Brucella neotomae Infection in Humans, Costa Rica

Several species of Brucella are known to be zoonotic, but B. neotomae infection has been thought to be limited to wood rats. In 2008 and 2011, however, B. neotomae was isolated from cerebrospinal fluid of 2 men with neurobrucellosis. The nonzoonotic status of B. neotomae should be reassessed

Brucella ceti infection in dolphins from the Western Mediterranean sea

Publishe

Casos de brucelosis canina presentados en la provincia de Pichincha, Ecuador

La brucelosis canina, ampliamente distribuida en el mundo y con reportes de seroprevalencia de entre 6 % a 35 %, es considerada un problema de salud pública. Esta infección es producida por cuatro de las trece especies del género Brucella: B. canis, B. abortus, B. melitensis y B. suis, dependiendo de la cepa circulante en el área geográfica de estudio. El contagio ocurre principalmente por ingestión, inhalación o contacto con fetos abortados, placenta, secreciones vaginales o semen y también por vía transmamaria y placentaria. La aparición de casos de brucelosis canina marcó el inicio de este estudio con el análisis de la enfermedad mediante identificación del agente causal en dos escenarios epidemiológicos diferentes. En el primer escenario (2008), se tomaron sueros de 151 caninos de 34 fincas de la provincia de Pichincha (Ecuador), que se analizaron para detección de anticuerpos contra cepas lisas mediante Rosa de Bengala, sero-aglutinación lenta en tubo (SAT-EDTA) y ELISA indirecto. En el segundo escenario (2017 a 2019) y utilizando el Rapid test kit (C. Bru Ab Test) para detección de anticuerpos contra cepas rugosas, se analizaron diez sueros de caninos provenientes de clínicas veterinarias de la provincia de Pichincha (Ecuador). Para ambos escenarios se realizó biotipificación y genotipificación.Trabajo publicado en Cagliada, Maria del Pilar Lilia y Galosi, Cecilia Mónica (comps.). I Congreso de Microbiología Veterinaria. Libro de resúmenes. La Plata: Facultad de Ciencias Veterinarias, 2021.Facultad de Ciencias Veterinaria

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 Glade 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

Brucella Genetic Variability in Wildlife Marine Mammals Populations Relates to Host Preference and Ocean Distribution

Intracellular bacterial pathogens probably arose when their ancestor adapted from a free-living environment to an intracellular one, leading to clonal bacteria with smaller genomes and less sources of genetic plasticity. Still, this plasticity is needed to respond to the challenges posed by the host. Members of the Brucella genus are facultative-extracellular intracellular bacteria responsible for causing brucellosis in a variety of mammals. The various species keep different host preferences, virulence, and zoonotic potential despite having 97–99% similarity at genome level. Here, we describe elements of genetic variation in Brucella ceti isolated from wildlife dolphins inhabiting the Pacific Ocean and the Mediterranean Sea. Comparison with isolates obtained from marine mammals from the Atlantic Ocean and the broader Brucella genus showed distinctive traits according to oceanic distribution and preferred host. Marine mammal isolates display genetic variability, represented by an important number of IS711 elements as well as specific IS711 and SNPs genomic distribution clustering patterns. Extensive pseudogenization was found among isolates from marine mammals as compared with terrestrial ones, causing degradation in pathways related to energy, transport of metabolites, and regulation/ transcription. Brucella ceti isolates infecting particularly dolphin hosts, showed further degradation of metabolite transport pathways as well as pathways related to cell wall/membrane/envelope biogenesis and motility. Thus, gene loss through pseudogenization is a source of genetic variation in Brucella, whichinturn, relates to adaptation to different hosts.This is relevant to understand the natural history of bacterial diseases, their zoonotic potential, and the impact of human interventions such as domestication.Comisión Nacional para la Gestión de la Biodiversidad/[R-028-203-OT]/CONAGEBIO/Costa RicaMinisterio de Ciencia, Tecnología y Telecomunicaciones/[FV-004-13]/MICITT/Costa RicaWellcome Trust/[098051]/WT/LondresWellcome Trust/[106690/Z/14/Z]/WT/LondresUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET

A Sinorhizobium meliloti and Agrobacterium tumefaciens ExoR ortholog is not crucial for Brucella abortus virulence

Brucella is a facultative extracellular-intracellular pathogen that belongs to the Alphaproteo- bacteria class. Precise sensing of environmental changes and a proper response mediated by a gene expression regulatory network are essential for this pathogen to survive. The plant-related Alphaproteobacteria Sinorhizobium meliloti and Agrobacterium tumefaciens also alternate from a free to a host-associated life, where a regulatory invasion switch is needed for this transition. This switch is composed of a two-component regulatory system (TCS) and a global inhibitor, ExoR. In B. abortus, the BvrR/BvrS TCS is essential for intra- cellular survival. However, the presence of a TCS inhibitor, such as ExoR, in Brucella is still unknown. In this work, we identified a genomic sequence similar to S. meliloti exoR in the B. abortus 2308W genome, constructed an exoR mutant strain, and performed its characteri- zation through ex vivo and in vivo assays. Our findings indicate that ExoR is related to the BvrR phosphorylation state, and is related to the expression of known BvrR/BrvS gene tar- gets, such as virB8, vjbR, and omp25 when grown in rich medium or starving conditions. Despite this, the exoR mutant strain showed no significant differences as compared to the wild-type strain, related to resistance to polymyxin B or human non-immune serum, intracel- lular replication, or infectivity in a mice model. ExoR in B. abortus is related to BvrR/BvrS as observed in other Rhizobiales; however, its function seems different from that observed for its orthologs described in A. tumefaciens and S. meliloti.Brucella es un patógeno extracelular-intracelular facultativo que pertenece a la clase de las alfaproteobacterias. Para que este patógeno sobreviva, es esencial que detecte con precisión los cambios ambientales y dé una respuesta adecuada mediada por una red de regulación de la expresión génica. Las alfaproteobacterias Sinorhizobium meliloti y Agrobacterium tumefaciens, relacionadas con las plantas, también alternan entre una vida libre y otra asociada al hospedador, y para esta transición es necesario un interruptor regulador de la invasión. Este interruptor está compuesto por un sistema regulador de dos componentes (TCS) y un inhibidor global, ExoR. En B. abortus, el TCS BvrR/BvrS es esencial para la supervivencia intracelular. supervivencia intracelular. Sin embargo, la presencia de un inhibidor del TCS, como ExoR, en Brucella es aún desconocida. En este trabajo, identificamos una secuencia genómica similar a la de ExoR de S. meliloti en el genoma de B.abortus 2308W, construimos una cepa mutante de ExoR y realizamos su caracterización mediante ensayos ex vivo e in vivo. Nuestros resultados indican que ExoR está relacionado con la estado de fosforilación de BvrR, y está relacionado con la expresión de objetivos génicos conocidos de BvrR/BrvS, como virB8, vjbR y omp25 cuando se cultiva en medio rico o en condiciones de inanición. A pesar de esto, la cepa mutante exoR no mostró diferencias significativas en comparación con la cepa de tipo salvaje, relacionadas con la resistencia a la polimixina B o al suero humano no inmune, la replicación intracelular o la infectividad en un modelo de ratón. El ExoR en B. abortus está relacionado con el BvrR/BvrS como como se ha observado en otros Rhizobiales; sin embargo, su función parece diferente de la observada para sus ortólogos descritos en A. tumefaciens y S. meliloti.This work was supported by Fondos del Sistema FEES/CONARE [02-2020, 0652-19 to C. G-V], Fondos FIDA, Universidad Nacional [SIA 0047-17 to C. G-V], Espacio Universitario de Estudios Avanzados, UCREA [B8762] from the Presidency of University of Costa Rica, and the Vice Presidency for Research, University of Costa Rica [C0456 to E.C-O]. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.Escuela de Medicina Veterinari

A bvrR/bvrS Non-Polar Brucella abortus Mutant Confirms the Role of the Two-Component System BvrR/BvrS in Virulence and Membrane Integrity

Brucella abortus is a bacterial pathogen causing bovine brucellosis worldwide. This facultative extracellular–intracellular pathogen can be transmitted to humans, leading to a zoonotic disease. The disease remains a public health concern, particularly in regions where livestock farming is present. The two-component regulatory system BvrR/BvrS was described by isolating the attenuated transposition mutants bvrR::Tn5 and bvrS::Tn5, whose characterization led to the understanding of the role of the system in bacterial survival. However, a phenotypic comparison with deletion mutants has not been performed because their construction has been unsuccessful in brucellae and difficult in phylogenetically related Rhizobiales with BvrR/BvrS orthologs. Here, we used an unmarked gene excision strategy to generate a B. abortus mutant strain lacking both genes, called B. abortus ∆bvrRS. The deletion was verified through PCR, Southern blot, Western blot, Sanger sequencing, and whole-genome sequencing, confirming a clean mutation without further alterations at the genome level. B. abortus ∆bvrRS shared attenuated phenotypic traits with both transposition mutants, confirming the role of BvrR/BvrS in pathogenesis and membrane integrity. This B. abortus ∆bvrRS with a non-antimicrobial marker is an excellent tool for continuing studies on the role of BvrR/BvrS in the B. abortus lifestyle.Brucella abortus es un patógeno bacteriano causante de la brucelosis bovina en todo el mundo. Este patógeno extracelular-intracelular facultativo puede transmitirse al ser humano, dando lugar a una enfermedad zoonótica. La enfermedad sigue siendo motivo de preocupación para la salud pública, sobre todo en las regiones donde hay ganadería. El sistema regulador de dos componentes BvrR/BvrS se describió aislando los mutantes de transposición atenuados mutantes de transposición atenuados bvrR::Tn5 y bvrS::Tn5, cuya caracterización condujo a la comprensión de el papel del sistema en la supervivencia bacteriana. Sin embargo, la comparación fenotípica con los mutantes de deleción no se ha realizado porque su construcción no ha tenido éxito en brucelas y ha sido difícil en Rhizobiales filogenéticamente relacionados con ortólogos de BvrR/BvrS. Aquí hemos utilizado una estrategia de para generar una cepa mutante de B. abortus que carece de ambos genes, denominada B. abortus ∆bvrRS. La deleción se verificó mediante PCR, Southern blot, Western blot, secuenciación de Sanger y secuenciación del genoma completo. secuenciación del genoma completo, confirmando una mutación limpia sin más alteraciones a nivel genoma. B. abortus ∆bvrRS compartió rasgos fenotípicos atenuados con ambos mutantes de transposición, confirmando el papel de BvrR/BvrS en la patogénesis y la integridad de la membrana. Este B. abortus ∆bvrRS con un marcador no antimicrobiano es una herramienta excelente para continuar los estudios sobre el papel de BvrR/BvrS en el estilo de vida de B. abortus.Universidad Nacional, Costa RicaEscuela de Medicina Veterinari

Brucella abortus strain 2308 Wisconsin genome: importance of the definition of reference strains

Brucellosis is a bacterial infectious disease affecting a wide range of mammals and a neglected zoonosis caused by species of the genetically homogenous genus Brucella. As in most studies on bacterial diseases, research in brucellosis is carried out by using reference strains as canonical models to understand the mechanisms underlying host pathogen interactions. We performed whole genome sequencing (WGS) analysis of the reference strain Brucella abortus 2308 routinely used in our laboratory, including manual curated annotation accessible as an editable version at www.wikipedia.Comparison of this genome with two publically available 2308 genomes showed significant differences, particularly indels related to insertional elements, suggesting variability related to the transposition of these elements within the same strain. Considering the outcome of high resolution genomic techniques in the bacteriology field, the conventional concept of strain definition needs to be revised

Brucella abortus senses the intracellular environment through the BvrR/Bvrs two-component system, which allows B. abortus to adapt to its replicative nich

Brucella abortus is a facultative extracellular-intracellular pathogen belonging to a group of Alphaproteobacteria that establishes close interactions with animal cells. This bacterium enters host cells in a membrane-bound compartment, avoiding the lysosomal route and reaching the endoplasmic reticulum through the action of the type IV secretion system, VirB. In this work, we demonstrate that the BvrR/BvrS two-component system senses the intracellular environment to mount the transcriptional response required for intracellular life adaptation. By combining a method to purify intracellularly extracted bacteria with a strategy that allows direct determination of BvrR phosphorylation, we showed that upon entrance to host cells, the regulatory protein BvrR was activated (BvrR-P) by phosphorylation at aspartate 58. This activation takes place in response to intracellular cues found in early compartments, such as low pH and nutrient deprivation. Furthermore, BvrR activation was followed by an increase in the expression of VjbR and VirB. The in vitro activation of this BvrR-P/VjbR/VirB virulence circuit rescued B. abortus from the inhibition of intracellular replication induced by bafilomycin treatment of cells, demonstrating the relevance of this mechanism for intracellular bacterial survival and replication. All together, our results indicate that B. abortus senses the transition from the extracellular to the intracellular milieu through BvrR/BvrS, allowing the bacterium to transit safely to its replicative niche. These results serve as a working model for understanding the role of this family of two-component systems in the adaptation to intracellular life of AlphaproteobacteriaBrucella abortus es un patógeno facultativo extracelular-intracelular perteneciente a un grupo de alfaproteobacterias que establece estrechas interacciones con las células animales. Esta bacteria entra en las células huéspedes en un compartimento unido a la membrana, evitando la ruta lisosómica y llegando al retículo endoplásmico por la acción del sistema de secreción de tipo IV, VirB. En este trabajo, demostramos que el sistema de dos componentes BvrR/BvrS detecta el entorno intracelular para montar la respuesta transcripcional necesaria para la adaptación de la vida intracelular. Combinando un método para purificar las bacterias extraídas intracelularmente con una estrategia que permite la determinación directa de la fosforilación de BvrR, demostramos que al entrar en las células huéspedes, la proteína reguladora BvrR fue activada (BvrR-P) por la fosforilación en el aspartato 58. Esta activación tiene lugar en respuesta a las señales intracelulares encontradas en los primeros compartimentos, como el bajo pH y la privación de nutrientes. Además, a la activación de BvrR le siguió un aumento de la expresión de VjbR y VirB. La activación in vitro de este circuito de virulencia BvrR-P/VjbR/VirB rescató a B. abortus de la inhibición de la replicación intracelular inducida por el tratamiento de las células con bafilomicina, lo que demuestra la importancia de este mecanismo para la supervivencia y la replicación de las bacterias intracelulares. En conjunto, nuestros resultados indican que B. abortus percibe la transición del medio extracelular al intracelular a través de BvrR/BvrS, permitiendo a la bacteria transitar con seguridad a su nicho de replicación. Estos resultados sirven como modelo de trabajo para comprender el papel de esta familia de sistemas de dos componentes en la adaptación a la vida intracelular de las alfaproteobacterias.Universidad Nacional, Costa RicaEscuela de Medicina Veterinari