Two Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains CCM 4915 and CCM 4916), isolated from clinical specimens of the common vole during an epizootic in the Czech Republic in 2001, were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA () and gene sequence similarities, both isolates were allocated to the genus . Affiliation to was confirmed by DNA–DNA hybridization studies. Both strains reacted equally with M-monospecific antiserum and were lysed by the bacteriophages Tb, Wb, F1 and F25. Biochemical profiling revealed a high degree of enzyme activity and metabolic capabilities not observed in other species. The and genes of isolates CCM 4915 and CCM 4916 were indistinguishable. Whereas was identical to of brucellae from certain pinniped marine mammals, clustered with of terrestrial brucellae. Analysis of the gene downstream region identified strains CCM 4915 and CCM 4916 as of terrestrial origin. Both strains harboured five to six copies of the insertion element IS, displaying a unique banding pattern as determined by Southern blotting. In comparative multilocus VNTR (variable-number tandem-repeat) analysis (MLVA) with 296 different genotypes, the two isolates grouped together, but formed a separate cluster within the genus . Multilocus sequence typing (MLST) analysis using nine different loci also placed the two isolates separately from other brucellae. In the IS-based AMOS PCR, a 1900 bp fragment was generated with the -specific primers, revealing that the insertion element had integrated between a putative membrane protein and , encoding a methyltransferase, an integration site not observed in other brucellae. Isolates CCM 4915 and CCM 4916 could be clearly distinguished from all known species and their biovars by means of both their phenotypic and molecular properties, and therefore represent a novel species within the genus , for which the name sp. nov. with the type strain CCM 4915 (=BCCN 07-01=CAPM 6434) is proposed.


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  1. Al Dahouk, S., Tomaso, H., Prenger-Berninghoff, E., Splettstoesser, W. D., Scholz, H. C. & Neubauer, H.(2005). Identification of Brucella species and biotypes using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Crit Rev Microbiol 31, 191–196.[CrossRef] [Google Scholar]
  2. Al Dahouk, S., Le Flèche, P., Noeckler, K., Jacques, I., Grayon, M., Scholz, H. C., Tomaso, H., Vergnaud, G. & Neubauer, H.(2007). Evaluation of Brucella MLVA typing for human brucellosis. J Microbiol Methods 69, 137–145.[CrossRef] [Google Scholar]
  3. Altenburger, P., Kämpfer, P., Makristathis, A., Lubitz, W. & Busse, H.-J.(1996). Classification of bacteria isolated from a medieval wall painting. J Biotechnol 47, 39–52.[CrossRef] [Google Scholar]
  4. Alton, G. G., Jones, L. M., Angus, R. D. & Verger, J. M.(1988).Techniques for the Brucellosis Laboratory (Techniques et Pratiques). Paris: INRA.
  5. Bricker, B. J. & Halling, S. M.(1994). Differentiation of Brucella abortus bv. 1, 2, and 4, Brucella melitensis, Brucella ovis and Brucella suis bv. 1 by PCR. J Clin Microbiol 32, 2660–2666. [Google Scholar]
  6. Busse, H. J. & Auling, G.(1988). Polyamine pattern as a chemotaxonomic marker within the Proteobacteria. Syst Appl Microbiol 11, 1–8.[CrossRef] [Google Scholar]
  7. Clavareau, C., Wellemans, V., Walravens, K., Tryland, M., Verger, J.-M., Grayon, M., Cloeckaert, A., Letesson, J. J. & Godfroid, J.(1998). Phenotypic and molecular characterization of a Brucella strain isolated from a minke whale (Balaeonoptera acutorostrata). Microbiology 144, 3267–3273.[CrossRef] [Google Scholar]
  8. Cloeckaert, A., Verger, J. M., Grayon, M. & Grepinet, O.(1995). Restriction site polymorphism of the genes encoding the major 25 kDa and 36 kDa outer-membrane proteins of Brucella. Microbiology 141, 2111–2121.[CrossRef] [Google Scholar]
  9. Cloeckaert, A., Grayon, M. & Grepinet, O.(2000). An IS711 element downstream of the bp26 gene is a specific marker of Brucella spp. isolated from marine mammals. Clin Diagn Lab Immunol 7, 835–839. [Google Scholar]
  10. Cloeckaert, A., Verger, J.-M., Grayon, M., Paquet, J. Y., Garin-Bastuji, B., Foster, G. & Godfroid, J.(2001). Classification of Brucella spp. isolated from marine mammals by DNA polymorphism at the omp2 locus. Microbes Infect 3, 729–738.[CrossRef] [Google Scholar]
  11. Corbel, M. J. & Brinley-Morgan, W. J.(1975). Proposal for minimal standards for descriptions of new species and biotypes of the genus Brucella. Int J Syst Bacteriol 25, 83–89.[CrossRef] [Google Scholar]
  12. Corbel, M. J. & Brinley-Morgan, W. J.(1984). Genus Brucella. In Bergey's Manual of Systematic Bacteriology, vol. 1, pp. 377–388. Edited by N. R. Krieg & J. G. Holt. Baltimore: Williams & Wilkins.
  13. Ferrao-Beck, L., Cardoso, R., Munoz, P. M., de Miguel, M. J., Albert, D., Ferreira, A. C., Marin, C. M., Thiebaud, M., Jacques, I. & other authors(2006). Development of a multiplex PCR assay for polymorphism analysis of Brucella suis biovars causing brucellosis in swine. Vet Microbiol 115, 269–277.[CrossRef] [Google Scholar]
  14. Foster, G., Osterman, B., Godfroid, J., Jacques, I. & Cloeckaert, A.(2007).Brucella ceti sp. nov. and Brucella pinnipedialis sp. nov. for Brucella strains with cetaceans and seals as their preferred hosts. Int J Syst Evol Microbiol 57, 2688–2693.[CrossRef] [Google Scholar]
  15. Garcia-Yoldi, D., Marin, C. M., de Miguel, M. J., Munoz, P. M., Vizmanos, J. L. & Lopez-Goni, I.(2006). Multiplex PCR assay for the identification and differentiation of all Brucella species and the vaccine strains Brucella abortus S19 and RB51 and Brucella melitensis Rev1. Clin Chem 52, 779–781.[CrossRef] [Google Scholar]
  16. Garcia-Yoldi, D., Le Flèche, P., Marin, C. M., De Miguel, M. J., Munoz, P. M., Vergnaud, G. & Lopez-Goni, I.(2007). Assessment of genetic stability of Brucella melitensis Rev 1 vaccine strain by multiple-locus variable-number tandem repeat analysis. Vaccine 25, 2858–2862.[CrossRef] [Google Scholar]
  17. Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors)(1994).Methods for General and Molecular Bacteriology. Washington, DC: American Society for Microbiology.
  18. Hubálek, Z., Scholz, H. C., Sedláček, I., Melzer, F., Sanogo, Y. & Nesvadbová, J.(2007). Brucellosis of the common vole (Microtus arvalis). Vector Borne Zoonotic Dis 7, 679–688.[CrossRef] [Google Scholar]
  19. Kämpfer, P., Rosselló-Mora, R., Scholz, H. C., Welinder-Olsson, C., Falsen, E. & Busse, H.-J.(2006). Description of Pseudochrobactrum gen. nov., with the two species Pseudochrobactrum asaccharolyticum sp. nov. and Pseudochrobactrum saccharolyticum sp. nov. Int J Syst Evol Microbiol 56, 1823–1829.[CrossRef] [Google Scholar]
  20. Kämpfer, P., Scholz, H., Huber, B., Thummes, K., Busse, H.-J., Maas, E. W. & Falsen, E.(2007). Description of Pseudochrobactrum kiredjianiae sp. nov. Int J Syst Evol Microbiol 57, 755–760.[CrossRef] [Google Scholar]
  21. Le Flèche, P., Jacques, I., Grayon, M., Al Dahouk, S., Bouchon, P., Denoeud, F., Nockler, K., Neubauer, H., Guilloteau, L. A. & Vergnaud, G.(2006). Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay. BMC Microbiol 6, 9[CrossRef] [Google Scholar]
  22. Neubauer, H., Molitor, M., Rahalison, L., Aleksic, S., Backes, H., Chanteau, S. & Meyer, H.(2000). A miniaturised semiautomated system for identification of Yersinia species within the genus Yersinia. Clin Lab 46, 561–567. [Google Scholar]
  23. Osterman, B. & Moriyón, I.(2006). International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Brucella. Minutes of the meeting, 17 September 2003, Pamplona, Spain. Int J Syst Evol Microbiol 56, 1173–1175.[CrossRef] [Google Scholar]
  24. Ratushna, V. G., Sturgill, D. M., Ramamoorthy, S., Reichow, S. A., He, Y., Lathigra, R., Sriranganathan, N., Halling, S. M., Boyle, S. M. & other authors(2006). Molecular targets for rapid identification of Brucella spp. BMC Microbiol 6, 13[CrossRef] [Google Scholar]
  25. Stolz, A., Busse, H.-J. & Kämpfer, P.(2007).Pseudomonas knackmussii sp. nov. Int J Syst Evol Microbiol 57, 572–576.[CrossRef] [Google Scholar]
  26. Tindall, B. J.(1990a). A comparative study of the lipid composition of Halobacterium saccharovorum from various sources. Syst Appl Microbiol 13, 128–130.[CrossRef] [Google Scholar]
  27. Tindall, B. J.(1990b). Lipid composition of Halobacterium lacusprofundi. FEMS Microbiol Lett 66, 199–202.[CrossRef] [Google Scholar]
  28. Verger, J.-M., Grimont, F., Grimont, P. A. D. & Grayon, M.(1985).Brucella, a monospecific genus as shown by deoxyribonucleic acid hybridization. Int J Syst Bacteriol 35, 292–295.[CrossRef] [Google Scholar]
  29. Vizcaíno, N., Caro-Hernandez, P., Cloeckaert, A. & Fernandez-Lago, L.(2004). DNA polymorphism in the omp25/omp31 family of Brucella spp.: identification of a 1.7-kb inversion in Brucella cetaceae and of a 15.1-kb genomic island, absent from Brucella ovis, related to the synthesis of smooth lipopolysaccharide. Microbes Infect 6, 821–834.[CrossRef] [Google Scholar]
  30. Whatmore, A. M., Shankster, S. J., Perrett, L. L., Murphy, T. J., Brew, S. D., Thirlwall, R. E., Cutler, S. J. & MacMillan, A. P.(2006). Identification and characterization of variable-number tandem-repeat markers for typing of Brucella spp. J Clin Microbiol 44, 1982–1993.[CrossRef] [Google Scholar]
  31. Whatmore, A. M., Perrett, L. L. & Macmillan, A. P.(2007). Characterisation of the genetic diversity of Brucella by multilocus sequencing. BMC Microbiol 7, 34[CrossRef] [Google Scholar]
  32. Ziemke, F., Höfle, M. G., Lalucat, J. & Rosselló-Mora, R.(1998). Reclassification of Shewanella putrefaciens Owen's genomic group II as Shewanella baltica sp. nov. Int J Syst Bacteriol 48, 179–186.[CrossRef] [Google Scholar]

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