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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

A Gram-negative, non-motile, non-spore-forming coccoid bacterium (strain BO1 T ) was isolated recently from a breast implant infection of a 71-year-old female patient with clinical signs of brucellosis. Affiliation of strain BO1T to the genus Brucella was confirmed by means of polyamine pattern, polar lipid profile, fatty acid profile, quinone system, DNA-DNA hybridization studies and by insertion sequence 711 (IS711)-specific PCR. Strain BO1 T harboured four to five copies of the Brucella-specific insertion element IS711, displaying a unique banding pattern, and exhibited a unique 16S rRNA gene sequence and also grouped separately in multilocus sequence typing analysis. Strain BO1 T reacted with Brucella M-monospecific antiserum. Incomplete lysis was detected with bacteriophages Tb (Tbilisi), F1 and F25. Biochemical profiling revealed a high degree of enzymic activity and metabolic capabilities. In multilocus VNTR (variable-number tandem-repeat) analysis, strain BO1 T showed a very distinctive profile and clustered with the other 'exotic' Brucella strains, including strains isolated from marine mammals, and Brucella microti, Brucella suis biovar 5 and Brucella neotomae. Comparative omp2a and omp2b gene sequence analysis revealed the most divergent omp2 sequences identified to date for a Brucella strain. The recA gene sequence of strain BO1 T differed in seven nucleotides from the Brucella recA consensus sequence. Using the Brucella species-specific multiplex PCR assay, strain BO1T displayed a unique banding pattern not observed in other Brucella species. From the phenotypic and molecular analysis it became evident that strain BO1 T was clearly different from all other Brucella species, and therefore represents a novel species within the genus Brucella. Because of its unexpected isolation, the name Brucella inopinata with the type strain BO1 T (5BCCN

The genus Yersinia has been used as a model system to study pathogen evolution. Using whole-genome sequencing of all Yersinia species, we delineate the gene complement of the whole genus and define patterns of virulence evolution. Multiple distinct ecological specializations appear to have split pathogenic strains from environmental, nonpathogenic lineages. This split demonstrates that contrary to hypotheses that all pathogenic Yersinia species share a recent common pathogenic ancestor, they have evolved independently but followed parallel evolutionary paths in acquiring the same virulence determinants as well as becoming progressively more limited metabolically. Shared virulence determinants are limited to the virulence plasmid pYV and the attachment invasion locus ail. These acquisitions, together with genomic variations in metabolic pathways, have resulted in the parallel emergence of related pathogens displaying an increasingly specialized lifestyle with a spectrum of virulence potential, an emerging theme in the evolution of other important human pathogens.genomics metabolic streamlining | pathoadaptation | Enterobacteriaceae B acterial species are defined on the basis of phenotypic characteristics, such as cellular morphology and biochemical characteristics, as well as DNA-DNA hybridization and 16S rRNA comparison. Using high-throughput whole-genome approaches we can now move beyond classic methods and develop population frameworks to reconstruct accurate inter-and intraspecies relationships and gain insights into the complex patterns of gene flux that define different taxonomic groups.Bacterial whole-genome sequencing has revealed enormous heterogeneity in gene content, even between members of the same species. From a bacterial perspective the acquisition of new genes provides the flexibility to adapt and exploit novel niches and opportunities. From a human perspective, integration of genes by bacteria has been directly linked to the emergence of new pathogenic clones, often from formerly harmless lineages (1, 2). In addition to gene gain, gene loss is also strongly associated with host restriction in acutely pathogenic bacterial species, such as Yersinia pestis and Salmonella enterica serovars, including Salmonella Typhi (3-5), where gene loss can remove functions unnecessary in the new niche (6). These specialist pathogens show a much higher frequency of functional gene loss than closely related host generalist pathogens, such as Yersinia pseudotuberculosis (7).Previous Yersinia genome studies (8, 9) have examined the evolution of pathogenicity by comparing strains from a selection of species or species subtypes within the genus, limiting our understanding of the evolutionary context of individual species. The majority of the Yersinia species are found in the environment and do not cause disease in mammals. Three species are known as human pathogens: the plague bacillus Y. pestis and the enteropathogens Yersinia enterocolitica and Y. pseudotuberculosis. SignificanceOur past understanding of pathogen evo...

Two Gram-negative, non-motile, non-spore-forming coccoid bacteria (strains F8/08-60 T and F8/ 08-61) isolated from clinical specimens obtained from baboons (Papio spp.) that had delivered stillborn offspring were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA gene sequence similarities, both strains, which possessed identical sequences, were assigned to the genus Brucella. This placement was confirmed by extended multilocus sequence analysis (MLSA), where both strains possessed identical sequences, and whole-genome sequencing of a representative isolate. All of the above analyses suggested that the two strains represent a novel lineage within the genus Brucella. The strains also possessed a unique profile when subjected to the phenotyping approach classically used to separate species of the genus Brucella, reacting Abbreviations: MLSA, multilocus sequence analysis; MLVA, multilocus variable number of tandem repeat analysis; RTD, routine test dilution.The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, omp2a and omp2b gene sequences of strains F8/08-60 T and F8/08-61 are HG932316 and HG932317 (16S rRNA gene), KJ493822 and KJ493823 (omp2a) and KJ510540 and KJ510541 (omp2b), respectively.A supplementary table and a supplementary figure are available with the online Supplementary Material.

Yersinia pestis, the etiologic agent of the disease plague, has been implicated in three historical pandemics. These include the third pandemic of the 19th and 20th centuries, during which plague was spread around the world, and the second pandemic of the 14th–17th centuries, which included the infamous epidemic known as the Black Death. Previous studies have confirmed that Y. pestis caused these two more recent pandemics. However, a highly spirited debate still continues as to whether Y. pestis caused the so-called Justinianic Plague of the 6th–8th centuries AD. By analyzing ancient DNA in two independent ancient DNA laboratories, we confirmed unambiguously the presence of Y. pestis DNA in human skeletal remains from an Early Medieval cemetery. In addition, we narrowed the phylogenetic position of the responsible strain down to major branch 0 on the Y. pestis phylogeny, specifically between nodes N03 and N05. Our findings confirm that Y. pestis was responsible for the Justinianic Plague, which should end the controversy regarding the etiology of this pandemic. The first genotype of a Y. pestis strain that caused the Late Antique plague provides important information about the history of the plague bacillus and suggests that the first pandemic also originated in Asia, similar to the other two plague pandemics.