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1 Bundeswehr Institute of Microbiology, Neuherbergstrasse 11, D-80937 Munich, Germany
2 Institute of Vertebrate Biology, Academy of Sciences, Kvetna 8, CZ-60365 Brno, Czech Republic
3 Czech Collection of Microorganisms, Faculty of Science, Masaryk University, Brno, Czech Republic
4 Centre d'Etudes du Bouchet, BP3, 91710 Vert le Petit, France
5 Université Paris-Sud, Institut de Génétique et Microbiologie, Orsay, F-91405, France; CNRS, Orsay F-91405, France
6 Friedrich Loeffler Institute, Institute of Bacterial Infections and Zoonoses, German Reference Center for Brucellosis, Jena, Germany
7 Institute for Applied Microbiology, Justus-Liebig-Universität Giessen, IFZ, Heinrich-Buff- Ring 26-32, D-35392 Giessen, Germany
8 INRA, UR1282, Infectiologie Animale et Santé Publique, IASP, Nouzilly, F-37380, France
9 Veterinary Laboratories Agency, Woodham Lane, Addlestone KT15 3NB, UK
10 Culture Collection, Department of Clinical Bacteriology, University of Göteborg, Guldhedsg. 10 S-41346 Göteborg, Sweden
11 Federal Institute for Risk Assessment, Diedersdorfer Weg 1, D-12277 Berlin, Germany
12 Institut für Bakteriologie, Mykologie und Hygiene, Veterinärmedizinische Universität, A-1210 Wien, Austria
Correspondence
Holger C. Scholz
Holger1Scholz{at}bundeswehr.org
Two Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains CCM 4915T and CCM 4916), isolated from clinical specimens of the common vole Microtus arvalis during an epizootic in the Czech Republic in 2001, were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA (rrs) and recA gene sequence similarities, both isolates were allocated to the genus Brucella. Affiliation to Brucella was confirmed by DNA–DNA hybridization studies. Both strains reacted equally with Brucella 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 Brucella species. The omp2a and omp2b genes of isolates CCM 4915T and CCM 4916 were indistinguishable. Whereas omp2a was identical to omp2a of brucellae from certain pinniped marine mammals, omp2b clustered with omp2b of terrestrial brucellae. Analysis of the bp26 gene downstream region identified strains CCM 4915T and CCM 4916 as Brucella of terrestrial origin. Both strains harboured five to six copies of the insertion element IS711, 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 Brucella. Multilocus sequence typing (MLST) analysis using nine different loci also placed the two isolates separately from other brucellae. In the IS711-based AMOS PCR, a 1900 bp fragment was generated with the Brucella ovis-specific primers, revealing that the insertion element had integrated between a putative membrane protein and cboL, encoding a methyltransferase, an integration site not observed in other brucellae. Isolates CCM 4915T and CCM 4916 could be clearly distinguished from all known Brucella species and their biovars by means of both their phenotypic and molecular properties, and therefore represent a novel species within the genus Brucella, for which the name Brucella microti sp. nov. with the type strain CCM 4915T (=BCCN 07-01T=CAPM 6434T) is proposed.
The GenBank/EMBL/DDBJ accession numbers for the gene sequences omp22, omp25, omp25b, omp31 and omp31b of strain CCM 4915T are AM712379, AM712381, AM712383, AM712385 and AM712387, respectively.
Present address: RWTH University of Aachen, Department of Internal Medicine III, Pauwelsstraße 30, D-52074 Aachen, Germany.
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