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Abstract Background A commercial biotyping system (Taxa Profile™, Merlin Diagnostika) testing the metabolization of various substrates by bacteria was used to determine if a set of phenotypic features will allow the identification of members of the genus Brucella and their differentiation into species and biovars. Results A total of 191 different amines, amides, amino acids, other organic acids and heterocyclic and aromatic substrates (Taxa Profile™ A), 191 different mono-, di-, tri- and polysaccharides and sugar derivates (Taxa Profile™ C) and 95 amino peptidase- and protease-reactions, 76 glycosidase-, phosphatase- and other esterase-reactions, and 17 classic reactions (Taxa Profile™ E) were tested with the 23 reference strains representing the currently known species and biovars of Brucella and a collection of 60 field isolates. Based on specific and stable reactions a 96-well "Brucella identification and typing" plate (Micronaut™) was designed and re-tested in 113 Brucella isolates and a couple of closely related bacteria. Brucella species and biovars revealed characteristic metabolic profiles and each strain showed an individual pattern. Due to their typical metabolic profiles a differentiation of Brucella isolates to the species level could be achieved. The separation of B. canis from B. suis bv 3, however, failed. At the biovar level, B. abortus bv 4, 5, 7 and B. suis bv 1-5 could be discriminated with a specificity of 100%. B. melitensis isolates clustered in a very homogenous group and could not be resolved according to their assigned biovars. Conclusions The comprehensive testing of metabolic activity allows cluster analysis within the genus Brucella. The biotyping system developed for the identification of Brucella and differentiation of its species and biovars may replace or at least complement time-consuming tube testing especially in case of atypical strains. An easy to handle identification software facilitates the applicability of the Micronaut™ system for microbiology laboratories.

Abstract Background The classification of Brucella into species and biovars relies on phenotypic characteristics and sometimes raises difficulties in the interpretation of the results due to an absence of standardization of the typing reagents. In addition, the resolution of this biotyping is moderate and requires the manipulation of the living agent. More efficient DNA-based methods are needed, and this work explores the suitability of multiple locus variable number tandem repeats analysis (MLVA) for both typing and species identification. Results Eighty tandem repeat loci predicted to be polymorphic by genome sequence analysis of three available Brucella genome sequences were tested for polymorphism by genotyping 21 Brucella strains (18 reference strains representing the six 'classical' species and all biovars as well as 3 marine mammal strains currently recognized as members of two new species). The MLVA data efficiently cluster the strains as expected according to their species and biovar. For practical use, a subset of 15 loci preserving this clustering was selected and applied to the typing of 236 isolates. Using this MLVA-15 assay, the clusters generated correspond to the classical biotyping scheme of Brucella spp. The 15 markers have been divided into two groups, one comprising 8 user-friendly minisatellite markers with a good species identification capability (panel 1) and another complementary group of 7 microsatellite markers with higher discriminatory power (panel 2). Conclusion The MLVA-15 assay can be applied to large collections of Brucella strains with automated or manual procedures, and can be proposed as a complement, or even a substitute, of classical biotyping methods. This is facilitated by the fact that MLVA is based on non-infectious material (DNA) whereas the biotyping procedure itself requires the manipulation of the living agent. The data produced can be queried on a dedicated MLVA web service site.

Background: A commercial biotyping system (Taxa Profile™, Merlin Diagnostika) testing the metabolization of various substrates by bacteria was used to determine if a set of phenotypic features will allow the identification of members of the genus Brucella and their differentiation into species and biovars., Results: A total of 191 different amines, amides, amino acids, other organic acids and heterocyclic and aromatic substrates (Taxa Profile™ A), 191 different mono-, di-, tri- and polysaccharides and sugar derivates (Taxa Profile™ C) and 95 amino peptidase- and protease-reactions, 76 glycosidase-, phosphatase- and other esterase-reactions, and 17 classic reactions (Taxa Profile™ E) were tested with the 23 reference strains representing the currently known species and biovars of Brucella and a collection of 60 field isolates. Based on specific and stable reactions a 96-well "Brucella identification and typing" plate (Micronaut™) was designed and re-tested in 113 Brucella isolates and a couple of closely related bacteria.Brucella species and biovars revealed characteristic metabolic profiles and each strain showed an individual pattern. Due to their typical metabolic profiles a differentiation of Brucella isolates to the species level could be achieved. The separation of B. canis from B. suis bv 3, however, failed. At the biovar level, B. abortus bv 4, 5, 7 and B. suis bv 1-5 could be discriminated with a specificity of 100%. B. melitensis isolates clustered in a very homogenous group and could not be resolved according to their assigned biovars., Conclusions: The comprehensive testing of metabolic activity allows cluster analysis within the genus Brucella. The biotyping system developed for the identification of Brucella and differentiation of its species and biovars may replace or at least complement time-consuming tube testing especially in case of atypical strains. An easy to handle identification software facilitates the applicability of the Micronaut™ system for microbiology laboratories.

Background: The classification of Brucella into species and biovars relies on phenotypic characteristics and sometimes raises difficulties in the interpretation of the results due to an absence of standardization of the typing reagents. In addition, the resolution of this biotyping is moderate and requires the manipulation of the living agent. More efficient DNA-based methods are needed, and this work explores the suitability of multiple locus variable number tandem repeats analysis (MLVA) for both typing and species identification., Results: Eighty tandem repeat loci predicted to be polymorphic by genome sequence analysis of three available Brucella genome sequences were tested for polymorphism by genotyping 21 Brucella strains (18 reference strains representing the six 'classical' species and all biovars as well as 3 marine mammal strains currently recognized as members of two new species). The MLVA data efficiently cluster the strains as expected according to their species and biovar. For practical use, a subset of 15 loci preserving this clustering was selected and applied to the typing of 236 isolates. Using this MLVA-15 assay, the clusters generated correspond to the classical biotyping scheme of Brucella spp. The 15 markers have been divided into two groups, one comprising 8 user-friendly minisatellite markers with a good species identification capability (panel 1) and another complementary group of 7 microsatellite markers with higher discriminatory power (panel 2)., Conclusion: The MLVA-15 assay can be applied to large collections of Brucella strains with automated or manual procedures, and can be proposed as a complement, or even a substitute, of classical biotyping methods. This is facilitated by the fact that MLVA is based on non-infectious material (DNA) whereas the biotyping procedure itself requires the manipulation of the living agent. The data produced can be queried on a dedicated MLVA web service site.