Two Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains CCM 4915 T 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 4915 T 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 4915 T 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 Abbreviations: MLST, multilocus sequence typing; MLVA, multilocus VNTR (variable-number tandem-repeat) analysis; RTD, routine test dilution.The GenBank/EMBL/DDBJ accession numbers for the gene sequences omp22, omp25, omp25b, omp31 and omp31b of strain CCM 4915

Lactobacillus apis sp. nov., from the stomach of honeybees (Apis mellifera), having an in vitro inhibitory effect on the causative agents of American and European foulbrood At the present time, the bacterial genus Lactobacillus comprises more than 100 species and subspecies (Hammes & Hertel, 2009) and represents a large group of Grampositive bacteria within the phylum Firmicutes. Lactobacilli are well known as the main representatives of the lactic acid bacteria that occur in various carbohydrate-rich environments such as plant-derived materials, products of the dairy industry, the reproductive tract of women and the digestive tracts of mammals and insects (Hammes & Hertel, 2009;Forsgren et al., 2010). Lactobacilli are important members of healthy gastrointestinal tracts of animals, and some of them are used frequently as probiotics because of their beneficial influences on mammalian and human health (Zubillaga et al., 2001).It is supposed that lactobacilli, other lactic acid bacteria and bifidobacteria have a beneficial effect on honeybee health. For this reason, attempts to describe the occurrence of these bacteria in the digestive tract of these important pollinators have increased in recent years (Olofsson & Vásquez, 2008). Novel species of lactobacilli have been detected from the digestive tract of Apis mellifera and ApisThe GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, atpA, hsp60, pheS, rpoA and tuf gene sequences of strain R4B T are KF386017, JQ363701, JQ363676, JQ363685, JQ363694 and JQ363711, respectively. The accession number for the 16S rRNA gene sequence of strain R4C is KF386018.

Direct matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) bacterial cell or lysate analysis appears to meet all the criteria required for a rapid and reliable analytical microorganism identification and taxonomical classification tool. Few-minute analytical procedure providing information extending up to sub-species level underlines the potential of the MALDI-MS profiling in comparison with other methods employed in the field. However, the quality of MALDI-MS profiles and consequently the performance of the method are influenced by numerous factors, which involve particular steps of the sample preparation procedure. This review is aimed at advances in development and optimization of the MALDI-MS profiling methodology. Approaches improving the quality of the MALDI-MS profiles and universal feasibility of the method are discussed.

A systemic disease occurred in a wild population of the common vole Microtus arvalis in South Moravia (Czech Republic) during the years 1999-2003. Acute infections were characterized by edema of extremities, occasionally with colliquating abscesses, arthritis, lymphadenitis, perforations of the skin resulting from colliquated abscesses, orchitis, and peritoneal granulomas. From the clinical samples, small Gram-negative coccobacilli were isolated and identified as Ochrobactrum intermedium by API 20NE and colistin sensitivity profiles. However, subsequent rrs (16S rRNA) and recA (recombinase A) gene sequencing analysis of two isolates (CCM 4915=CAPM 6434; CCM 4916=CAPM 6435) identified them as Brucella sp. with sequence identities of 100% to other Brucella spp. Analysis of the omp2a/b genes confirmed the two isolates as Brucella. In AMOS polymerase chain reaction (PCR), a 2000-bp fragment was generated that was not seen in other brucellae. Experimental infection of outbred ICR mice with these isolates resulted in a mortality rate of 50%. Based on the results of the molecular investigations and the mortality observed in experimentally infected mice we conclude that the epizootic was caused by Brucella sp. and not by Ochrobactrum intermedium. The study demonstrates the limitations of commercial biochemical test systems in accurately differentiating among Ochrobactrum and Brucella.

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Two strains of Gram-negative bacteria isolated from soil by selective enrichment with nitroaromatics were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA gene sequence analysis, the two strains were found to belong to the genus Pseudomonas, within the Gammaproteobacteria. Strain 1B4T shared the highest sequence similarity with Pseudomonas koreensis DSM 16610T (99.5 %) and Pseudomonas jessenii CCM 4840T (99.3 %), and strain 2B2T with Pseudomonas asplenii DSM 17133T (98.9 %), Pseudomonas fuscovaginae DSM 7231T (98.9 %) and Pseudomonas putida DSM 291T (98.7 %). On the basis of phylogenetic analysis, DNA–DNA hybridization and phenotype, including chemotaxonomic characteristics, two novel species, Pseudomonas moraviensis sp. nov. with the type strain 1B4T (=CCM 7280T=DSM 16007T) and Pseudomonas vranovensis sp. nov. with the type strain 2B2T (=CCM 7279T=DSM 16006T), are proposed. The description of P. asplenii was emended on the basis of additional data obtained in this study.

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