Construction and evaluation of an ORFeome-based Brucella whole-genome DNA microarray.

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.

#EUROmicroMOOC: using Twitter to share trends in Microbiology worldwide.

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.

Evaluation of a multiplex PCR assay (Bruce-ladder) for molecular typing of all Brucella species, including the vaccine strains.

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.

Restriction site polymorphisms in the genes encoding new members of group 3 outer membrane protein family of Brucella spp.

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 232bp 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.

Brucella group 3 outer membrane proteins contain a heat-modifiable protein.

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.

Regulation of Brucella virulence by the two-component system BvrR/BvrS.

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.

Evaluation of a PCR test for the diagnosis of Brucella ovis infection in semen samples from rams.

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.

Structure and properties of the outer membranes of Brucella abortus and Brucella melitensis.

The brucellae are Gram-negative bacteria characteristically able to multiply facultatively within phagocytic cells and which cause a zoonosis of world-wide importance. This article reviews the structure and topology of the main components (lipopolysaccharide, native hapten polysaccharide, free lipids and proteins) of the outer membranes of Brucella abortus and B. melitensis, as well as some distinctive properties (permeability and interactions with cationic peptides) of these membranes. On these data, an outer membrane model is proposed in which, as compared to other Gram-negatives, there is a stronger hydrophobic anchorage for the lipopolysaccharide, free lipids, porin proteins and lipoproteins, and a reduced surface density of anionic groups, which could be partially or totally neutralized by ornithine lipids. This model accounts for the permeability of Brucella to hydrophobic permeants and for its resistance to the bactericidal oxygen-independent systems of phagocytes.

Improved method for purification of bacterial DNA from bovine milk for detection of Brucella spp. by PCR.

Different methods of extraction of bacterial DNA from bovine milk to improve the direct detection of Brucella by PCR were evaluated. We found that the use of a lysis buffer with high concentrations of Tris, EDTA, and NaCl, high concentrations of sodium dodecyl sulfate and proteinase K, and high temperatures of incubation was necessary for the efficient extraction of Brucella DNA. The limit of detection by PCR was 5 to 50 Brucella CFU/ml of milk.

Identification of 2,3-dihydroxybenzoic acid as a Brucella abortus siderophore.

Brucella abortus grown in low-iron medium or in the presence of iron chelators [ethylenediamine-di(o-hydroxyphenylacetic acid) and 2,2-dipyridyl] showed reduced cell yields and released a material positive in chemical and biological assays for catechols. This material was purified from culture fluids of B. abortus 2308 by chromatography on agarose-iminodiacetic acid-Fe3+ and identified as 2,3-dihydroxybenzoic acid (2,3-DHBA) by thin-layer chromatography, paper electrophoresis, and UV-visible nuclear magnetic resonance and mass spectroscopy. No other major catechols were observed at different stages of growth, and 2,3-DHBA was also produced upon iron limitation by representative strains of B. abortus biotypes 1, 5, 6, and 9. Both synthetic 2,3-DHBA and the natural catechol relieved the growth inhibition of B. abortus 2308 by ethylenediamine-di(o-hydroxyphenylacetic acid), and 2,3-DHBA promoted 55Fe uptake by B. abortus 2308 by an energy-dependent mechanism. Two other monocatechols tested, 2,3-dihydroxybenzoyl-Ser and 2,3-dihydroxybenzoyl-Gly, also promoted 55Fe uptake. More complex catechol siderophores (agrobactin and enterobactin), hydroxamate siderophores (aerobactin, ferrichrome, and deferriferrioxamine mesylate [Desferal]), and an EDTA-related siderophore (rhizobactin) failed to mediate 55Fe uptake. B. abortus cells grown in low-iron medium or in medium with iron had similar rates of iron uptake when supplied with 55Fe-2,3-DHBA, and the release of 2,3-DHBA under iron starvation was not associated with the expression of new outer membrane proteins. These results suggest an uptake system in which only the synthesis of the siderophore is regulated by the iron available for growth.

Brucella abortus siderophore 2,3-dihydroxybenzoic acid protects brucellae from killing by macrophages.

Addition of 2,3-dihydroxybenzoic acid, a siderophore produced by Brucella abortus, to macrophage cultures prevented intracellular killing of brucellae during the first 12 h after infection and increased the number of intracellular brucellae recovered at 48 h after infection. The protective effect could be demonstrated with inflammatory macrophages, interferon-gamma-activated macrophages and with macrophages supplemented with iron, shown elsewhere to facilitate killing of B abortus.

[Confirmation by molecular typing of cross-contamination in a mycobacteria laboratory]. / Confirmación mediante tipificación molecular de contaminaciones cruzadas en el laboratorio de micobacterias.

BACKGROUND: Detection of cross-contamination in the laboratory by restriction fragments length polymorphism (RFLP) analysis of Mycobacterium tuberculosis strains. MATERIAL AND METHODS: 224 strains isolated during a five years period were characterized by IS6110 fingerprinting performed (RFLP) by standardized protocols. RESULTS: Four groups of isolates with smear-negative specimens and low number of colony form its units were detected. They were processed in the same batch and day than other smear-positive specimens with identical RFLP patterns. Fifteen patients were involved and the review of four patients' charts showed that they did not have the typical manifestations of tuberculosis. CONCLUSIONS: When M. tuberculosis isolates were obtained from smear-negative specimens, the results of specimens processed in the same batch and the patients' charts should be reviewed. If with these data the possibility of cross-contamination is suspected, the isolates must be analyzed by molecular typing methods.

Specific detection of Brucella DNA by PCR.

A PCR assay with primers derived from the 16S rRNA sequence of Brucella abortus was developed. Nine different combinations between six primers were tested. One pair of primers, which amplified a 905-bp fragment, was selected. As little as 80 fg of Brucella DNA was detected by this method. DNAs from all of the representative strains of the species and biovars of Brucella and from 23 different Brucella isolates were analyzed and yielded exclusively the 905-bp fragment. No amplification was detected with DNAs from 10 strains phylogenetically related to Brucella spp., 5 gram-negative bacteria showing serological cross-reactions with Brucella spp., and 36 different clinical isolates of non-Brucella species. Only Ochrobactrum anthropi biotype D yielded a PCR product of 905 bp, suggesting a closer relationship between Brucella spp. and O. anthropi biotype D. The specificity and high sensitivity of the PCR assay may provide a valuable tool for the diagnosis of brucellosis.

Evaluation of the relatedness of Brucella spp. and Ochrobactrum anthropi and description of Ochrobactrum intermedium sp. nov., a new species with a closer relationship to Brucella spp.

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).

Factors affecting detection of Brucella melitensis by BACTEC NR730, a nonradiometric system for hemocultures.

The detection of Brucella bacteremia by subculture does not always correlate with a positive signal in the BACTEC NR730 nonradiometric system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.). The effect of the inoculum size, pH, sodium polyanetholesulfonate, carbon sources (i-erythritol, sodium pyruvate, monosodium glutamate, D-glucose, and L-alanine), and urea in the release of CO2 was evaluated by using the reference strain Brucella melitensis 16M. In standard NR6 vials with or without blood, inocula 5 to 10 times larger (at least 265 CFU per vial) than those usually found in the blood of patients with brucellosis were necessary to produce a positive growth value (GV) in 4 days or less, and similar results were obtained with vials supplemented with the substrates listed above. GVs were consistently lower in vials with sodium polyanetholesulfonate than in vials without this agent. Vials with no blood inoculated with 265 CFU per vial showed turbidity 1 day before GVs became positive, proving that the major limiting detection factor was the low level of release of CO2 and not an inadequate growth medium. In NR6 vials buffered to pH 6.2, GVs became positive faster and were higher than those in standard vials. NR6 vials at pH 6.2 with 0.3% sodium pyruvate yielded a positive GV in the first day of bacterial turbidity.

Comparative activity of azithromycin and doxycycline against Brucella spp. infection in mice.

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.

Evaluation of PCR and indirect enzyme-linked immunosorbent assay on milk samples for diagnosis of brucellosis in dairy cattle.

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.

The two-component system BvrR/BvrS essential for Brucella abortus virulence regulates the expression of outer membrane proteins with counterparts in members of the Rhizobiaceae.

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 cell-associated alpha-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 alpha-Proteobacteria play a role in bacterial surface control and host cell interactions.

GTPases of the Rho subfamily are required for Brucella abortus internalization in nonprofessional phagocytes: direct activation of Cdc42.

Members of the genus Brucella are intracellular alpha-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.