Search

The COVID-19 pandemic has imposed several challenges and strains at all levels of the educational system, especially as a consequence of lockdown and social distance measures. After a period of exclusive use of the online educational environment, educators have adapted to the new circumstances and, by a combination of different strategies, have fought to overcome the limitations and deficiencies of virtual learning. Student motivation, productivity, and creativity continue to be the main pedagogical issues that have to be reached with the new didactic tools developed during the pandemic. At the same time, this pandemic has shown the importance of the inclusion of microbiology as a core element of the educational curriculum and the opportunity to raise public awareness of the importance of microbes to everyday life.

Internet y las redes sociales han democratizado la comunicación de contenidos sobre salud y han multiplicado la difusión pública de informaciones relacionadas con ese tema. Muchas de las informaciones sanitarias proceden de fuentes acreditadas y son plenamente solventes, lo que permite a la ciudadanía acceder fácilmente a información de calidad que promueve comportamientos responsables. Sin embargo, al mismo tiempo, en las redes circulan cada vez más contenidos sobre salud de procedencia desconocida y fiabilidad dudosa. Buena parte de esos mensajes son, de hecho, intencionadamente engañosos. Las redes, en definitiva, son una moneda de dos caras: dan acceso a contenidos sanitarios de calidad, pero exponen asimismo a la ciudadanía a diversos riesgos.

Following the declaration, in March 2020, of the Covid-19 pandemic, there was an escalation of disinformation, involving multiple actors and reaching global dimensions. In this article, we analyze the possible causes and characteristics of the spread of disinformation on this issue. Disinformation about science can be explained by the distance that separates scientific knowledge from common knowledge and the difficult relationship between science and the media. The pandemic has multiplied the number of scientific publications and has accelerated publication rates, which has contributed to the dissemination of provisional, erroneous, or totally false information. A process of politicization has also developed, which has led to misinformation. In addition, the need to confront this health crisis has led society to demand accurate information from science, despite the fact that in many cases there is only uncertainty. The experience of this pandemic highlights the importance of providing citizens with accessible and rigorous knowledge that creates confidence in science. To achieve this, it is necessary to have specialized professionals capable of providing rigorous information, not only on the results but also on the research processes.

Se presenta un analisis de contenido de todos los bulos (N=292) relacionados con la pandemia Covid-19 identificados por las tres plataformas de verificacion acreditadas en Espana, durante el primer mes del estado de alarma decretado por el Gobierno (14 marzo 2020 – 13 abril 2020). El estudio muestra que los bulos sobre el coronavirus fueron diseminados principalmente en las redes sociales y, entre ellas, sobre todo en las cerradas, como la aplicacion movil de mensajeria WhatsApp. Tambien detecta las particularidades formales y de contenido mas frecuentes de los contenidos falsificados. Los resultados revelan que la pandemia, ademas de generar un gran numero de bulos sobre salud y ciencia, casi un tercio de la muestra, tambien propicio la difusion de numerosos contenidos falsos de tema politico y gubernamental. El articulo explora los formatos, fuentes y territorios de procedencia de los bulos. Mas alla de sus resultados empiricos, este estudio realiza contribuciones teoricas en el marco de los emergentes estudios sobre desordenes informativos. En concreto, aporta una definicion propia de bulo, asi como una tipologia en la que se identifican cuatro tipos de bulos: broma, exageracion, descontextualizacion y engano. A partir de esos cuatro tipos, se propone un ‘diagrama de gravedad de los bulos’.

Twitter is one of the most popular social media networks that, in recent years, has been increasingly used by researchers as a platform to share science and discuss ongoing work. Despite its popularity, Twitter is not commonly used as a medium to teach science. Here, we summarize the results of #EUROmicroMOOC: the first worldwide Microbiology Massive Open Online Course taught in English using Twitter. Content analytics indicated that more than 3 million users saw posts with the hashtag #EUROmicroMOOC, which resulted in over 42 million Twitter impressions worldwide. These analyses demonstrate that free Microbiology MOOCs shared on Twitter are valuable educational tools that reach broad audiences throughout the world. We also describe our experience teaching an entire Microbiology course using Twitter and provide recommendations when using social media to communicate science to a broad audience., J.L.T. is supported by an NSERC postdoctoral fellowship and a Pasteur–Roux fellowship.

Social networks have been used to teach and engage people about the importance of science. The integration of social networks in the daily routines of faculties and scientists is strongly recommended to increase their personal brand, improve their skills, enhance their visibility, share and communicate science to society, promote scientific culture, and even as a tool for teaching and learning. Here we review the use of Twitter in science and comment on our previous experience of using this social network as a platform for a Massive Online Open Course (MOOC) in Spain and Latin America. We propose to extend this strategy to a pan-European Microbiology MOOC in the near future.

López-Goñi, Ignacio et al., Social networks are already being exploited for searching, storing, and sharing knowledge, demonstrating that they are an efficient vehicle for social learning. Consequently, they could be implemented as a competent tool for formal learning. Twitter is among the 10 most popular online social networks, integrating a community of over 500 million users around the world. Twitter has already been used in several educational programs and evaluated as a positive teaching experience with an outstanding potential in academic and educational environments (1–6). However, there are scarce examples available in the literature about its use in science teaching and communication. In this work, we present and analyze the application of Twitter to create an online space for communication and learning of basic microbiology. With this aim, a team of professionals in the field has imparted, to our knowledge, the first worldwide open access microbiology course via Twitter. Here we assess the results of our experience of using this social network as a tool for teaching, promoting, and communicating scientific knowledge to a wide audience.

The assessment of the genetic stability is one of the essential elements to guarantee the biological quality of live anti-bacteria vaccines. Live attenuated Brucella melitensis Rev 1 is the most effective vaccine against brucellosis in small ruminants. Thirty-six B. melitensis Rev 1 vaccine strains isolated from human or animal sources from different geographic regions, from different commercial batches or laboratory collections were typed by the multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) recently described for Brucella spp. Our results demonstrated that B. melitensis Rev 1 group as assayed by MLVA is genetically very homogeneous. We believe that MLVA methodology could be an essential assay to guarantee the quality and stability of live anti-bacterial vaccines being produced worldwide and can be included as in vitro control.

The bacteria of the Brucella genus are responsible for a worldwide zoonosis called brucellosis. They belong to the α-proteobacteria group, as many other bacteria that live in close association with a eukaryotic host. Importantly, the Brucellae are mainly intracellular pathogens, and the molecular mechanisms of their virulence are still poorly understood. Using the complete genome sequence of Brucella melitensis, we generated a database of protein-coding open reading frames (ORFs) and constructed an ORFeome library of 3091 Gateway Entry clones, each containing a defined ORF. This first version of the Brucella ORFeome (v1.1) provides the coding sequences in a user-friendly format amenable to high-throughput functional genomic and proteomic experiments, as the ORFs are conveniently transferable from the Entry clones to various Expression vectors by recombinational cloning. The cloning of the Brucella ORFeome v1.1 should help to provide a better understanding of the molecular mechanisms of virulence, including the identification of bacterial protein-protein interactions, but also interactions between bacterial effectors and their host's targets.

Brucella abortus rough lipopolysaccharide (LPS) mutants were obtained by transposon insertion into two wbk genes ( wbkA [putative glycosyltransferase; formerly rfbU ] and per [perosamine synthetase]), into manB ( pmm [phosphomannomutase; formerly rfbK ]), and into an unassigned gene. Consistent with gene-predicted roles, electrophoretic analysis, 2-keto-3- manno -d- octulosonate measurements, and immunoblots with monoclonal antibodies to O-polysaccharide, outer and inner core epitopes showed no O-polysaccharide expression and no LPS core defects in the wbk mutants. The rough LPS of manB mutant lacked the outer core epitope and the gene was designated manB core to distinguish it from the wbk manB O-Ag . The fourth gene (provisionally designated wa** ) coded for a putative glycosyltransferase involved in inner core synthesis, but the mutant kept the outer core epitope. Differences in phage and polymyxin sensitivity, exposure or expression of outer membrane protein, core and lipid A epitopes, and lipid A acylation demonstrated that small changes in LPS core caused significant differences in B. abortus outer membrane topology. In mice, the mutants showed different degrees of attenuation and induced antibodies to rough LPS and outer membrane proteins. Core-defective mutants and strain RB51 were ineffective vaccines against B. abortus in mice. The mutants per and wbkA induced protection but less than the standard smooth vaccine S19, and controls suggested that anti O-polysaccharide antibodies accounted largely for the difference. Whereas no core-defective mutant was effective against B. ovis , S19, RB51, and the wbkA and per mutants afforded similar levels of protection. These results suggest that rough Brucella vaccines should carry a complete core for maximal effectiveness.

The activities of a short therapeutic regimen with azithromycin and the classic treatment doxycycline with streptomycin were compared and evaluated in mice infected with Brucella melitensis. In a chronic model, starting therapy 31 days after challenge, azithromycin (10 days, 50 mg/kg/day) significantly reduced the infection (2.9 logs, day 48 post-infection). The effectiveness of doxycycline (21 days, 50 mg/kg/12 hourly) was greater than azithromycin (4.1 logs of reduction, day 48 post-infection), and when doxycycline was administered for a period of 45 days, all the animals were bacteriologically cured from day 78. The combination with streptomycin (14 days, 10 mg/kg/day) did not improve the effect of any of the regimens. In an acute model infection, treatments with doxycycline or doxycycline-streptomycin, for a period of 3 days, starting 1 day after lethal challenge, were able to protect all the mice. In contrast, only 50% of the mice treated with azithromycin survived the challenge. In conclusion, although a short oral treatment with azithromycin was able to reduce the infection significantly, it was not able to cure the animals as effectively as the classic regimen with doxycycline administered for a longer period of time.

Members of the genus Brucella are intracellular -Proteobacteria responsible for brucellosis, a chronic disease of humans and animals. Little is known about Brucella virulence mechanisms, but the abilities of these bacteria to invade and to survive within cells are decisive factors for causing disease. Transmission electron and fluorescence microscopy of infected nonprofessional phagocytic HeLa cells revealed minor membrane changes accompanied by discrete recruitment of F-actin at the site of Brucella abortus entry. Cell uptake of B. abortus was negatively affected to various degrees by actin, actin-myosin, and microtubule chemical inhibitors. Modulators of MAPKs and protein-tyrosine kinases hampered Brucella cell internalization. Inactivation of Rho small GTPases using clostridial toxins TcdB-10463, TcdB-1470, TcsL-1522, and TcdA significantly reduced the uptake of B. abortus by HeLa cells. In contrast, cytotoxic necrotizing factor from Escherichia coli, known to activate Rho, Rac, and Cdc42 small GTPases, increased the internalization of both virulent and non-virulent B. abortus. Expression of dominant-positive Rho, Rac, and Cdc42 forms in HeLa cells promoted the uptake of B. abortus, whereas expression of dominant-negative forms of these GTPases in HeLa cells hampered Brucella uptake. Cdc42 was activated upon cell contact by virulent B. abortus, but not by a noninvasive isogenic strain, as proven by affinity precipitation of active Rho, Rac, and Cdc42. The polyphasic approach used to discern the molecular events leading to Brucella internalization provides new alternatives for exploring the complexity of the signals required by intracellular pathogens for cell invasion. Los miembros del género Brucella son intracelulares -Proteobacterias responsables de la brucelosis, una enfermedad crónica de los humanos y animales. Se sabe poco sobre los mecanismos de virulencia de la Brucella, pero la capacidad de estas bacterias para invadir y sobrevivir dentro de las células son factores decisivos para causar la enfermedad. La microscopía electrónica de transmisión y la microscopía de fluorescencia de las células HeLa fagocíticas no profesionales infectadas revelaron cambios menores en la membrana acompañados de un reclutamiento discreto de F-actina en el lugar de entrada de Brucella abortus. La captación celular de B. abortus se vio afectada negativamente en diversos grados por la actina, la actina-miosina y los inhibidores químicos de los microtúbulos. Los moduladores de los MAPK y las proteínas-tirosina-cinasas obstaculizaron la internalización de las células de Brucella. La inactivación de las pequeñas GTPasas Rho usando las toxinas clostridiales TcdB-10463, TcdB-1470, TcsL-1522, y TcdA redujo significativamente la captación de B. abortus por las células HeLa. En cambio, el factor necrosante citotóxico de Escherichia coli, conocido por activar las pequeñas GTPasas Rho, Rac y Cdc42, aumentó la internalización de la B. abortus tanto virulenta como no virulenta. La expresión de las formas dominantes-positivas de Rho, Rac y Cdc42 en las células HeLa promovió la captación de B. abortus, mientras que la expresión de las formas dominantes-negativas de estas GTPasas en las células HeLa obstaculizó la captación de Brucella. El Cdc42 se activó al contacto con la célula por la virulenta B. abortus, pero no por una cepa isogénica no invasiva, como se demostró por la precipitación por afinidad de Rho, Rac y Cdc42 activos. El enfoque polifásico utilizado para discernir los acontecimientos moleculares que conducen a la internalización de Brucella proporciona nuevas alternativas para explorar la complejidad de las señales que requieren los patógenos intracelulares para la invasión celular. Universidad Nacional, Costa Rica Escuela de Medicina Veterinaria

Multiple-locus variable-number tandem-repeat analysis (MLVA), multiplex PCR, and PCR-restriction fragment length polymorphism analysis were compared for typing Brucella suis isolates. A perfect concordance was obtained among these molecular assays. However, MLVA was the only method to demonstrate brucellosis outbreaks and to confirm that wildlife is a reservoir for zoonotic brucellosis.

The genus Brucella consists of 6 recognized bacterial species (1) and 2 proposed new species recently isolated from marine mammals (2). Some species have several biovars or biotypes, distinguishable by time-consuming analysis of phenotypic characteristics (3). Such analyses are subject to variable interpretations and must be performed by skilled technicians who are at high risk because most clinical Brucella strains are highly pathogenic. Accurate typing procedures are critical for eradication and control of disease-causing organisms, but DNA-based techniques are challenging because the various Brucella species and strains have a high degree of genetic homology, as demonstrated by whole-genome comparative analyses of the 3 Brucella genomes sequenced, Brucella melitensis (4), Brucella suis (5), and Brucella abortus (6). Unique fragments occur among the different Brucella genomes, however, which allowed us to develop a rapid, specific single-test-tube assay for molecular identification of all known Brucella species. We used the following species- or strain-specific genetic differences to design PCR primers: ( a ) a 25-kb DNA deletion leading to the loss of omp31 gene in the reference strains of all B. abortus biovars (6)(7); ( b ) a 15-kb deletion comprising …

Soler-Llorens, Pedro et al., 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., This work was funded by Ministerio de Economía y Competitividad of Spain (reference project AGL2011-30453-C04) and Fundación para la Investigación Médica Aplicada (FIMA). Financial support to PS-L from Ministerio de Economía y Competitividad of Spain (reference BES-2009-015656), AZ-B from Universidad Pública de Navarra, and BSR from CSIC and European Social Fund (Programa JAE-Doc) are also gratefully acknowledged.

Brucella melitensis Rev 1 is the best vaccine available for the prophylaxis of small ruminant brucellosis and, indirectly, for reducing human brucellosis. However, Rev 1 shows anomalously high rates of spontaneous dissociation from smooth (S) to rough (R) bacteria, the latter being inefficacious as vaccines. This S-R instability results from the loss of the O-polysaccharide. To overcome this problem, we investigated whether some recently described mechanisms promoting mutations in O-polysaccharide genes were involved in Rev 1 S-R dissociation. We found that a proportion of Rev 1 R mutants result from genome rearrangements affecting the wbo O-polysaccharide loci of genomic island GI-2 and the wbkA O-polysaccharide glycosyltransferase gene of the wbk region. Accordingly, we mutated the GI-2 int gene and the wbk IS transposase involved in those arrangements, and found that these Rev 1 mutants maintained the S phenotype and showed lower dissociation levels. Combining these two mutations resulted in a strain (Rev 2) displaying a 95% decrease in dissociation with respect to parental Rev 1 under conditions promoting dissociation. Rev 2 did not differ from Rev 1 in the characteristics used in Rev 1 typing (growth rate, colonial size, reactivity with O-polysaccharide antibodies, phage, dye and antibiotic susceptibility). Moreover, Rev 2 and Rev 1 showed similar attenuation and afforded similar protection in the mouse model of brucellosis vaccines. We conclude that mutations targeting genes and DNA sequences involved in spontaneous O-polysaccharide loss enhance the stability of a critical vaccine phenotype and complement the empirical stabilization precautions taken during S Brucella vaccine production. © 2013 Mancilla et al.; licensee BioMed Central Ltd., This work was funded by MINECO (reference project AGL2011-30453-C04) of Spain, the FIMA foundation and the European Union’s FP7/2007-2013 (grant agreement n° 221948, ICONZ - Integrated control of Neglected Zoonoses) and CSIC JAE-Doc program (FSE)., We also acknowledge institutional support from the Unit of Information Resources for Research at the “Consejo Superior de Investigaciones Científicas” (CSIC) for the article-processing charges contribution.

The brucellae are Gram-negative pathogens that cause brucellosis, a zoonosis of worldwide importance. The genus Brucella includes smooth and rough species that differ in that they carry smooth and rough lipopolysaccharides, respectively. Brucella abortus , B. melitensis , and B. suis are typical smooth species. However, these smooth brucellae dissociate into rough mutants devoid of the lipopolysaccharide O-polysaccharide, a major antigen and a virulence determinant encoded in regions wbo (included in genomic island-2) and wbk . We demonstrate here the occurrence of spontaneous recombination events in those three Brucella species leading to the deletion of a 5.5-kb fragment carrying the wbkA glycosyltranferase gene and to the appearance of rough mutants. Analysis of the recombination intermediates suggested homologous recombination between the IS Bm1 insertion sequences flanking wbkA as the mechanism generating the deletion. Excision of wbkA was reduced but not abrogated in a recA -deficient mutant, showing the existence of both RecA-dependent and -independent processes. Although the involvement of the IS Bm1 copies flanking wbkA suggested a transpositional event, the predicted transpositional joint could not be detected. This absence of detectable transposition was consistent with the presence of polymorphism in the inverted repeats of one of the IS Bm1 copies. The spontaneous excision of wbkA represents a novel dissociation mechanism of smooth brucellae that adds to the previously described excision of genomic island-2. This IS Bm1 -mediated wbkA excision and the different %GC levels of the excised fragment and of other wbk genes suggest that the Brucella wbk locus is the result of at least two horizontal acquisition events.

Background Brucellosis is a zoonosis caused by Brucella spp., a group of highly homogeneous bacteria. The insertion sequence IS711 is characteristic of these bacteria, and occurs in variable numbers and positions, but always constant within a given species. This species-associated polymorphism is used in molecular typing and identification. Field isolates of B. abortus, the most common species infecting cattle, typically carry seven IS711 copies (one truncated). Thus far, IS711 transposition has only been shown in vitro and only for B. ovis and B. pinnipedialis, two species carrying a high number of IS711 copies, but never in other Brucella species, neither in vitro nor in field strains. Results We found several B. abortus strains isolated from milk and aborted fetuses that carried additional IS711 copies in two hitherto undescribed insertion sites: one in an intergenic region near to the 3' end of a putative lactate permease gene and the other interrupting the sequence of a marR transcriptional regulator gene. Interestingly, the second type of insertion was identified in isolates obtained repeatedly from the same herd after successive brucellosis outbreaks, an observation that proves the stability and virulence of the new genotype under natural conditions. Sequence analyses revealed that the new copies probably resulted from the transposition of a single IS711 copy common to all Brucella species sequenced so far. Conclusions Our results show that the replicative transposition of IS711 can occur under field conditions. Therefore, it represents an active mechanism for the emergence of genetic diversity in B. abortus thus contributing to intra-species genetic polymorphism.

Brucellais a Gram-negative bacterium that causes a worldwide-distributed zoonosis. The genus includes smooth (S) and rough (R) species that differ in the presence or absence, respectively, of the O-polysaccharide of lipopolysaccharide. In S brucellae, the O-polysaccharide is a critical diagnostic antigen and a virulence determinant. However, S brucellae spontaneously dissociate into R forms, a problem in antigen and S vaccine production. Spontaneous R mutants ofBrucella abortus,Brucella melitensis, andBrucella suiscarried the chromosomal scar corresponding to genomic island 2 (GI-2) excision, an event causing the loss of thewboAandwboBO-polysaccharide genes, and the predicted excised circular intermediate was identified inB. abortus,B. melitensis, andB. suiscultures. Moreover, disruption of a putative phage integrase gene inB. abortusGI-2 caused a reduction in O-polysaccharide loss rates under conditions promoting S-R dissociation. However, spontaneous R mutants not carrying the GI-2 scar were also detected. These results demonstrate that the phage integrase-related GI-2 excision is a cause of S-R brucella dissociation and that other undescribed mechanisms must also be involved. In the RBrucellaspecies, previous works have shown thatBrucella ovisbut notBrucella canislacks GI-2, and a chromosomal scar identical to those in R mutants was observed. These results suggest that the phage integrase-promoted GI-2 excision played a role inB. ovisspeciation and are consistent with other evidence, suggesting that this species andB. canishave emerged as two independent lineages.

This is an open-access article distributed under the terms of the Creative Commons Attribution License., [Background]: The two-component BvrR/BvrS system is essential for Brucella abortus virulence. It was shown previously that its dysfunction alters the expression of some major outer membrane proteins and the pattern of lipid A acylation. To determine the genes regulated by BvrR/BvrS, we performed a whole-genome microarray analysis using B. abortus RNA obtained from wild type and bvrR mutant cells grown in the same conditions. [Methodology/Principal Findings]: A total of 127 differentially expressed genes were found: 83 were over expressed and 44 were less expressed in the bvrR mutant. Two operons, the phosphotransferase system and the maltose transport system, were down-regulated. Several genes involved in cell envelope or outer membrane biogenesis were differentially expressed: genes for outer membrane proteins (omp25a, omp25d), lipoproteins, LPS and fatty acid biosynthesis, stress response proteins, chaperones, flagellar genes, and twelve genes encoding ABC transport systems. Ten genes related with carbon metabolism (pckA and fumB among others) were up-regulated in the bvrR mutant, and denitrification genes (nirK, norC and nosZ) were also regulated. Notably, seven transcriptional regulators were affected, including VjbR, ExoR and OmpR that were less expressed in the bvrR mutant. Finally, the expression of eleven genes which have been previously related with Brucella virulence was also altered. [Conclusions/Significance]: All these data corroborate the impact of BvrR/BvrS on cell envelope modulation, confirm that this system controls the carbon and nitrogen metabolism, and suggest a cross-talk among some regulators to adjust the Brucella physiology to the shift expected to occur during the transit from the extracellular to the intracellular niche., This work was supported by the Ministerio de Ciencia y Tecnolog­ia of Spain (AGL2008-04514 to ILG, and BIO2007-63656 to FJS) and Instituto de Salud Carlos III (PI050894 to JMG-L). Fellowship support to CV for the Gobierno Vasco and to MCR for Fundacion Marques de Valdecilla-IFIMAV are gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The genus Brucella contains bacteria producing a zoonosis of large sanitary and economical impact. The complete nucleotide sequence of eight Brucella isolates is currently available. This information can be used for high throughput approaches to the biology of this genus such as the construction of comprehensive collections of ORF clones or ORFeomes. The ORFeome of Brucella melitensis was a first contribution to this goal. Using the Brucella ORFeome as starting material we have amplified each ORF and printed them in duplicate onto coated glass slides along with the appropriate positive and negative controls. Quality control of the microarray was performed by image analysis after ethidium bromide staining. This Brucella DNA microarray was used to determine the global transcriptional profile of Brucella abortus grown under laboratory conditions. Two sets of genes representing strongly and poorly expressed genes have been defined. The occurrence of several genes of the same operon in the same data set has been taken as additional proof of the significance of the results. The two sets have been validated by RT-PCR of retrotranscribed RNA. Among the more abundant transcripts we found ribosomal proteins, Krebs cycle and oxidative phosphorylation enzymes. virB, flagellar components and other genes related with virulence and intracellular growth were in the poorly transcribed set. This report demonstrated the usefulness of the ORFeome for the construction of a PCR product microarray for the analysis of global gene expression in Brucella and also applicable to other microorganisms. The results provided here represent a comprehensive description of the global transcriptional profile of B. abortus grown under laboratory conditions and, at the same time, validate the use of this Brucella microarray for the study of the biology and pathogenesis of Brucella through the analysis of gene expression under any experimental conditions.

Background: The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines. Methodology/Principal Findings: To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies. Conclusions/Significance: The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are suitable for the control of brucellosis in endemic areas. This work was funded by the European Commission (Research Contract QLK2-CT-2002-00918) and the Ministerio de Ciencia y Tecnología of Spain (Proyecto AGL2004-01162/GAN).

An evaluation of a multiplex PCR assay (Bruce-ladder) was performed in seven laboratories using 625 Brucella strains from different animal and geographical origins. This robust test can differentiate in a single step all of the classical Brucella species, including those found in marine mammals and the S19, RB51, and Rev.1 vaccine strains.

The Brucella abortus two-component regulatory system BvrR/BvrS controls the expression of outer membrane proteins (Omp) Omp3a (Omp25) and Omp3b (Omp22). Disruption of bvrS or bvrR generates avirulent mutants with altered cell permeability, higher sensitivity to microbicidal peptides, and complement. Consequently, the role of Omp3a and Omp3b in virulence was examined. Similar to bvrS or bvrR mutants, omp3a and omp3b mutants displayed increased attachment to cells, indicating surface alterations. However, they showed unaltered permeability; normal expression of Omp10, Omp16, Omp19, Omp2b, and Omp1; native hapten polysaccharide; and lipopolysaccharide and were resistant to complement and polymyxin B at ranges similar to those of the wild-type (WT) counterpart. Likewise, omp3a and omp3b mutants were able to replicate in murine macrophages and in HeLa cells, were resistant to the killing action of human neutrophils, and persisted in mice, like the WT strain. Murine macrophages infected with the omp3a mutant generated slightly higher levels of tumor necrosis factor alpha than the WT, whereas the bvrS mutant induced lower levels of this cytokine. Since the absence of Omp3a or Omp3b does not result in attenuation, it can be concluded that BvrR/BvrS influences additional Brucella properties involved in virulence. Our results are discussed in the light of previous works suggesting that disruption of omp3a generates attenuated Brucella strains, and we speculate on the role of group 3 Omps. El sistema regulador de dos componentes Brucella abortus BvrR/BvrS controla la expresión de las proteínas de la membrana externa (Omp) Omp3a (Omp25) y Omp3b (Omp22). La interrupción de la bvrS o bvrR genera mutantes avirulentos con permeabilidad celular alterada, mayor sensibilidad a los péptidos microbicidas y al complemento. Por consiguiente, se examinó el papel de Omp3a y Omp3b en la virulencia. De manera similar a los mutantes bvrS o bvrR, los mutantes omp3a y omp3b mostraron un mayor apego a las células, lo que indica alteraciones en la superficie. Sin embargo, mostraron permeabilidad inalterada; expresión normal de Omp10, Omp16, Omp19, Omp2b y Omp1; polisacárido hapteno nativo; y lipopolisacárido y fueron resistentes al complemento y a la polimixina B en rangos similares a los de su homólogo de tipo silvestre (WT). Asimismo, los mutantes omp3a y omp3b pudieron replicarse en macrófagos murinos y en células HeLa, fueron resistentes a la acción asesina de los neutrófilos humanos y persistieron en ratones, como la cepa WT. Murine Los macrófagos infectados con el mutante omp3a generaron niveles ligeramente más altos del factor de necrosis tumoral alfa que el TW, mientras que el mutante bvrS indujo niveles más bajos de esta citoquina. Dado que la ausencia de Omp3a u Omp3b no produce atenuación, se puede concluir que el BvrR/BvrS influye en las propiedades adicionales de Brucella implicadas en la virulencia. Nuestros resultados se discuten a la luz de trabajos anteriores que sugieren que la interrupción de omp3a genera cepas de Brucella atenuadas, y especulamos sobre el papel del grupo 3 Omps. Universidad Nacional, Costa Rica Escuela de Medicina Veterinaria

Thirty-seven Brucella reference and field strains representing all the species and their biovars were analysed by PCR–RFLP to determine the degree of variation in the genes encoding the new members of group 3 outer membrane protein (Omp) family. Analysis of the omp22 and omp25c/omp25d genes indicated that the restriction patterns were identical for all species and biovars with all restriction enzymes tested, except for Brucella ovis that showed a short 30 bp deletion close to omp22 gene, and for B. abortus biovar 6 and B. ovis that lacked a DdeI site and a HinfI site, respectively, in the omp25c/omp25d genes. Analysis of PCR products of the omp31b gene digested with 20 restriction enzymes revealed that this gene has a greater level of DNA polymorphism than the other genes encoding the new members of group 3 Omp family. A deletion of 232 bp was detected in fourteen B melitensis strains from different hosts and from different geographic origins, confirming that this feature is indeed a hallmark of B. melitensis. PCR–RFLP analysis of omp31b with DdeI allowed us to identify species-specific markers for B. abortus, B. melitensis, and B. ovis. Finally, by PCR analysis, Southern blot hybridization and DNA sequencing we showed that a large deletion of 15 kb, comprising the entire omp25b gene and 21 more genes, is present in all B. ovis strains, thus confirming previous observations from other authors.

International audience; - Brucellosis control and eradication requires serological tests and vaccines. Effective classical vaccines (S19 in cattle and Rev 1 in small ruminants), however, induce antibodies to the O-polysaccharide of the lipopolysaccharide which may be difficult to distinguish from those resulting from infection and may thus complicate diagnosis. Rough attenuated mutants lack the O-polysaccharide and would solve this problem if eliciting protective immunity; the empirically obtained rough mutants 45/20 and RB51 have been used as vaccines. Strain 45/20 is reportedly unstable and it is not presently used. RB51 is increasingly used instead of S19 in some countries but it is rifampicin resistant and its effectiveness is controversial. Some controlled experiments have found good or absolute protection in adult cattle vaccinated orally (full dose) or subcutaneously (reduced dose) and in one field experiment, RB51 was reported to afford absolute protection to calves and to perform better than S19. Controlled experiments in calves, however, have shown reduced doses of RB51 to be ineffective, full doses only partially effective, and RB51 less effective than S19 against severe challenges. Moreover, other observations suggest that RB51 is ineffective when prevalence is high. RB51 is not useful in sheep and evidence in goats is preliminary and contradictory. Rough mutants obtained by molecular biology methods on the knowledge of the genetics and structure of Brucella lipopolysaccharide may offer alternatives. The B. abortus manBcore (rfbK) mutant seems promising in cattle, and analyses in mice suggest that mutations affecting only the O-polysaccharide result in better vaccines than those affecting both core and O-polysaccharide. Possible uses of rough vaccines also include boosting immunity by revaccination but solid evidence on its effectiveness, safety and practicality is not available.

The sensitivity and specificity of a PCR assay with primers derived from the insertion sequence IS6501 was compared with that of bacteriological culture and serological tests for the diagnosis of Brucella ovis infection in rams. No amplifications were detected with DNAs from the strains phylogenetically related to Brucella and from the seven bacterial species considered as the main etiologic agents of epididymitis in rams. In addition, the specificity of the PCR was 100% when testing semen samples from Brucella-free rams. The comparison of the semen culture and PCR results from 192 semen samples tested, showed a proportion of agreement of 0.91 between both tests. The PCR-based test described has sensitivity similar to that of semen culture and could be used as a complementary test for the direct diagnosis of Brucella ovis in semen samples of rams.

The Brucella BvrR/BvrS two-component regulatory system is highly similar to the regulatory and sensory proteins of Sinorhizobium and Agrobacterium necessary for endosymbiosis and pathogenicity in plants, and very similar to a putative system present in the animal pathogen Bartonella. Mutations in the bvrR or bvrS genes hamper the penetration of B. abortus in non-phagocytic cells and impairs intracellular trafficking and virulence. In contrast to virulent Brucella, BvrR/BvrS mutants do not recruit small GTPases of the Rho subfamily required for actin polymerization and penetration to cells. Dysfunction of the BvrR/BvrS system alters the outer membrane permeability, the expression of several group 3 outer membrane proteins and the pattern of lipid A acylation. Constructs of virulent B. abortus chimeras containing heterologous LPS from the bvrS− mutant demonstrated an altered permeability to cationic peptides similar to that of the BvrR/BvrS mutants. We hypothesize that the Brucella BvrR/BvrS is a system devoted to the homeostasis of the outer membrane and, therefore in the interface for cell invasion and mounting the required structures for intracellular parasitism. l sistema regulador de dos componentes Brucella BvrR / BvrS es muy similar a las proteínas reguladoras y sensoriales de Sinorhizobium y Agrobacterium necesarias para la endosimbiosis y patogenicidad en plantas, y muy similar a un sistema putativo presente en el patógeno animal Bartonella . Las mutaciones en los genes bvrR o bvrS dificultan la penetración de B. abortus en células no fagocíticas y altera el tráfico intracelular y la virulencia. En contraste con la virulenta Brucella, Los mutantes BvrR / BvrS no reclutan pequeñas GTPasas de la subfamilia Rho requeridas para la polimerización de actina y la penetración en las células. La disfunción del sistema BvrR / BvrS altera la permeabilidad de la membrana externa, la expresión de varias proteínas de la membrana externa del grupo 3 y el patrón de acilación del lípido A. Las construcciones de virulenta B. abortus quimeras que contienen LPS heterólogos a partir de los BVRS - mutantes demostraron una permeabilidad alterada a péptidos catiónicos similares a la de los / BVRS mutantes BvrR. Nuestra hipótesis es que Brucella BvrR / BvrS es un sistema dedicado a la homeostasis de la membrana externa y, por lo tanto, en la interfaz para la invasión celular y el montaje de las estructuras necesarias para el parasitismo intracelular. Universidad Nacional, Costa Rica Escuela de Medicina Veterinaria

The Brucella BvrR/BvrS two-component regulatory system is homologous to the ChvI/ChvG systems of Sinorhizobium meliloti and Agrobacterium tumefaciens necessary for endosymbiosis and pathogenicity in plants. BvrR/BvrS controls cell invasion and intracellular survival. Probing the surface of bvrR and bvrS transposon mutants with monoclonal antibodies showed all described major outer membrane proteins (Omps) but Omp25, a protein known to be involved in Brucella virulence. Absence of Omp25 expression was confirmed by two-dimensional electrophoresis of envelope fractions and by gene reporter studies. The electrophoretic analysis also revealed reduction or absence in the mutants of a second set of protein spots that by matrix-assisted laser desorption ionization MS and peptide mass mapping were identified as a non-previously described Omp (Omp3b). Because bvrR and bvrS mutants are also altered in cell-surface hydrophobicity, permeability, and sensitivity to surface-targeted bactericidal peptides, it is proposed that BvrR BvrS controls cell envelope changes necessary to transit between extracellular and intracellular environments. A genomic search revealed that Omp25 (Omp3a) and Omp3b belong to a family of Omps of plant and animal cellassociated -Proteobacteria, which includes Rhizobium leguminosarum RopB and A. tumefaciens AopB. Previous work has shown that RopB is not expressed in bacteroids, that AopB is involved in tumorigenesis, and that dysfunction of A. tumefaciens ChvI/ChvG alters surface properties. It is thus proposed that the BvrR/BvrS and Omp3 homologues of the cell-associated -Proteobacteria play a role in bacterial surface control and host cell interactions. El sistema regulador de dos componentes Brucella BvrR / BvrS es homólogo a los sistemas ChvI / ChvG de Sinorhizobium meliloti y Agrobacterium tumefaciens necesarios para la endosimbiosis y patogenicidad en plantas. BvrR / BvrS controla la invasión celular y la supervivencia intracelular. El sondeo de la superficie de los mutantes de transposón bvrR y bvrS con anticuerpos monoclonales que muestran todas las proteínas de la membrana externa (Omps) descritas, excepto Omp25, una proteína que se sabe que está involucrada en Brucellavirulencia. La ausencia de expresión de Omp25 se confirma mediante electroforesis bidimensional de fracciones de la envoltura y mediante estudios de reporteros de genes. El análisis electroforético también reducción o ausencia en los mutantes de un segundo conjunto de manchas de proteína que mediante EM de ionización por desorción láser asistida por matriz y mapeo de masa de péptidos se identificaron como un Omp no descrito previamente (Omp3b). Porque bvrR y bvrSLos mutantes también se alteran en la hidrofobicidad, permeabilidad y sensibilidad de la superficie celular a los péptidos bactericidas cambios dirigidos a la superficie, se propone que BvrR / BvrS controle los de la envoltura celular necesarios para transitar entre entornos extracelulares e intracelulares. Una búsqueda genómica resultante que Omp25 (Omp3a) y Omp3b pertenecen a una familia de Omps de α- Proteobacterias asociadas a células vegetales y animales, que incluyen Rhizobium leguminosarum RopB y A. tumefaciens AopB. Trabajos anteriores han demostrado que RopB no se expresa en bacteroides, que AopB está involucrado en la tumorigénesis y que la disfunción de A. tumefaciensChvI / ChvG altera las propiedades de la superficie. Por tanto, se propone que los homólogos BvrR / BvrS y Omp3 de las α- proteobacterias asociadas a células desempeñan un papel en el control de la superficie bacteriana y las interacciones de la célula huésped. Escuela de Medicina Veterinaria

Brucella abortus is an intracellular pathogen that replicates within a membrane-bounded compartment. In this study, we have examined the intracellular pathway of the virulent B. abortus strain 2308 (S2308) and the attenuated strain 19 (S19) in HeLa cells. At 10 min after inoculation, both bacterial strains are transiently detected in phagosomes characterized by the presence of early endosomal markers such as the early endosomal antigen 1. At ;1 h postinoculation, bacteria are located within a compartment positive for the lysosome associated membrane proteins (LAMPs) and the endoplasmic reticulum (ER) marker sec61b but negative for the mannose 6-phosphate receptors and cathepsin D. Interestingly, this compartment is also positive for the autophagosomal marker monodansylcadaverin, suggesting that S2308 and S19 are located in autophagic vacuoles. At 24 h after inoculation, attenuated S19 is degraded in lysosomes, while virulent S2308 multiplies within a LAMP- and cathepsin D-negative but sec61b- and protein disulfide isomerase-positive compartment. Furthermore, treatment of infected cells with the pore-forming toxin aerolysin from Aeromonas hydrophila causes vacuolation of the bacterial replication compartment. These results are compatible with the hypothesis that pathogenic B. abortus exploits the autophagic machinery of HeLa cells to establish an intracellular niche favorable for its replication within the ER Brucella abortus es un patógeno intracelular que se replica dentro de un compartimento delimitado por una membrana. En este estudio, hemos examinado la vía intracelular de la cepa virulenta de B. abortus 2308 (S2308) y la cepa atenuada 19 (S19) en células HeLa. A los 10 minutos de la inoculación, ambas cepas bacterianas se detectan transitoriamente en fagosomas caracterizados por la presencia de marcadores endosomales tempranos como el antígeno endosomal temprano 1. A ∼1 h después de la inoculación, las bacterias se localizan dentro de un compartimento positivo para las proteínas de membrana asociadas al lisosoma (LAMP) y el marcador sec61β del retículo endoplásmico (RE), pero negativo para los receptores de manosa 6-fosfato y la catepsina D. Curiosamente, este compartimento también es positivo para el marcador autofagosomal monodansilcadaverina, lo que sugiere que S2308 y S19 se localizan en vacuolas autofágicas. A las 24 horas de la inoculación, el S19 atenuado se degrada en los lisosomas, mientras que el S2308 virulento se multiplica en un compartimento negativo para LAMP y catepsina D, pero positivo para sec61β y proteína disulfuro isomerasa. Además, el tratamiento de las células infectadas con la toxina formadora de poros aerolisina de Aeromonas hydrophila provoca la vacuolización del compartimento de replicación bacteriana. Estos resultados son compatibles con la hipótesis de que el B. abortus patógeno explota la maquinaria autofágica de las células HeLa para establecer un nicho intracelular favorable para su replicación dentro del RE. Universidad Nacional, Costa Rica Escuela de Medicina Veterinaria

Two mutants showing increased sensitivity to polycations and surfactants were obtained by transposon mutagenesis of virulent Brucella abortus 2308 Nal(r). These mutants showed no obvious in vitro growth defects and produced smooth-type lipopolysaccharides. However, they hardly multiplied or persisted in mouse spleens, displayed reduced invasiveness in macrophages and HeLa cells, lost the ability to inhibit lysosome fusion and were unable to replicate intracellularly. Subsequent DNA analyses identified a two-component regulatory system [Brucella virulence related (Bvr)] with a regulatory (BvrR) and sensory (BvrS) protein. Cloning on bvrR in the BvrR-deficient mutant restored the resistance to polycations and, in part, the invasiveness to polycations and, in part, the invasiveness and the ability to multiply intracellularly. BvrR and BvrS were highly similar (87-89% and 70-80% respectively) to the regulatory and sensory proteins of the chromosomally encoded Rhizobium meliloti Chvl-ExoS and Agrobacterium tumefaciens Chvl-ChvG systems previously shown to be critical for endosymbiosis and pathogenicity in plants. Divergence among the three sensory proteins was located mostly within a periplasmic domain probably involved in stimulus sensing. As B. abortus, R. meliloti and A. tumefaciens are phylogenetically related, these observations suggest that these systems have a common ancestor that has evolved to sense stimuli in plant and animal microbial environments. Dos mutantes que muestran una mayor sensibilidad a los policationes y a los tensioactivos se obtuvieron por mutagénesis de transposones de la Brucella abortus 2308 Nal(r) virulenta. Estos mutantes no mostraron defectos evidentes de crecimiento in vitro y produjeron lipopolisacáridos de tipo suave. Sin embargo, apenas se multiplicaron o persistieron en el bazo de los ratones, mostraron una menor capacidad de invasión en macrófagos y células HeLa, perdieron la capacidad de inhibir la fusión de lisosomas y fueron incapaces de replicarse intracelularmente. Los análisis posteriores del ADN identificaron un sistema regulador de dos componentes [Brucella virulence related (Bvr)] con una proteína reguladora (BvrR) y otra sensorial (BvrS). La clonación en bvrR en el mutante deficiente en BvrR restauró la resistencia a los policationes y, en parte, la capacidad de invasión a los policationes y, en parte, la capacidad de invasión y de multiplicación intracelular. BvrR y BvrS eran muy similares (87-89% y 70-80% respectivamente) a las proteínas reguladoras y sensoriales de los sistemas Rhizobium meliloti Chvl-ExoS y Agrobacterium tumefaciens Chvl-ChvG codificados cromosómicamente, que habían demostrado ser críticos para la endosimbiosis y la patogenicidad en las plantas. La divergencia entre las tres proteínas sensoriales se localizó sobre todo en un dominio periplásmico probablemente implicado en la detección de estímulos. Dado que B. abortus, R. meliloti y A. tumefaciens están filogenéticamente relacionados, estas observaciones sugieren que estos sistemas tienen un ancestro común que ha evolucionado para percibir estímulos en entornos microbianos vegetales y animales. Universidad Nacional, Costa Rica Escuela de Medicina Veterinaria

The relatedness of Brucella spp. and Ochrobactrum anthropi was studied by protein profiling, Western blot, immunoelectrophoresis and 16S rRNA analysis. Whole-cell and soluble proteins of brucellae and O. anthropi showed serological cross-reactivities quantitatively and qualitatively more intense than those existing with similar extracts of Agrobacterium spp. Numerical analysis of Western blot profiles of whole-cell extracts showed that O. anthropi LMG 3301 was closer to Brucella spp. than to O. anthropi LMG 3331T, a result not obtained by protein profiling. These differences were not observed by Western blot with soluble fractions, and immunoelectrophoretic analyses suggested that this was due to destruction of conformational epitopes in Western blot procedures with the subsequent simplification of antigenic profile. Analysis of the 16S rRNA sequences of strains previously used in the species definition confirmed that strain LMG 3301, and also LMG 3306, were closer to the brucellae, and that LMG 3331T was in a separate cluster. The LMG 3301 and the LMG 3331T clusters could also be separated by their different colistin sensitivity and by PCR with 16S rRNA Brucella primers, and both methods showed strains of both clusters among clinical isolates classified as O. anthropi by conventional tests. These results and those of previous DNA-DNA hybridization studies [Holmes, B., Popoff, M., Kiredjian, M. & Kersters, K. (1988). Int J Syst Bacteriol 38, 406-416] show that the LMG 3301 cluster and related clinical isolates should be given a new species status for which the name Ochrobactrum intermedium sp. nov. is proposed (type strain is LMG 3301T=NCTC 12171T = CNS 2-75T).

A study was performed to evaluate the previously described PCR (C. Romero, C. Gamazo, M. Pardo, and I. López-Goñi, J. Clin. Microbiol. 33:615-617, 1995) for the diagnosis of brucellosis in dairy cattle. Milk samples from 56 Brucella milk culture-positive cattle and from 37 cattle from Brucella-free herds were examined for Brucella DNA by PCR and for specific antibodies by an indirect enzyme-linked immunosorbent assay (ELISA). The specificities of both tests were 100% when testing the milk samples from Brucella-free cattle. The milk samples from 49 infected cattle were positive by PCR (87.5% sensitivity), and 55 were positive by ELISA (98.2% sensitivity). A PCR-positive sample was negative by ELISA, and 7 ELISA-positive samples were PCR negative, yielding an observed proportion of agreement of 0.91 for the two tests. Although the results suggest that ELISA is a better screening test than PCR, the combined sensitivity of the two assays was 100%, and their simultaneous application could be more useful than one test alone for a rapid screening of brucellosis in dairy cattle.

Brucella melitensis and B. ovis outer membrane blebs contained a protein displaying a temperature-dependent molecular mass upshift from 25 kDa to 30 kDa. A fraction of the protein tightly bound to LPS did not show the molecular mass upshift which was also blocked by exposure of the protein to Zwittergent 314. The B. melitensis heat-modifiable protein and Escherichia coli OmpA shared antigenic determinants. These data indicate that the Brucella group 3 outer membrane proteins belonged to the OmpA family of proteins.