Genomic Diversity and Zoonotic Potential of Brucella neotomae.

After reports in 2017 of Brucella neotomae infections among humans in Costa Rica, we sequenced 12 strains isolated from rodents during 1955-1964 from Utah, USA. We observed an exact strain match between the human isolates and 1 Utah isolate. Independent confirmation is required to clarify B. neotomae zoonotic potential.

Pseudochrobactrum algeriensis sp. nov., isolated from lymph nodes of Algerian cattle.

Three Gram-negative, rod-shaped, oxidase-positive, non-spore-forming, non-motile strains (C130915_07T, 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 (C19:cyclo, C18 : 1, C18 : 0, C16 : 1 and C16 : 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_07T=CECT30232T=LMG 32378T).

Brucella vulpis sp. nov., isolated from mandibular lymph nodes of red foxes (Vulpes vulpes).

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 new strains Brucella inopinata BO1 and Brucella species 83-210 behave biologically like classic infectious Brucella species and cause death in murine models of infection.

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.

Brucella papionis sp. nov., isolated from baboons (Papio spp.).

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)).

Mutants in the lipopolysaccharide of Brucella ovis are attenuated and protect against B. ovis infection in mice.

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.

Brucella abortus in Kazakhstan, population structure and comparison with worldwide genetic diversity.

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.

The two-component system PrlS/PrlR of Brucella melitensis is required for persistence in mice and appears to respond to ionic strength.

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.

The genome sequence of Brucella pinnipedialis B2/94 sheds light on the evolutionary history of the genus Brucella.

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.

Novel IS711 chromosomal location useful for identification of marine mammal Brucella genotype ST27, which is associated with zoonotic infection.

We report a novel IS711 chromosomal location that is specific for the Brucella genotype ST27 previously associated with Pacific marine mammals and human zoonotic infection in New Zealand and Peru. Our data support the previous observation that this peculiar genotype is distinct from those commonly isolated from the Atlantic and currently classified within the species B. ceti and B. pinnipedialis.

Whole-Genome Sequence of a Brucella pinnipedialis Sequence Type 54 Strain Isolated from a Hooded Seal (Cystophora cristata) from the North Atlantic Ocean, Norway.

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.

Novel IS711-specific chromosomal locations useful for identification and classification of marine mammal Brucella strains.

We report five new IS711 chromosomal locations that are specific for marine mammal Brucella groups of strains and useful for their identification and classification. Our data support their current classification into two species, Brucella ceti and B. pinnipedialis, with subgroups in each, but also the possibility of additional species.

Omp2b Porin Alteration in the Course of Evolution of Brucella spp.

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.

DNA polymorphism analysis of Brucella lipopolysaccharide genes reveals marked differences in O-polysaccharide biosynthetic genes between smooth and rough Brucella species and novel species-specific markers.

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, manA(O-Ag), manB(O-Ag), manC(O-Ag), wbkF and wbkD) and wbo (wboA and wboB), and core genes manB(core) 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), manB(O-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 manB(O-Ag) for O-polysaccharide synthesis and contribute to explain the lipopolysaccharide structure of rough and smooth Brucella species.

Real-time PCR for identification of Brucella spp.: a comparative study of IS711, bcsp31 and per target genes.

Culture is considered as the reference standard assay for diagnosis of Brucella spp. in humans and animals but it is time-consuming and hazardous. In this study, we evaluated the performances of newly designed real-time PCR assays using TaqMan probes and targeting the 3 following specific genes: (i) the insertion sequence IS711, (ii) bcsp31 and (iii) per genes for the detection of Brucella at genus level. The real-time PCR assays were compared to previously described conventional PCR assays targeting the same genes. The genus-specificity was evaluated on 26 Brucella strains, including all species and biovars. The analytical specificity was evaluated on a collection of 68 clinically relevant, phylogenetically related or serologically cross-reacting micro-organisms. The analytical sensitivity was assessed using decreasing DNA quantities of Brucella ovis, B. melitensis bv. 1, B. abortus bv. 1 and B. canis reference strains. Finally, intra-assay repeatability and inter-assay reproducibility were assessed. All Brucella species DNA were amplified in the three tests. However, the earliest signal was observed with the IS711 real-time PCR, where it varied according to the IS711 copy number. No cross-reactivity was observed in all three tests. Real-time PCR was always more sensitive than conventional PCR assays. The real-time PCR assay targeting IS711 presented an identical or a greater sensitivity than the two other tests. In all cases, the variability was very low. In conclusion, real-time PCR assays are easy-to-use, produce results faster than conventional PCR systems while reducing DNA contamination risks. The IS711-based real-time PCR assay is specific and highly sensitive and appears as an efficient and reproducible method for the rapid and safe detection of the genus Brucella.

Taxonomic Organization of the Family Brucellaceae Based on a Phylogenomic Approach.

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.

Corrigendum: Genetic and Phenotypic Characterization of the Etiological Agent of Canine Orchiepididymitis Smooth Brucella sp. BCCN84.3.

[This corrects the article DOI: 10.3389/fvets.2019.00175.].

Genetic and Phenotypic Characterization of the Etiological Agent of Canine Orchiepididymitis Smooth Brucella sp. BCCN84.3.

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.

Identification of novel DNA fragments and partial sequence of a genomic island specific of Brucella pinnipedialis.

Since the 1990s, Brucella strains have been isolated from a wide variety of marine mammals and were recently recognized as two different species, i.e. Brucella pinnipedialis for pinniped isolates and Brucella ceti for cetacean isolates. The aim of this study was to identify specific DNA fragments of marine mammal Brucella strains using a previously described infrequent restriction site-PCR (IRS-PCR) method but with three new couples of restriction enzymes applied on a larger panel of marine mammal Brucella isolates (n=74) and one human isolate from New Zealand likely from marine mammal origin. This study revealed five DNA fragments specific of Brucella strains isolated from marine mammals. Among them two new DNA fragments were specific of B. pinnipedialis but were not detected in hooded seal isolates. DNA fragment I identified in the previous IRS-PCR study and fragment VI of this study were located on a cloned and sequenced 6kb SacI fragment. Its nucleotide sequence revealed that it is likely part of a putative genomic island. Sequence analysis showed that it carries four ORFs coding for putative metabolic functions. Although hooded seal isolates are classified within B. pinnipedialis it was shown in this study that they do not carry this genomic island and this raises the question about their evolutionary history within B. pinnipedialis.