B. selleck screening library ceti and B. pinnipedialis showed significantly different carbohydrate utilization patterns. B. neotomae was the only species tested negative for d-Ala-pNA (DANA), Gly-pNA (GNA), Leu-pNA (LNA), Lys-pNA (KNA), Lys-βNA (K), and Gly-Gly-βNA (GG). Like B. neotomae the two yet unidentified strains isolated from foxes were negative for DANA and GNA. Despite of genetic consistency with the genus Brucella (data not shown) these two strains completely differed in their metabolic profile from the species described to date. The panel of 93 discriminating reactions was re-evaluated

for its usefulness in the identification of Brucella and the differentiation of its species and biovars using a broad spectrum of well characterized field strains. Both inter- and intra-assay variability

were ascertained to be negligible. Results of the cluster analysis of the 113 strains investigated regarding their ability to metabolize the 93 selected substances supported our findings in the smaller collection of Brucella reference strains (Figure 3). Based on the metabolic profiles determined by the Brucella specific 96-well Micronaut™ plate, B. melitensis and B. abortus isolates fell into two distinct groups (Figure 3). B. suis (except for biovar 5) could be found in another group but the biovars 1, and 3 and 4 gathered together with B. inopinata and B. canis isolates, respectively. B. suis bv 2 could be separated by its substrate assimilation pattern. B. suis bv 5 showed PRT062607 mw metabolic traits similar to B. ovis, B. neotomae and the marine mammal strains. Each Brucella strain investigated revealed an individual metabolic profile. Figure 3 Cluster analysis of Brucella field isolates based on biochemical reactions. Cluster analysis of 113 Brucella strains including the

reference strains and two isolates of a potentially new species that originated from Austrian foxes based on 93 biochemical PtdIns(3,4)P2 reactions tested with the newly developed Brucella specific Micronaut™ microtiter plate. Hierarchical cluster analysis was performed by the Ward’s linkage algorithm using the binary coded data based on the empirically set cut-off. Using the newly developed Brucella specific Micronaut™ biotyping assay, B. abortus bv 4, 5, and 7, B. suis bv 1-5, B. ovis, B. neotomae, B. pinnipedialis, B. ceti, B. microti, and B. inopinata could be discriminated within the genus with a specificity of 100% (Table 1). In contrast, members of the three B. melitensis biovars formed a homogenous group. Although the metabolic activity of B. melitensis strains did not correlate with the classical biotyping scheme, VE-821 datasheet subgroups within the species could still be defined (Figure 3). Gram-negative microorganisms other than brucellae e.g. Ochrobactrum intermedium, O. anthropi, Yersinia enterocolitica O:9, and Acinetobacter lwoffii showed differing oxidative metabolic profiles and could clearly be distinguished from Brucella spp.