Rapid identification of Brucella isolates to the species level by real time PCR based single nucleotide polymorphism (SNP) analysis

<p>Abstract</p> <p>Background</p> <p>Brucellosis, caused by members of the genus <it>Brucella</it>, remains one of the world's major zoonotic diseases. Six species have classically been recognised within the family <it>Brucella </it>largely based on a combination of classical microbiology and host specificity, although more recently additional isolations of novel <it>Brucella </it>have been reported from various marine mammals and voles. Classical identification to species level is based on a biotyping approach that is lengthy, requires extensive and hazardous culturing and can be difficult to interpret. Here we describe a simple and rapid approach to identification of <it>Brucella </it>isolates to the species level based on real-time PCR analysis of species-specific single nucleotide polymorphisms (SNPs) that were identified following a robust and extensive phylogenetic analysis of the genus.</p> <p>Results</p> <p>Seven pairs of short sequence Minor Groove Binding (MGB) probes were designed corresponding to SNPs shown to possess an allele specific for each of the six classical <it>Brucella </it>spp and the marine mammal <it>Brucella</it>. Assays were optimised to identical reaction parameters in order to give a multiple outcome assay that can differentiate all the classical species and <it>Brucella </it>isolated from marine mammals. The scope of the assay was confirmed by testing of over 300 isolates of <it>Brucella</it>, all of which typed as predicted when compared to other phenotypic and genotypic approaches. The assay is sensitive being capable of detecting and differentiating down to 15 genome equivalents. We further describe the design and testing of assays based on three additional SNPs located within the 16S rRNA gene that ensure positive discrimination of <it>Brucella </it>from close phylogenetic relatives on the same platform.</p> <p>Conclusion</p> <p>The multiple-outcome assay described represents a new tool for the rapid, simple and unambiguous characterisation of <it>Brucella </it>to the species level. Furthermore, being based on a robust phylogenetic framework, the assay provides a platform that can readily be extended in the future to incorporate newly identified <it>Brucella </it>groups, to further type at the subspecies level, or to include markers for additional useful characteristics.</p

Marine Mammal Brucella Genotype Associated with Zoonotic Infection

Marine Mammal Brucella Genotype Associated with Zoonotic Infectio

Multiplex Assay Based on Single-Nucleotide Polymorphisms for Rapid Identification of Brucella Isolates at the Species Level▿ †

The genus Brucella includes a number of species that are major animal pathogens worldwide and significant causes of zoonotic infections of humans. Traditional methods of identifying Brucella to the species level can be time-consuming, can be subjective, and can pose a hazard to laboratory personnel in the absence of suitable biocontainment facilities. Using a robust phylogenetic framework, a number of single-nucleotide polymorphisms (SNPs) that define particular species within the genus were identified. These SNPs were used to develop a multiplex SNP detection assay, based on primer extension technology, that can rapidly and unambiguously identify an isolate as a member of one of the six classical Brucella species or as a member of the recently identified marine mammal group

Оксана Козловская работает по специальности

Brucellosis is one of the major bacterial zoonoses worldwide. In the past decade, an increasing number of atypical Brucella strains and species have been described. Brucella microti in particular has attracted attention, because this species not only infects mammalian hosts but also persists in soil. An environmental reservoir may pose a new public health risk leading to the reemergence of brucellosis. In a polyphasic approach, comprising conventional microbiological techniques and extensive biochemical and molecular techniques, all currently available Brucella microti strains were characterized. While differing in their natural habitats and host preferences, B. microti isolates were found to possess identical 16S rRNA, recA, omp2a and omp2b gene sequences, and identical multilocus sequence analysis (MLSA) profiles at 21 different genomic loci. Only highly variable microsatellite markers of multiple-locus variable-number tandem repeat (VNTR) analysis comprising 16 loci (MLVA-16) showed intraspecies discriminatory power. In contrast, biotyping demonstrated striking differences within the genetically homologous species. The majority of the mammalian isolates agglutinated only with monospecific anti-M serum, whereas soil isolates agglutinated with anti-A, anti-M, and anti-R sera. Bacteria isolated from animal sources were lysed by phages F1, F25, Tb, BK2, Iz, and Wb whereas soil isolates usually were not. Rough strains of environmental origin were lysed only by R/C B. microti exhibited high metabolic activities similar to those closely related soil organisms such as Ochrobactrum spp. Each strain was tested with 93 different substrates and showed an individual metabolic profile. In summary, the adaptation of Brucella microti to a specific habitat or host seems to be matter of gene regulation rather than a matter of gene configuration

Brucella vulpis sp. nov., a novel Brucella species isolated from mandibular lymph nodes of red foxes (Vulpes vulpes) in Austria

International audienceTwo slow-growing, Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains F60T and F965), isolated in Austria from mandibular lymph nodes of two red foxes (Vulpes vulpes), were subjected to a polyphasic taxonomic analysis. In a recent study, both isolates were assigned to the genus Brucella but could not be attributed to any of the existing species. Hence, we have analysed both strains in further detail to determine their exact taxonomic position and genetic relatedness to other members of the genus Brucella. The genome sizes of F60T and F965 were 3 236 779 and 3 237 765 bp, respectively. Each genome consisted of two chromosomes, with a DNA G+C content of 57.2 %. A genome-to-genome distance of >80 %, an average nucleotide identity (ANI) of 97 % and an average amino acid identity (AAI) of 98 % compared with the type species Brucella melitensis confirmed affiliation to the genus. Remarkably, 5 % of the entire genetic information of both strains was of non-Brucella origin, including as-yet uncharacterized bacteriophages and insertion sequences as well as ABC transporters and other genes of metabolic function from various soil-living bacteria. Core-genome-based phylogenetic reconstructions placed the novel species well separated from all hitherto-described species of the genus Brucella, forming a long-branched sister clade to the classical species of Brucella. In summary, based on phenotypic and molecular data, we conclude that strains F60T and F965 are members of a novel species of the genus Brucella, for which the name Brucella vulpis sp. nov. is proposed, with the type strain F60T ( = BCCN 09-2T = DSM 101715T)

Novel Brucella Strain (BO1) Associated with a Prosthetic Breast Implant Infection▿

We report the microbiological, biochemical, and molecular characterization of an unusual Brucella strain (BO1) isolated from a breast implant wound in a 71-year-old woman with clinical symptoms consistent with brucellosis. Initial phenotypic analysis, including biochemical and antimicrobial susceptibility testing, cellular fatty acid analysis, and molecular analysis based on DNA-DNA reassociation and the presence of multiple copies of IS711 element suggested that the isolate was a Brucella-like organism, but species determination using microbiological algorithms was unsuccessful. Furthermore, molecular data based on 16S rRNA gene sequencing and multilocus sequence analysis demonstrated that BO1 was an unusual Brucella strain and not closely related to any currently described Brucella species. However, comparison with equivalent sequences in Ochrobactrum spp. confirms that the isolate is much more closely related to Brucella than to Ochrobactrum spp., and thus the isolate likely represents an atypical and novel strain within the genus Brucella

Brucella papii sp. nov. isolated from baboons (Papio spp.)

International audienceTwo 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 only with Brucella A monospecific antiserum, being sensitive to the dyes thionin and fuchsin, being lysed by bacteriophage Wb, Bk2 and Fi phage at routine test dilution (RTD) but only partially sensitive to bacteriophage Tb, and with no requirement for CO2 and no production of H2S but strong urease activity. Biochemical profiling revealed a pattern of enzyme activity and metabolic capabilities distinct from existing species of the genus Brucella. Molecular analysis of the omp2 locus genes showed that both strains had a novel combination of two highly similar omp2b gene copies. The two strains shared a unique fingerprint profile of the multiple-copy Brucella-specific element IS711. Like MLSA, a multilocus variable number of tandem repeat analysis (MLVA) showed that the isolates clustered together very closely, but represent a distinct group within the genus Brucella. Isolates F8/08-60(T) and F8/08-61 could be distinguished clearly from all known species of the genus Brucella and their biovars by both phenotypic and molecular properties. Therefore, by applying the species concept for the genus Brucella suggested by the ICSP Subcommittee on the Taxonomy of Brucella, they represent a novel species within the genus Brucella, for which the name Brucella papionis sp. nov. is proposed, with the type strain F8/08-60(T) ( = NCTC 13660(T) = CIRMBP 0958(T))