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We have functionally and structurally defined an essential protein phosphorelay that regulates expression of genes required for growth, division, and intracellular survival of the global zoonotic pathogen Brucella abortus . Our study delineates phosphoryl transfer through this molecular pathway, which initiates from the sensor kinase CckA and proceeds through the ChpT phosphotransferase to two regulatory substrates: CtrA and CpdR. Genetic perturbation of this system results in defects in cell growth and division site selection, and a specific viability deficit inside human phagocytic cells. Thus, proper control of B. abortus division site polarity is necessary for survival in the intracellular niche. We further define the structural foundations of signaling from the central phosphotransferase, ChpT, to its response regulator substrate, CtrA, and provide evidence that there are at least two modes of interaction between ChpT and CtrA, only one of which is competent to catalyze phosphoryltransfer. The structure and dynamics of the active site on each side of the ChpT homodimer are distinct, supporting a model in which quaternary structure of the 2:2 ChpT–CtrA complex enforces an asymmetric mechanism of phosphoryl transfer between ChpT and CtrA. Our study provides mechanistic understanding, from the cellular to the atomic scale, of a conserved transcriptional regulatory system that controls the cellular and infection biology of B. abortus . More generally, our results provide insight into the structural basis of two-component signal transduction, which is broadly conserved in bacteria, plants, and fungi. Brucella abortus is an intracellular bacterial pathogen that inflicts a significant health burden on both humans and their livestock on a global scale. We demonstrate that an essential regulatory system controls the growth and morphology of B. abortus , and that this system is required for survival inside mammalian host cells. Using experimental and computational tools of structural biology, we further define how the protein components of this regulatory pathway interact at the atomic scale. Our results provide evidence for multiple, asymmetric modes of binding between essential pathway proteins that control transcription. The multimodal molecular interactions we observe provide evidence for new layers of allosteric control of this conserved gene regulatory system.

The Pantanal region, the largest floodplain in the world, has a huge biodiversity and is an important livestock center. Bovine brucellosis has been reported in the region over the last three decades, posing implications for cattle industry as well as for the maintenance of biodiversity. We aimed to investigate the presence of B. abortus S19 vaccine strain DNA in unvaccinated domestic and wild ungulates from the Brazilian Pantanal. Fifty-two heifers, 63 ovine, 24 domestic pigs, 28 feral pigs, and three Pampas deer were sampled. Brucella spp. was detected through bcsp31 PCR of blood samples in 45.3% (77/170) of the sampled animals, of which 36.4% (28/77) showed positivity in ery PCR corresponding to B. abortus S19 strain. Feral pigs presented the highest occurrence of positive samples in bcsp31 PCR (75%), followed by ovine (47.6%), domestic pigs (41.7%), and unvaccinated heifers (30.8%). We did not observe positivity in Pampas deer. Our results strongly suggest that vaccination against bovine brucellosis may promote spill-over of B. abortus S19 strain in the Pantanal region. Moreover, our data indicate that wild strains of Brucella circulates in the Pantanal Biome.

Brucellosis is a zoonotic disease caused by bacteria of the Brucella species. This infectious disease represents a significant public health and economic challenge in many regions of the world, including Ecuador. Brucella abortus is the most common species in cattle. Transmission mainly occurs through direct contact with secretions, aborted fetuses, or contaminated reproductive fluids. In this study, to evaluate the circulating strains of Brucella in continental Ecuador, Brucella strains were cultured and isolated from retromammary lymph nodes and milk samples collected over the past three years from six Ecuadorian provinces within the National Brucellosis Program of Ecuador. Brucella cultures were performed on two specific media, CITA and Farrell, followed by molecular identification using PCR and multiple-locus variable-number tandem repeat analysis 16 (MLVA-16) diagnostic techniques. Out of a total of 25 retromammary lymph nodes collected at slaughterhouses and 50 milk samples obtained from serologically positive animals on farms, Brucella was isolated from 35 milk samples and 19 retromammary lymph node samples and identified as Brucella abortus by PCR. Subsequent MLVA-16 genotyping enabled accurate discrimination among the Brucella strains present in Ecuador. This study confirmed the presence of Brucella abortus strains of biovars 1 and 4 and, for the first time, detected the presence of biovar 2 in Ecuador. The isolation and accurate detection of Brucella, along with the implementation of advanced genotyping techniques, such as MLVA, are crucial for future epidemiological studies, outbreak tracing, and the development of control strategies to mitigate animal and human infection in Ecuador.

This study aimed to determine the susceptibility profile of Brazilian Brucella abortus isolates from cattle to eight antimicrobial agents that are recommended for the treatment of human brucellosis and to correlate the susceptibility patterns with origin, biotype and MLVA16-genotype of the strains. Screening of 147 B. abortus strains showed 100% sensitivity to doxycycline and ofloxacin, one (0.68%) strain resistant to ciprofloxacin, two strains (1.36%) resistant to streptomycin, two strains (1.36%) resistant to trimethoprim-sulfamethoxazole and five strains (3.40%) resistant to gentamicin. For rifampicin, three strains (2.04%) were resistant and 54 strains (36.73%) showed reduced sensitivity. Two strains were considered multidrug resistant. In conclusion, the majority of B. abortus strains isolated from cattle in Brazil were sensitive to the antimicrobials commonly used for the treatment of human brucellosis; however, a considerable proportion of strains showed reduced susceptibility to rifampicin and two strains were considered multidrug resistant. Moreover, there was no correlation among the drug susceptibility pattern, origin, biotype and MLVA16-genotypes of these strains.

Brucella abortus S19 is a smooth strain used as live vaccine against bovine brucellosis. Smooth lipopolysaccharide (LPS) is responsible for its residual virulence and serological interference. Rough mutants defective of LPS are more attenuated but confers lower level of protection. We describe a modified B. abortus S19 strain, named as S19Δper, which exhibits intermediate rough phenotype with residual O-polysaccharide (OPS). Deletion of perosamine synthetase gene resulted in substantial attenuation of S19Δper mutant without affecting immunogenic properties. It mounted strong immune response in Swiss albino mice and conferred protection similar to S19 vaccine. Immunized mice produced higher levels of IFN-γ, IgG2a and thus has immune response inclined towards Th1 cell mediated immunity. Sera from immunized animals did not show agglutination reaction with RBPT antigen and thus could serve as DIVA (Differentiating Infected from Vaccinated Animals) vaccine. S19Δper mutant displayed more susceptibility to serum complement mediated killing and sensitivity to polymyxin B. Pregnant guinea pigs injected with S19Δper mutant completed full term of pregnancy and did not cause abortion, still birth or birth of weak offspring. S19Δper mutant with intermediate rough phenotype displayed remarkable resemblance to S19 vaccine strain with improved properties of safety, immunogenicity and DIVA capability for control of bovine brucellosis.

Background Brucellosis, a zoonotic disease in Türkiye, which has significant direct and indirect impacts on the healthcare system and livestock. This study, which aimed to investigate the differences among Brucella spp. isolates originating from different regions of Türkiye, for implications for public health and veterinary medicine. Method Twenty-one isolates from ruminants and two isolates from humans obtained from various regions of Türkiye were utilized in the study. The isolates were identified and biotyped using traditional microbiological procedures, and whole-genome sequencing (WGS) was performed. This was followed by single nucleotide polymorphism (SNP)--based phylogenetic analysis and WGS-based analysis of virulence and resistance genes. Additionally, phenotypic antimicrobial resistance and phage susceptibilities were determined. The obtained data were then compared for concordance, ensuring the validity and reliability of the results. Results Our study, employing culture methods, polymerase chain reaction (PCR), and WGS analyses, identified 11 Brucella melitensis (bv 3 ( n  = 9), one each bv 1 and bv 2) and 12 B. abortus (bv 3 ( n  = 11), bv 9 ( n  = 1)) isolates All B. abortus isolates were of bovine origin, while the B. melitensis isolates were from sheep ( n  = 7), goat ( n  = 1), ram ( n  = 1), and humans ( n  = 2). In the whole-genome SNP-based phylogenetic tree, all B. melitensis strains were found to be of the IIb subtype of genotype II associated with the Eastern Mediterranean lineage. Ten different genotypes were identified in the SNP analysis of the isolates, with a maximum SNP difference of 278 and a minimum SNP difference of 4 among these genotypes. According to the WGS-SNP-based phylogenetic tree of B. abortus isolates, they were grouped in clade C1. In the SNP analysis, where ten different genotypes were identified, the SNP difference among these genotypes was a maximum of 316 and a minimum of 6. In the in silico MLST analysis performed with WGS data, B. melitensis isolates were identified as ST8 and ST102 genotypes, while B. abortus isolates were identified as ST2 and ST3 genotypes. The dominant genotypes were ST8 for B. melitensis and ST2 for B. abortus , respectively. Virulence gene analysis conducted based on WGS data of the 23 B. abortus and B. melitensis isolates revealed 43 virulence gene-associated regions in all strains, irrespective of species, host, or isolation year. Although classical resistance-related genes were not detected by WGS-based antimicrobial resistance gene analysis, phenotypic resistance analysis revealed resistance to azithromycin, rifampin, and trimethoprim/sulfamethoxazole in B. abortus and B. melitensis isolates. Conclusion Both B. melitensis and B. abortus were circulating species in animals and human. The dominant genotypes were ST8 for B. melitensis and ST2 for B. abortus , respectively. All B. melitensis strains were found to be of the IIb subtype of genotype II associated with the Eastern Mediterranean lineage, while B. abortus isolates, they were grouped in clade C1. Further, a comprehensive study with a sufficient number of isolates covering all regions of Türkiye would provide more accurate information about the current epidemiological situation in the country.

In China, brucellosis is an endemic disease and the main sources of brucellosis in animals and humans are infected sheep, cattle and swine. Brucella melitensis (biovars 1 and 3) is the predominant species, associated with sporadic cases and outbreak in humans. Isolates of B. abortus, primarily biovars 1 and 3, and B. suis biovars 1 and 3 are also associated with sporadic human brucellosis. In this study, the genetic profiles of B. melitensis and B. abortus isolates from humans and animals were analyzed and compared by multi-locus variable-number tandem-repeat analysis (MLVA). Among the B. melitensis isolates, the majority (74/82) belonged to MLVA8 genotype 42, clustering in the 'East Mediterranean' group. Two B. melitensis biovar 1 genotype 47 isolates, belonging to the 'Americas' group, were recovered; both were from the Himalayan blue sheep (Pseudois nayaur, a wild animal). The majority of B. abortus isolates (51/70) were biovar 3, genotype 36. Ten B. suis biovar 1 field isolates, including seven outbreak isolates recovered from a cattle farm in Inner Mongolia, were genetically indistinguishable from the vaccine strain S2, based on MLVA cluster analysis. MLVA analysis provided important information for epidemiological trace-back. To the best of our knowledge, this is the first report to associate Brucella cross-infection with the vaccine strain S2 based on molecular comparison of recovered isolates to the vaccine strain. MLVA typing could be an essential assay to improve brucellosis surveillance and control programs.

Background Brucella abortus , a facultative intracellular pathogenic bacterium that usually causes diseases under animals and humans, can survive and replicate within phagocytic cells. Within the host cells, B. abortus has to adapt to low cytosolic magnesium ion (Mg 2+ ) environment, which is critical for bacterial survival and replication. To understand the fitness of B. abortus under the low Mg 2+ environment, transcriptome analysis was performed by RNA-seq. Results: 262 differentially expressed genes (DEGs, fold-change > 1.5 and p  < 0.05) of B. abortus , 123 significantly upregulated genes and 139 significantly downregulated genes, were identified under Mg 2+ starvation environment, highlighting that B. abortus probably employed large amounts of factors to support the adaptation of low Mg 2+ stress responses. Amongst them, two key genes, BAB_RS26550 (encoding putative protein, abbreviated as HP3) and BAB_RS26555 (encoding MgtC/SapB family protein, abbreviated as MgtC), was associated with the ATP hydrolysis to maintain the growth and metabolism of B. abortus under Mg 2+ starvation environment. Furthermore, the HP3 supported B. abortus to resist bactericidal polycations and polymyxin B, as well as influenced the biofilm formation of B. abortus . However, HP3 does not appear to have an appreciable effect on the B. abortus virulence. Conclusions: In this study, a first description of the pattern of B. abortus genetic expression in response to low Mg 2+ stress response provides insights into the intracellular behavior of B. abortus at the genetic level.

Brucellosis remains an underreported zoonotic disease in Ecuador. Its control program in cattle integrates diagnostic testing, vaccination, and eradication incentives, although participation is largely voluntary. Since 2025, vaccination has become compulsory nationwide. Human surveillance remains largely passive, and strain-level data are very limited. This study applied an integrated approach, combining serology (Rose Bengal and SAT-EDTA), microbiological culture, and molecular diagnostics, to assess the presence and diversity of Brucella spp. in cattle and pigs from six slaughterhouses in the northern Andean highlands. A total of 2054 cattle and 1050 pigs from Carchi, Imbabura, and Pichincha were sampled. Among cattle, 133 (6.5%; 95% CI: 5.5–7.6) were seropositive, and viable B. abortus strains were isolated from 17 (12.8%). Genus identification was confirmed by IS711-PCR, while species- and biovar-level differentiation was achieved with AMOS-PCR; additional assays targeting the ery gene and RB51 marker were used to distinguish field from vaccine strains. Biotyping and molecular analysis revealed a predominance of B. abortus biovar 4 (13/17 isolates) over biovar 1, all confirmed as field strains. In pigs, 10 animals (0.95%) tested seropositive, but no isolates were recovered, highlighting limitations of serology in swine. Most livestock, including the positives, originated locally, reinforcing the representativeness of our findings. The successful isolation and molecular characterization of B. abortus demonstrates the value of combining diagnostic strategies beyond serology. These results underscore the utility of active surveillance when supported by traceability systems; this approach may also contribute to guide interventions to reduce infection risk in livestock and humans.

Lipopolysaccharide is essential for most Gram-negative bacteria as it is a main component of the outer membrane. In the pathogen Brucella abortus , smooth lipopolysaccharide containing the O-antigen is required for virulence. Being part of the Rhizobiales, Brucella spp. display unipolar growth and lipopolysaccharide was shown to be incorporated at the active growth sites, i.e. the new pole and the division site. By localizing proteins involved in the lipopolysaccharide transport across the cell envelope, from the inner to the outer membrane, we show that the lipopolysaccharide incorporation sites are determined by the inner membrane complex of the lipopolysaccharide transport system. Moreover, we identify the main O-antigen ligase of Brucella spp. involved in smooth lipopolysaccharide synthesis. Altogether, our data highlight a layer of spatiotemporal organization of the lipopolysaccharide biosynthesis pathway and identify an original class of bifunctional O-antigen ligases. Pathogenic Brucella abortus , containing a mix of lipopolysaccharides with or without O-antigen, grows its envelope in a unipolar manner. Here, Servais et al , localize the LPS translocation machinery and identify the main O-antigen ligase in Brucella species, shedding light on the basic biology of this organism.