and were submitted to phylogenetic analysis, as described above. Phylogenetic trees obtained by analysis of 396 nt revealed that the overall topology was congruent with that of 16S rDNA-based trees except for the relative branching of V. caviae, which was supported by a very low bootstrap value in the dnaK tree. The three strains described here formed a distinct lineage, supported by a high bootstrap value, within the genus Veillonella (Fig. 2
). Pairwise similarity analysis showed low levels of dnaK sequence similarity between strain ADV 281.99T and other Veillonella species, from 79 % with V. ratti ATCC 177346T to 86·4 % with V. dispar ATCC 17748T, whereas strains ADV 281.99T and ADV 2216.03 were identical in dnaK sequence. The dnaK sequence of strain ADV 3198.03 shared only 98·4 % similarity with those of strains ADV 281.99T and ADV 2216.03. Regarding the sequence similarity level of 99·7 % between the two strains of V. parvula, the affiliation of the three strains in a single species is questionable. However, as no higher than 96 % similarity was observed between dnaK sequences of the type strains of the related species V. parvula and V. dispar, inclusion of strain ADV 3198.03 in the same species as strains ADV 281.99T and ADV 2216.03 appeared reasonable.
|
Colony and cell morphology
The three strains grew on Columbia blood agar at 37 °C under anaerobic conditions, forming small colonies of 13 mm diameter, which were smooth, opaque and greyish-white. The three isolates were Gram-negative, non-motile, non-sporulating, tiny cocci that were spherical or slightly elongated and were organized singly, in pairs or occasionally in short chains. General morphology of the cells was observed after negative staining as described previously (Marchandin et al., 2003a
), confirming that the cells were spherical, either single or in short chains of two to four cells. Most cells were 0·30·5 µm in diameter. The convoluted nature of the cell surface and the diplococcal shape of the cells were observed (Fig. 3a
) and were in accordance with the observations of Bladen & Mergenhagen (1964)
for other Veillonella species. Ultrastructure of thin sections was also observed by electron microscopy (EM) (Fig. 3b
) and revealed typical Gram-negative surface layers, which consisted of an outer membrane, a thin peptidoglycan layer and a convoluted cytoplasmic membrane (Fig. 3c
), as described previously for several representatives of the family Acidaminococacceae, e.g. Veillonella spp. (Bladen & Mergenhagen, 1964
), Megasphaera spp. (Marchandin et al., 2003a
), Selenomonas ruminantium (Kamio & Takahashi, 1980
; Kalmokoff et al., 2000
) and Centipeda periodontii (Males et al., 1984
).
|
Cellular fatty acid analysis
Cellular fatty acid composition was analysed by GC according to Veys et al. (1989)
. Briefly, strains were grown anaerobically in 10 ml TGYH (Carlier et al., 2002) for 4872 h and methyl esters were chromatographed on a fused-silica capillary column (25 mx0·25 mm ID) coated with 5 % methyl phenyl silicone. The cellular fatty acid composition of the three clinical strains was very similar to that of other species of the genus Veillonella (Table 1
). It is also noteworthy that all species studied contained a unidentified compound, which eluted between 17 : 0 iso and
-cis-9, 10-methylenehexadecanoate (17 : 0
-cyclic 9, 10) (Table 1
). With the exception of V. caviae DSM 20738T, the only notable difference with the other type strains consisted of a smaller percentage of 13 : 0 and a higher amount of cis-9-octadecenoic acid (18 : 1
9cis). Also, levels of 18 : 0 that were observed for these new isolates tended to be slightly higher than those obtained for the other species.
|
Description of Veillonella montpellierensis sp. nov.
Veillonella montpellierensis (mont.pel.li.er.en'sis. N.L. fem. adj. montpellierensis pertaining to Montpellier in the south of France, where the type strain and two other strains supporting the description of the species were isolated).
Cells are coccoid (0·30·5 µm in diameter) and occur singly, in pairs or in short chains. Gram-negative, non-motile, non-sporulating and with a convoluted surface. Colonies on Columbia blood agar are 13 mm in diameter and appear smooth, opaque and greyish-white. Strictly anaerobic and oxidase-negative. Nitrate is reduced. Gas is produced. Major metabolic end products are acetic and propionic acids. Major cellular fatty acids are 12 : 0, 13 : 0, 14 : 0, 15 : 0, 16 : 1
9cis, 16 : 0, 18 : 1
9cis and 18 : 0. Strains contained an unidentified compound, which eluted between 17 : 0 iso and
-cis-9, 10-methylenehexadecanoate (17 : 0
-cyclic 9, 10). Can be differentiated from other species of the genus Veillonella by 16S rDNA and dnaK sequencing.
The type strain is ADV 281.99T (=CIP 107992T=CCUG 48299T) and reference strains are ADV 2216.03 and ADV 3198.03. Found in human clinical samples.
| ACKNOWLEDGEMENTS |
|---|
| REFERENCES |
|---|
|
|
|---|
Carlier, J.-P. (1985). Gas chromatography of fermentation products: its application in diagnosis of anaerobic bacteria. Bull Inst Pasteur 83, 5769.
Carlier, J.-P., Marchandin, H., Jumas-Bilak, E., Lorin, V., Henry, C., Carrière, C. & Jean-Pierre, H. (2002). Anaeroglobus geminatus gen. nov., sp. nov., a novel member of the family Veillonellaceae. Int J Syst Evol Microbiol 52, 983986.[Abstract]
Felsenstein, J. (1993). PHYLIP (phylogeny inference package), version 3.5c. Department of Genetics, University of Washington, Seattle, USA.
Garrity, G. M. & Holt, J. G. (2001). Taxonomic outline of the Archaea and Bacteria. In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 1, pp. 155166. Edited by D. R. Boone & R. W. Castenholz. New York: Springer.
Holdeman, L. V., Cato, E. P. & Moore, W. E. C. (editors) (1977). Anaerobe Laboratory Manual, 4th edn. Blacksburg, VA: Virginia Polytechnic Institute and State University.
Houston, S., Taylor, D. & Rennie, R. (1997). Prosthetic valve endocarditis due to Veillonella dispar: successful medical treatment following penicillin desensitization. Clin Infect Dis 24, 10131014.[Medline]
Kalmokoff, M. L., Austin, J. W., Whitford, M. F. & Teather, R. M. (2000). Characterization of a major envelope protein from the rumen anaerobe Selenomonas ruminantium OB268. Can J Microbiol 46, 295303.[CrossRef][Medline]
Kamio, Y. & Takahashi, H. (1980). Outer membrane proteins and cell surface structure of Selenomonas ruminantium. J Bacteriol 141, 899907.
Kishino, H. & Hasegawa, M. (1989). Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in Hominoidea. J Mol Evol 29, 170179.[CrossRef][Medline]
Kluge, A. G. & Farris, F. S. (1969). Quantitative phyletics and the evolution of anurans. Syst Zool 18, 132.
Kolenbrander, P. E. & Moore, L. V. H. (1992). The genus Veillonella. In The Prokaryotes, 2nd edn, pp. 20342047. Edited by H. G. Balows, M. Trüper, W. Dworkin, W. Harder & K.-H. Schleifer. New York: Springer.
Liu, J. W., Wu, J. J., Wang, L. R., Teng, L. J. & Huang, T. C. (1998). Two fatal cases of Veillonella bacteremia. Eur J Clin Microbiol Infect Dis 17, 6264.[CrossRef][Medline]
Males, B. M., Berthold, P., Dougherty, P. A. & Listgarten, M. A. (1984). Helical flagellation in Centipeda periodontii, a Gram-negative, anaerobic bacillus from periodontitis lesions. J Gen Microbiol 130, 185191.
Marchandin, H., Jean-Pierre, H., Carrière, C., Canovas, F., Darbas, H. & Jumas-Bilak, E. (2001). Prosthetic joint infection due to Veillonella dispar. Eur J Clin Microbiol Infect Dis 20, 340342.[CrossRef][Medline]
Marchandin, H., Jumas-Bilak, E., Gay, B., Teyssier, C., Jean-Pierre, H., Siméon de Buochberg, M., Carrière, C. & Carlier, J.-P. (2003a). Phylogenetic analysis of some Sporomusa sub-branch members isolated from human clinical specimens: description of Megasphaera micronuciformis sp. nov. Int J Syst Evol Microbiol 53, 547553.
Marchandin, H., Teyssier, C., Siméon de Buochberg, M., Jean-Pierre, H., Carrière, C. & Jumas-Bilak, E. (2003b). Intra-chromosomal heterogeneity between the four 16S rRNA gene copies in the genus Veillonella: implications for phylogeny and taxonomy. Microbiology 149, 14931501.
Mays, T. D., Holdeman, L. V., Moore, W. E. C., Rogosa, M. & Johnson, J. L. (1982). Taxonomy of the genus Veillonella Prévot. Int J Syst Bacteriol 32, 2836.
Morgenstern, B. (2002). A simple and space-efficient fragment-chaining algorithm for alignment of DNA and protein sequences. Appl Math Lett 15, 1116.
Olsen, G. J., Matsuda, H., Hagstrom, R. & Overbeek, R. (1994). fastDNAml: a tool for construction of phylogenetic trees of DNA sequences using maximum likelihood. Comput Appl Biosci 10, 4148.
Rogosa, M. (1965). The genus Veillonella. IV. Serological groupings, and genus and species emendations. J Bacteriol 90, 704709.
Rogosa, M. (1971). Transfer of Veillonella Prévot and Acidaminococcus Rogosa from Neisseriaceae to Veillonellaceae fam. nov., and the inclusion of Megasphaera Rogosa in Veillonellaceae. Int J Syst Bacteriol 21, 231233.
Rogosa, M. (1984). Anaerobic Gram-negative cocci. In Bergey's Manual of Systematic Bacteriology, vol. 1, pp. 680685. Edited by N. R. Krieg & J. G. Holt. Baltimore: Williams & Wilkins.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406425.[Abstract]
Sato, T., Matsuyama, J., Sato, M. & Hoshino, E. (1997a). Differentiation of Veillonella atypica, Veillonella dispar and Veillonella parvula using restricted fragment-length polymorphism analysis of 16S rDNA amplified by polymerase chain reaction. Oral Microbiol Immunol 12, 350353.[Medline]
Sato, T., Sato, M., Matsuyama, J. & Hoshino, E. (1997b). PCR-restriction fragment length polymorphism analysis of genes coding for 16S rRNA in Veillonella spp. Int J Syst Bacteriol 47, 12681270.
Singh, N. & Yu, V. L. (1992). Osteomyelitis due to Veillonella parvula: case report and review. Clin Infect Dis 14, 361363.[Medline]
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846849.
Veys, A., Callewaert, W., Waelkens, E. & Van den Abbeele, K. (1989). Application of gas-liquid chromatography to the routine identification of nonfermenting gram-negative bacteria in clinical specimens. J Clin Microbiol 27, 15381542.
This article has been cited by other articles:
![]() |
U. Henssge, T. Do, D. R. Radford, S. C. Gilbert, D. Clark, and D. Beighton Emended description of Actinomyces naeslundii and descriptions of Actinomyces oris sp. nov. and Actinomyces johnsonii sp. nov., previously identified as Actinomyces naeslundii genospecies 1, 2 and WVA 963 Int J Syst Evol Microbiol, March 1, 2009; 59(3): 509 - 516. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. Kraatz and D. Taras Veillonella magna sp. nov., isolated from the jejunal mucosa of a healthy pig, and emended description of Veillonella ratti Int J Syst Evol Microbiol, December 1, 2008; 58(12): 2755 - 2761. [Abstract] [Full Text] [PDF] |
||||
![]() |
N. Arif, T. Do, R. Byun, E. Sheehy, D. Clark, S. C. Gilbert, and D. Beighton Veillonella rogosae sp. nov., an anaerobic, Gram-negative coccus isolated from dental plaque Int J Syst Evol Microbiol, March 1, 2008; 58(3): 581 - 584. [Abstract] [Full Text] [PDF] |
||||
![]() |
N. Arif, E.C. Sheehy, T. Do, and D. Beighton Diversity of Veillonella spp. from Sound and Carious Sites in Children Journal of Dental Research, March 1, 2008; 87(3): 278 - 282. [Abstract] [Full Text] [PDF] |
||||
![]() |
R. Byun, J.-P. Carlier, N. A. Jacques, H. Marchandin, and N. Hunter Veillonella denticariosi sp. nov., isolated from human carious dentine Int J Syst Evol Microbiol, December 1, 2007; 57(12): 2844 - 2848. [Abstract] [Full Text] [PDF] |
||||
![]() |
F. Morio, H. Jean-Pierre, L. Dubreuil, E. Jumas-Bilak, L. Calvet, G. Mercier, R. Devine, and H. Marchandin Antimicrobial Susceptibilities and Clinical Sources of Dialister Species Antimicrob. Agents Chemother., December 1, 2007; 51(12): 4498 - 4501. [Abstract] [Full Text] [PDF] |
||||
![]() |
D. Marriott, D. Stark, and J. Harkness Veillonella parvula Discitis and Secondary Bacteremia: a Rare Infection Complicating Endoscopy and Colonoscopy? J. Clin. Microbiol., February 1, 2007; 45(2): 672 - 674. [Abstract] [Full Text] [PDF] |
||||
![]() |
E. Jumas-Bilak, H. Jean-Pierre, J.-P. Carlier, C. Teyssier, K. Bernard, B. Gay, J. Campos, F. Morio, and H. Marchandin Dialister micraerophilus sp. nov. and Dialister propionicifaciens sp. nov., isolated from human clinical samples Int J Syst Evol Microbiol, November 1, 2005; 55(6): 2471 - 2478. [Abstract] [Full Text] [PDF] |
||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |