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The genus Brucella comprises a monophyletic group including 6 classical species showing clonal evolution: B. abortus, B. suis, B. melitensis, B. canis, B. ovis, and B. neotomae (1,2). The zoonotic [...]

Brucella abortus is the main causative agent of brucellosis in cattle, leading to severe economic consequences in agriculture and affecting public health. The zoonotic nature of the infection increases the need to control the spread and dynamics of outbreaks in animals with the incorporation of high resolution genotyping techniques. Based on such methods, B. abortus is currently divided into three clades, A, B, and C. The latter includes subclades C1 and C2. This study presents the results of whole-genome sequencing of 49 B. abortus strains isolated in Kazakhstan between 1947 and 2015 and of 36 B. abortus strains of various geographic origins isolated from 1940 to 2004. In silico Multiple Locus Sequence Typing (MLST) allowed to assign strains from Kazakhstan to subclades C1 and to a much lower extend C2. Whole-genome Single-Nucleotide Polymorphism (wgSNP) analysis of the 46 strains of subclade C1 with strains of worldwide origins showed clustering with strains from neighboring countries, mostly North Caucasia, Western Russia, but also Siberia, China, and Mongolia. One of the three Kazakhstan strains assigned to subclade C2 matched the B. abortus S19 vaccine strain used in cattle, the other two were genetically close to the 104 M vaccine strain. Bayesian phylodynamic analysis dated the introduction of B. abortus subclade C1 into Kazakhstan to the 19th and early 20th centuries. We discuss this observation in view of the history of population migrations from Russia to the Kazakhstan steppes. [ABSTRACT FROM AUTHOR]

Abstract Background Since the discovery of the Malta fever agent, Brucella melitensis, in the 19th century, six terrestrial mammal-associated Brucella species were recognized over the next century. More recently the number of novel Brucella species has increased and among them, isolation of species B. pinnipedialis and B. ceti from marine mammals raised many questions about their origin as well as on the evolutionary history of the whole genus. Results We report here on the first complete genome sequence of a Brucella strain isolated from marine mammals, Brucella pinnipedialis strain B2/94. A whole gene-based phylogenetic analysis shows that five main groups of host-associated Brucella species rapidly diverged from a likely free-living ancestor close to the recently isolated B. microti. However, this tree lacks the resolution required to resolve the order of divergence of those groups. Comparative analyses focusing on a) genome segments unshared between B. microti and B. pinnipedialis, b) gene deletion/fusion events and c) positions and numbers of Brucella specific IS711 elements in the available Brucella genomes provided enough information to propose a branching order for those five groups. Conclusions In this study, it appears that the closest relatives of marine mammal Brucella sp. are B. ovis and Brucella sp. NVSL 07-0026 isolated from a baboon, followed by B. melitensis and B. abortus strains, and finally the group consisting of B. suis strains, including B. canis and the group consisting of the single B. neotomae species. We were not able, however, to resolve the order of divergence of the two latter groups.

Abstract Background The lipopolysaccharide is a major antigen and virulence factor of Brucella, an important bacterial pathogen. In smooth brucellae, lipopolysaccharide is made of lipid A-core oligosaccharide and N-formylperosamine O-polysaccharide. B. ovis and B. canis (rough species) lack the O-polysaccharide. Results The polymorphism of O-polysaccharide genes wbkE, manAO-Ag, manBO-Ag, manCO-Ag, wbkF and wbkD) and wbo (wboA and wboB), and core genes manBcore and wa** was analyzed. Although most genes were highly conserved, species- and biovar-specific restriction patterns were found. There were no significant differences in putative N-formylperosamyl transferase genes, suggesting that Brucella A and M serotypes are not related to specific genes. In B. pinnipedialis and B. ceti (both smooth), manBO-Ag carried an IS711, confirming its dispensability for perosamine synthesis. Significant differences between smooth and rough species were found in wbkF and wbkD, two adjacent genes putatively related to bactoprenol priming for O-polysaccharide polymerization. B. ovis wbkF carried a frame-shift and B. canis had a long deletion partially encompassing both genes. In smooth brucellae, this region contains two direct repeats suggesting the deletion mechanism. Conclusion The results define species and biovar markers, confirm the dispensability of manBO-Ag for O-polysaccharide synthesis and contribute to explain the lipopolysaccharide structure of rough and smooth Brucella species.

The genus Brucella comprises major pathogenic species causing disease in livestock and humans, e.g. B. melitensis. In the past few years, the genus has been significantly expanded by the discovery of phylogenetically more distant lineages comprising strains from diverse wildlife animal species, including amphibians and fish. The strains represent several potential new species, with B. inopinata as solely named representative. Being genetically more distant between each other, relative to the "classical" Brucella species, they present distinct atypical phenotypes and surface antigens. Among surface protein antigens, the Omp2a and Omp2b porins display the highest diversity in the classical Brucella species. The genes coding for these proteins are closely linked in the Brucella genome and oriented in opposite directions. They share between 85 and 100% sequence identity depending on the Brucella species, biovar, or genotype. Only the omp2b gene copy has been shown to be expressed and genetic variation is extensively generated by gene conversion between the two copies. In this study, we analyzed the omp2 loci of the non-classical Brucella spp. Starting from two distinct ancestral genes, represented by Australian rodent strains and B. inopinata , a stepwise nucleotide reduction was observed in the omp2b gene copy. It consisted of a first reduction affecting the region encoding the surface L5 loop of the porin, previously shown to be critical in sugar permeability, followed by a nucleotide reduction in the surface L8 loop-encoding region. It resulted in a final omp2b gene size shared between two distinct clades of non-classical Brucella spp. (African bullfrog isolates) and the group of classical Brucella species. Further evolution led to complete homogenization of both omp2 gene copies in some Brucella species such as B. vulpis or B. papionis. The stepwise omp2b deletions seemed to be generated through recombination with the respective omp2a gene copy, presenting a conserved size among Brucella spp., and may involve short direct DNA repeats. Successive Omp2b porin alteration correlated with increasing porin permeability in the course of evolution of Brucella spp. They possibly have adapted their porin to survive environmental conditions encountered and to reach their final status as intracellular pathogen. [ABSTRACT FROM AUTHOR]

Deciphering the evolutionary history of pathogenic bacteria and their near neighbors may help to understand the genetic or ecological bases which led to their pathogenic behavior. The Brucellaceae family comprises zoonotic pathogenic species belonging to the genus Brucella as well as the environmental genus Ochrobactrum for which some species are considered as opportunistic pathogens. Here, we used a phylogenomic approach including a set of 145 Brucellaceae genomes representative of the family diversity and more than 40 genomes of the order Rhizobiales to infer the taxonomic relationships between the family's species. Our results clarified some unresolved phylogenetic ambiguities, conducting to the exclusion of Mycoplana spp. out of the family Brucellaceae and the positioning of all Brucella spp. as a single genomic species within the current Ochrobactrum species diversity. Additional analyses also revealed that Ochrobactrum spp. separate into two clades, one comprising mostly environmental species while the other one includes the species considered as pathogens (Brucella spp.) or opportunistic pathogens (mainly O. anthropi , O. intermedium , and O. pseudintermedium). Finally, we show that O. intermedium is undergoing a beginning of genome reduction suggestive of an ongoing ecological niche specialization, and that some lineages of O. intermedium and O. anthropi may shift toward an adaption to the human host. [ABSTRACT FROM AUTHOR]

Brucellosis, an infectious disease caused by Brucella, is one of the most extended bacterial zoonosis in the world and an important cause of economic losses and human suffering. The lipopolysaccharide (LPS) of Brucella plays a major role in virulence as it impairs normal recognition by the innate immune system and delays the immune response. The LPS core is a branched structure involved in resistance to complement and polycationic peptides, and mutants in glycosyltransferases required for the synthesis of the lateral branch not linked to the O-polysaccharide (O-PS) are attenuated and have been proposed as vaccine candidates. For this reason, the complete understanding of the genes involved in the synthesis of this LPS section is of particular interest. The chemical structure of the Brucella LPS core suggests that, in addition to the already identified WadB and WadC glycosyltransferases, others could be implicated in the synthesis of this lateral branch. To clarify this point, we identified and constructed mutants in 11 ORFs encoding putative glycosyltransferases in B. abortus. Four of these ORFs, regulated by the virulence regulator MucR (involved in LPS synthesis) or the BvrR/BvrS system (implicated in the synthesis of surface components), were not required for the synthesis of a complete LPS neither for virulence or interaction with polycationic peptides and/or complement. Among the other seven ORFs, six seemed not to be required for the synthesis of the core LPS since the corresponding mutants kept the O-PS and reacted as the wild type with polyclonal sera. Interestingly, mutant in ORF BAB1_0953 (renamed wadD) lost reactivity against antibodies that recognize the core section while kept the O-PS. This suggests that WadD is a new glycosyltransferase adding one or more sugars to the core lateral branch. WadD mutants were more sensitive than the parental strain to components of the innate immune system and played a role in chronic stages of infection. These results corroborate and extend previous work indicating that the Brucella LPS core is a branched structure that constitutes a steric impairment preventing the elements of the innate immune system to fight against Brucella. [ABSTRACT FROM AUTHOR]

Previous studies have shown the usefulness of MLVA16 as a rapid molecular identification and classification method for Brucella species and biovars including recently described novel Brucella species from wildlife. Most studies were conducted on a limited number of strains from limited geographic/host origins. The objective of this study was to assess genetic diversity of Brucella spp. by MLVA16 on a larger scale. Thus, 1404 animal or human isolates collected from all parts of the world over a period of 32 years (1974-2006) were investigated. Selection of the 1404 strains was done among the approximately 4000 strains collection of the BCCN (Brucella Culture Collection Nouzilly), based on classical biotyping and on the animal/human/geographic origin over the time period considered. MLVA16 was performed on extracted DNAs using high throughput capillary electrophoresis. The 16 loci were amplified in four multiplex PCR reactions. This large scale study firstly confirmed the accuracy of MLVA16 typing for Brucella species and biovar identification and its congruence with the recently described Extended Multilocus Sequence Analysis. In addition, it allowed identifying novel MLVA11 (based upon 11 slowly evolving VNTRs) genotypes representing an increase of 15% relative to the previously known Brucella MLVA11 genotypes. Cluster analysis showed that among the MLVA16 genotypes some were genetically more distant from the major classical clades. For example new major clusters of B. abortus biovar 3 isolated from cattle in Sub-Saharan Africa were identified. For other classical species and biovars this study indicated also genotypic expansion within the population structure of classical Brucella species. MLVA proves to be a powerful tool to rapidly assess genetic diversity of bacterial populations on a large scale, as here on a large collection of strains of the genomically homogeneous genus Brucella. The highly discriminatory power of MLVA appears of particular interest as a first step for selection of Brucella strains for whole-genome sequencing. The MLVA data of this study were added to the public Brucella MLVA database at http://microbesgenotyping.i2bc.paris-saclay.fr. Current version Brucella_4_3 comprises typing data from more than 5000 strains including in silico data analysis of public whole genome sequence datasets. [ABSTRACT FROM AUTHOR]

BACKGROUND: Recently, novel atypical Brucella strains isolated from humans and wild rodents have been reported. They are phenotypically close to Ochrobactrum species but belong to the genus Brucella, based on genetic relatedness, although genetic diversity is higher among the atypical Brucella strains than between the classic species. They were classified within or close to the novel species Brucella inopinata. However, with the exception of Brucella microti, the virulence of these novel strains has not been investigated in experimental models of infection. METHODS: The type species B. inopinata strain BO1 (isolated from a human) and Brucella species strain 83-210 (isolated from a wild Australian rodent) were investigated. A classic infectious Brucella reference strain, B. suis 1330, was also used. BALB/c, C57BL/6, and CD1 mice models and C57BL/6 mouse bone-marrow-derived macrophages (BMDMs) were used as infection models. RESULTS: Strains BO1 and 83-210 behaved similarly to reference strain 1330 in all mouse infection models: there were similar growth curves in spleens and livers of mice and similar intracellular replication rates in BMDMs. However, unlike strain 1330, strains BO1 and 83-210 showed lethality in the 3 mouse models. CONCLUSIONS: The novel atypical Brucella strains of this study behave like classic intracellular Brucella pathogens. In addition, they cause death in murine models of infection, as previously published for B. microti, another recently described environmental and wildlife species. [ABSTRACT FROM AUTHOR]

The brucellae are Gram-negative bacteria that cause an important zoonosis. Studies with the main Brucella species have shown that the O-antigens of the Brucella smooth lipopolysaccharide are α-(1→R2) and α-(1→3)-linked N-formyl-perosamine polysaccharides that carry M, A and C (A = M, A.M and A,M) epitopes relevant in serodiagnosis and typing. We report that, in contrast to the B. suis biovar 1 O-antigen used as a reference or to all described Brucella O-antigens, B. suis biovar 2 O-antigen failed to bind monoclonal antibodies of C (A =M), C (M.A) and M specificities. However, the biovar 2 O-antigen bound monoclonal antibodies to the Brucella A epitope, and to the C/Y epitope shared by brucellae and Yersinia enterocolitica O:9, a bacterium that carries an N-formyl-perosamine O-antigen in exclusively α-(1→2)-linkages. By 13C NMR spectroscopy, B. suis biovar 1 but not B. suis biovar 2 or Y. enterocolitica O:9 polysaccharide showed the signal characteristic of α-(1→3)-linked N-formyl-perosamine, indicating that biovar 2 may altogether lack this linkage. Taken together, the NMR spectroscopy and monoclonal antibody analyses strongly suggest a role for α-(1→3)-linked N-formyl-perosamine in the C (A =M) and C (M.A) epitopes. Moreover, they indicate that B. suis biovar 2 O-antigen lacks some lipopolysaccharide epitopes previously thought to be present in all smooth brucellae, thus representing a new brucella serovar that is M-negative, C-negative. Serologically and structurally this new serovar is more similar to Y. enterocolitica O:9 than to other brucellae. [ABSTRACT FROM AUTHOR]

Background: Since the discovery of the Malta fever agent, Brucella melitensis, in the 19th century, six terrestrial mammal-associated Brucella species were recognized over the next century. More recently the number of novel Brucella species has increased and among them, isolation of species B. pinnipedialis and B. ceti from marine mammals raised many questions about their origin as well as on the evolutionary history of the whole genus. Results: We report here on the first complete genome sequence of a Brucella strain isolated from marine mammals, Brucella pinnipedialis strain B2/94. A whole gene-based phylogenetic analysis shows that five main groups of host-associated Brucella species rapidly diverged from a likely free-living ancestor close to the recently isolated B. microti. However, this tree lacks the resolution required to resolve the order of divergence of those groups. Comparative analyses focusing on a) genome segments unshared between B. microti and B. pinnipedialis, b) gene deletion/fusion events and c) positions and numbers of Brucella specific IS711 elements in the available Brucella genomes provided enough information to propose a branching order for those five groups. Conclusions: In this study, it appears that the closest relatives of marine mammal Brucella sp. are B. ovis and Brucella sp. NVSL 07-0026 isolated from a baboon, followed by B. melitensis and B. abortus strains, and finally the group consisting of B. suis strains, including B. canis and the group consisting of the single B. neotomae species. We were not able, however, to resolve the order of divergence of the two latter groups. [ABSTRACT FROM AUTHOR]

Abstract: Marine mammal Brucella strains with different genomic characteristics according to distribution of IS711 elements in their genomes were analysed for their intracellular behaviour in human THP-1 macrophage-like cells. Seven different groups of marine mammal strains were identified including a human isolate from New Zealand presumably from marine origin. Entry and intracellular survival of strains representative of these groups in THP-1 human macrophage-like cells were analysed at several times of infection. Three patterns of infection were identified. The Brucella strain isolated from the human case from New Zealand, and two other groups of strains belonging to B. ceti or B. pinnipedialis were able to infect THP-1 macrophage cells to the same extent as the virulent strains B. suis 1330 or B. melitensis 16M. Three other groups of strains belonging to B. ceti or B. pinnipedialis were able to enter the cells as classical virulent strains but were eliminated after 48h. The last group was composed only of strains isolated from hooded seals (Cystophora cristata) and was even unable to enter and infect THP-1 macrophage cells. Thus, several groups of marine mammal Brucella strains appear to be non-infectious for human macrophages. [Copyright &y& Elsevier]

Background: The lipopolysaccharide is a major antigen and virulence factor of Brucella, an important bacterial pathogen. In smooth brucellae, lipopolysaccharide is made of lipid A-core oligosaccharide and N-formylperosamine O-polysaccharide. B. ovis and B. canis (rough species) lack the O-polysaccharide. Results: The polymorphism of O-polysaccharide genes wbkE, manAO-Ag, manBO-Ag, manCO-Ag, wbkF and wbkD) and wbo (wboA and wboB), and core genes manBcore and wa** was analyzed. Although most genes were highly conserved, species- and biovar-specific restriction patterns were found. There were no significant differences in putative N-formylperosamyl transferase genes, suggesting that Brucella A and M serotypes are not related to specific genes. In B. pinnipedialis and B. ceti (both smooth), manBO-Ag carried an IS711, confirming its dispensability for perosamine synthesis. Significant differences between smooth and rough species were found in wbkF and wbkD, two adjacent genes putatively related to bactoprenol priming for O-polysaccharide polymerization. B. ovis wbkF carried a frame-shift and B. canis had a long deletion partially encompassing both genes. In smooth brucellae, this region contains two direct repeats suggesting the deletion mechanism. Conclusion: The results define species and biovar markers, confirm the dispensability of manBOAg for O-polysaccharide synthesis and contribute to explain the lipopolysaccharide structure of rough and smooth Brucella species. [ABSTRACT FROM AUTHOR]

Abstract: Brucella species are Gram-negative bacteria which belong to α-Proteobacteria family. These organisms are zoonotic pathogens that induce abortion and sterility in domestic mammals and chronic infections in humans known as Malta fever. The virulence of Brucella is dependent upon its ability to enter and colonize the cells in which it multiplies. The genetic basis of this aspect is poorly understood. Signature-tagged mutagenesis (STM) was used to identify potential Brucella virulence factors. PCR amplification has been used in place of DNA hybridization to identify the STM-generated attenuated mutants. A library of 288 Brucella melitensis 16M tagged mini-Tn5 Km2 mutants, in 24 pools, was screened for its ability to colonize spleen, lymph nodes and liver of goats at three weeks post-i.v. infection. This comparative screening identified 7 mutants (approximately 5%) which were not recovered from the output pool in goats. Some genes were known virulence genes involved in biosynthesis of LPS (lpsA gene) or in intracellular survival (the virB operon). Other mutants included ones which had a disrupted gene homologous to flgF, a gene coding for the basal-body rod of the flagellar apparatus, and another with a disruption in a gene homologous to ppk which is involved in the biosynthesis of inorganic polyphosphate (PolyP) from ATP. Other genes identified encoded factors involved in DNA metabolism and oxidoreduction metabolism. Using STM and the caprine host for screening, potential virulence determinants in B. melitensis have been identified. [Copyright &y& Elsevier]

Twenty-one small Gram-negative motile coccobacilli were isolated from 15 systemically diseased African bullfrogs (Pyxicephalus edulis), and were initially identified as Ochrobactrum anthropi by standard microbiological identification systems. Phylogenetic reconstructions using combined molecular analyses and comparative whole genome analysis of the most diverse of the bullfrog strains verified affiliation with the genus Brucella and placed the isolates in a cluster containing B. inopinata and the other non-classical Brucella species but also revealed significant genetic differences within the group. Four representative but molecularly and phenotypically diverse strains were used for in vitro and in vivo infection experiments. All readily multiplied in macrophage-like murine J774-cells, and their overall intramacrophagic growth rate was comparable to that of B. inopinata BO1 and slightly higher than that of B. microti CCM 4915. In the BALB/c murine model of infection these strains replicated in both spleen and liver, but were less efficient than B. suis 1330. Some strains survived in the mammalian host for up to 12 weeks. The heterogeneity of these novel strains hampers a single species description but their phenotypic and genetic features suggest that they represent an evolutionary link between a soil-associated ancestor and the mammalian host-adapted pathogenic Brucella species. [ABSTRACT FROM AUTHOR]

Salmonella frequently causes human foodborne infections. Contaminated products from poultry infected with Salmonella enteritidis are mainly involved. This serovar is able to colonize the gastrointestinal tract and generally produces a chronic asymptomatic carrier state in poultry, except in very young birds. We have developed a model of S. enteritidis carriage in chicks and found that four chicken lines, B13, L2, PA12 and Y11 differ in their cecal colonization by S. enteritidis, whereas their systemic organs are similarly infected. We have monitored the serum and gut antibody responses of these four lines to S. enteritidis for 9 weeks post inoculation (pi). We confirm that S. enteritidis infected the spleens of the four chicken lines similarly, and that it often colonized the ceca at levels significantly higher in B13 and L2 chicks than those of the PA12 and Y11 chicks. The serum IgM and IgG antibody responses were high and the serum IgA antibody responses low. In contrast, the intestinal secretions contained mostly IgA antibodies. The serum IgM antibody values of the four chicken lines were similar. However, the B13 and L2 chicks often had significantly higher serum IgG and IgA antibody responses than PA12 and Y11 chicks. Only the B13 and L2 chicks showed high, persistent levels of IgA antibody in intestinal secretions. These results suggest that most antibody responses are related to cecal colonization by S. enteritidis. They also indicate that factors other than the antibody levels are involved in the control of this colonization. [Copyright &y& Elsevier]

Immunogenicity and protective activity against Brucella ovis of detergent-extracted recombinant Omp31 (rOmp31 extract) from Brucella melitensis produced in Escherichia coli, purified rough lipopolysaccharide from B. ovis (R-LPS) and a mixture of rOmp31 extract and R-LPS (rOmp31 extract + R-LPS) were assessed in BALB/c mice. The experimental vaccines were compared with a hot saline extract (HS extract) from B. ovis mainly composed of outer membrane proteins (OMPs) and R-LPS, and known to be protective in mice against a B. ovis infection. Serum antibodies to Omp31 and R-LPS were detected in the corresponding mice using Western blotting with B. ovis whole-cell lysates and ELISA with purified antigens. Protection was evaluated by comparing the levels of infection in the spleens of vaccinated mice challenged with B. ovis. A significantly lower number of B. ovis colony-forming units in spleens relative to unimmunized (saline injected) controls were considered as protection. Mice immunized with rOmp31 extract or rOmp31 extract mixed with R-LPS developed antibodies that bound to the B. ovis surface with similar titers. Vaccination with rOmp31 extract plus R-LPS provided the best protection level, which was comparable with that given by HS extract. Similar protection was also obtained with rOmp31 extract alone and, to a lesser degree, with R-LPS. Comparisons between groups showed that an extract from E. coli-pUC19 (devoid of Omp31) provided no protection relative to either HS extract, rOmp31 extract or rOmp31 extract mixed with R-LPS. In conclusion, the recombinant Omp31 associated or not with B. ovis R-LPS, could be an interesting candidate for a subcellular vaccine against B. ovis infection. [Copyright &y& Elsevier]

Three Gram-negative, rod-shaped, oxidase-positive, non-spore-forming, non-motile strains (C130915_07 T , C150915_16 and C150915_17) were isolated from lymph nodes of Algerian cows. On the basis of 16S rRNA gene and whole genome similarities, the isolates were almost identical and clearly grouped in the genus Pseudochrobactrum . This allocation was confirmed by the analysis of fatty acids (C 19:cyclo , C 18 : 1 , C 18 : 0 , C 16 : 1 and C 16 : 0 ) and of polar lipids (major components: phosphatidylethanolamine, ornithine-lipids, phosphatidylglycerol, cardiolipin and phosphatidylcholine, plus moderate amounts of phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine and other aminolipids). Genomic, physiological and biochemical data differentiated these isolates from previously described Pseudochrobactrum species in DNA relatedness, carbon assimilation pattern and growth temperature range. Thus, these organisms represent a novel species of the genus Pseudochrobactrum , for which the name Pseudochrobactrum algeriensis sp. nov. is proposed (type strain C130915_07 T =CECT30232 T =LMG 32378 T ).

Since the 1990s, Brucella strains have been isolated from a wide variety of marine mammal species. We report the first complete genome sequence of a Brucella strain isolated from a hooded seal ( Cystophora cristata ), Brucella pinnipedialis strain 23a-1 of sequence type 54, found in the North Atlantic Ocean surrounding Norway., (Copyright © 2021 Zygmunt et al.)

Members of the genus Brucella cluster in two phylogenetic groups: classical and non-classical species. The former group is composed of Brucella species that cause disease in mammals, including humans. A Brucella species, labeled as Brucella sp. BCCN84.3, was isolated from the testes of a Saint Bernard dog suffering orchiepididymitis, in Costa Rica. Following standard microbiological methods, the bacterium was first defined as " Brucella melitensis biovar 2." Further molecular typing, identified the strain as an atypical " Brucella suis ." Distinctive Brucella sp. BCCN84.3 markers, absent in other Brucella species and strains, were revealed by fatty acid methyl ester analysis, high resolution melting PCR and omp25 and omp2a/omp2b gene diversity. Analysis of multiple loci variable number of tandem repeats and whole genome sequencing demonstrated that this isolate was different from the currently described Brucella species. The smooth Brucella sp. BCCN84.3 clusters together with the classical Brucella clade and displays all the genes required for virulence. Brucella sp. BCCN84.3 is a species nova taxonomical entity displaying pathogenicity; therefore, relevant for differential diagnoses in the context of brucellosis. Considering the debate on the Brucella species concept, there is a need to describe the extant taxonomical entities of these pathogens in order to understand the dispersion and evolution.

Two 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).

The C epitope of Brucella O-polysaccharide (O-PS) has so far lacked definitive structural identity. Revised structures for this antigen revealed a unique capping perosamine tetrasaccharide consisting of a sequence of 1,2:1,3:1,2 interresidue linkages. Here, using synthetic oligosaccharide glycoconjugates, the α-1,3 linkage of the O-PS is shown to be an integral structural requirement of this epitope. Although A-dominant strains possess only one or two copies of the capping tetrasaccharide, this creates a unique pentasaccharide antigenic determinant with the linkage sequence 1,2:1,3:1,2:1,2 that is always present in major pathogenic Brucella species., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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 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))., (Crown Copyright 2014. Reproduced with the permission of the Controller of Her Majesty's Stationery Office/Queen's Printer for Scotland and AHVLA.)

Brucella spp. are Gram-negative bacteria that behave as facultative intracellular parasites of a variety of mammals. This genus includes smooth (S) and rough (R) species that carry S and R lipopolysaccharides (LPS), respectively. S-LPS is a virulence factor, and mutants affected in the S-LPS O-polysaccharide (R mutants), core oligosaccharide or both show attenuation. However, B. ovis is naturally R and is virulent in sheep. We studied the role of B. ovis LPS in virulence by mutating the orthologues of wadA, wadB and wadC, three genes known to encode LPS core glycosyltransferases in S brucellae. When mapped with antibodies to outer membrane proteins (Omps) and R-LPS, wadB and wadC mutants displayed defects in LPS structure and outer membrane topology but inactivation of wadA had little or no effect. Consistent with these observations, the wadB and wadC but not the wadA mutants were attenuated in mice. When tested as vaccines, the wadB and wadC mutants protected mice against B. ovis challenge. The results demonstrate that the LPS core is a structure essential for survival in vivo not only of S brucellae but also of a naturally R Brucella pathogenic species, and they confirm our previous hypothesis that the Brucella LPS core is a target for vaccine development. Since vaccine B. melitensis Rev 1 is S and thus interferes in serological testing for S brucellae, wadB mutant represents a candidate vaccine to be evaluated against B. ovis infection of sheep suitable for areas free of B. melitensis.

Bacterial adaptation to environmental conditions is essential to ensure maximal fitness in the face of several stresses. In this context, two-component systems (TCSs) represent a predominant signal transduction mechanism, allowing an appropriate response to be mounted when a stimulus is sensed. As facultative intracellular pathogens, Brucella spp. face various environmental conditions, and an adequate response is required for a successful infection process. Recently, bioinformatic analysis of Brucella genomes predicted a set of 15 bona fide TCS pairs, among which some have been previously investigated. In this report, we characterized a new TCS locus called prlS/R, for probable proline sensor-regulator. It encodes a hybrid histidine kinase (PrlS) with an unusual Na(+)/solute symporter N-terminal domain and a transcriptional regulator (belonging to the LuxR family) (PrlR). In vitro, Brucella spp. with a functional PrlR/S system form bacterial aggregates, which seems to be an adaptive response to a hypersaline environment, while a prlS/R mutant does not. We identified ionic strength as a possible signal sensed by this TCS. Finally, this work correlates the absence of a functional PrlR/S system with the lack of hypersaline-induced aggregation in particular marine Brucella spp.