#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.

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.

Characterization and expression of secA in Mycobacterium avium.

Mycobacterium avium is both a pathogen that infects several hosts such as humans, pigs, and birds, as well as a microorganism that is encountered in environmental sources (soil and water). Protein secretion by the bacterium is likely to influence its ability to overcome adverse and competitive conditions both within or outside the host. Using a combination of cloning and information available in the databank, we characterized the secA gene from M. avium, encoding for a major preprotein translocase subunit associated with the secretion system of prokaryotics. In addition, we cloned the secA promoter sequence in a reporter construct upstream of a promoterless gfp. It was determined that the secA of M. avium shares large homology with the secA of Mycobacterium tuberculosis but not with secA of Mycobacterium leprae. secA expression was determined to be greater at logarithmic growth phase although it was also expressed at low levels during the stationary phase. secA expression was also observed when the bacteria were incubated in water as well as within human monocyte-derived macrophages and in conditions that are associated with biofilm formation. Future evaluation of the sec pathway in M. avium might provide important information about secreted proteins that are required for survival in different environments.

Mycobacterium avium invades the intestinal mucosa primarily by interacting with enterocytes.

Previous studies have demonstrated that Mycobacterium avium can invade intestinal epithelial cells both in vitro and in vivo. When given to mice orally, M. avium preferentially interacts with the intestinal mucosa at the terminal ileum. We evaluated the mechanism(s) of M. avium binding and invasion of the intestinal mucosa using three different systems: (i) electron microscopy following administration of M. avium into an intestinal loop in mice, (ii) quantitative comparison of the bacterial load in Peyer's patch areas of the terminal ileum versus areas that do not contain Peyer's patches, and (iii) investigation of the ability of M. avium to cause disseminated infection following oral administration using B-cell-deficient mice, lacking Peyer's patches, in comparison with C57BL/6 black mice. By all approaches, M. avium was found to invade the intestinal mucosa by interacting primarily with enterocytes and not with M cells.

Cellular and molecular mechanisms of internalization of mycobacteria by host cells.

Mycobacteria are intracellular pathogens capable of invading mononuclear phagocytes, mucosal epithelial cells (including M cells) and Schwann cells. To enter cells, mycobacteria have been shown to interact with several molecules on macrophage and epithelial cell surfaces. This suggests adaptation to the host environment. In this review we address the strategies used by pathogenic mycobacteria to gain access to the intracellular environment.

Mycobacterium avium enters intestinal epithelial cells through the apical membrane, but not by the basolateral surface, activates small GTPase Rho and, once within epithelial cells, expresses an invasive phenotype.

Mycobacterium avium is a common pathogen in AIDS patients that is primarily (but not exclusively) acquired through the gastrointestinal tract, leading to the development of bacteraemia and disseminated disease. To cause infection through the gut, binding and invasion of the intestinal epithelial barrier are required. To characterize this process further, we determined the cell surface(s) (basolateral vs. apical membrane) that M. avium interacts with in intestinal mucosal cells in vitro. The level of binding and invasion of both HT-29 and Caco-2 intestinal cell monolayers by M. avium were similar when the assay was performed with control medium in the presence of Ca2+ (when only the apical surface was exposed), with Ca2+-depleted medium or with Ca2+-depleted medium + 1 mM EGTA (exposure of both apical and basolateral membranes), suggesting that the bacterium enters the apical surface of the epithelial lining. These observations were confirmed by assays in a transwell system and by using fluorescent microscopy. Real-time video microscopy showed that M. avium entry was not associated with membrane ruffling and the use of pharmacological inhibitors of the small GTPases demonstrated that M. avium invasion is dependent on the activation of the small GTPases Rho, but not on Rac or Cdc42. Passage of M. avium through HT-29 cells led to a phenotypic change (intracellular growth; IG) that was associated with a significantly greater (between five- and ninefold) ability to bind to and invade new monolayers of epithelial cells or macrophages when compared with the invasion by M. avium grown on agar (extracellular growth; EG). IG phenotype invasion of HT-29 cells also takes place only by the apical surface. M. avium enters intestinal epithelial cells by the apical surface and, once within the cells, changes phenotype, becoming more invasive towards both macrophages and other epithelial cells.

Characterization of IS666, a newly described insertion element of Mycobacterium avium.

The insertion sequence IS666 was isolated from Mycobacterium avium strain 101. IS666 is a 1474 bp insertion sequence belonging to the IS256 family, that includes IS6120 from Mycobacterium smegmatis, IS1166 and IS1295 from Rhodococcus sp. IGTS8, IST2 from Thiobacillus ferrooxidans, IS256 from Staphylococcus aureus, and ISRm3 from Rhizobium meliloti. IS666 has 24 bp imperfect inverted repeats that fit the consensus described for the family, and generates 9 bp duplications upon insertion into the host DNA with no apparent specificity in the target sequence. In contrast with its two closest homologues, IS1166 and IS6120, IS666 contains a single ORF that would codify a transposase of 434 aa. IS666 is restricted to M. avium, where it is present in 21% of the isolates in a number ranging between 1 to 7 copies.

Mycobacterium avium infection of epithelial cells results in inhibition or delay in the release of interleukin-8 and RANTES.

Mycobacterium avium is an opportunistic pathogen in AIDS patients, who acquire the infection mainly through the gastrointestinal tract. Previous studies in vitro have shown that M. avium invades epithelial cells of both intestinal and laryngeal origin. In addition, M. avium enters the intestinal mucosa of healthy mice. Because M. avium invasion of the intestinal mucosa in vivo initially is not accompanied by significant influx of inflammatory cells, we sought to determine whether M. avium would trigger chemokine release upon entry into epithelial cells by using HT-29 intestinal and HEp-2 laryngeal epithelial cell lines. Chemokine synthesis was measured both by the presence of specific mRNA and protein secretion in the cell culture supernatant as determined by enzyme-linked immunosorbent assay. Infection of HT-29 intestinal cells with M. avium did not induce the release of interleukin-8 (IL-8) or RANTES for up to 7 days postinfection. However, infection of HEp-2 cells resulted in the release of IL-8 and RANTES at 72 h. Similar findings were observed with other AIDS M. avium isolates belonging to different serovars. Secretion of IL-8 by HEp-2 cells was dependent upon bacterial uptake. In addition, prior infection with M. avium suppressed IL-8 production by HT-29 cells infected with Salmonella typhimurium. Our results suggest that M. avium infection of epithelial cells is associated with a delay in IL-8 and RANTES production which, in the case of HT-29, is prolonged up to 1 week. These findings may explain the weak inflammatory response after intestinal mucosa invasion in mice and are probably related with the ability of the bacterium to evade the host's immune response.

Mycobacterium avium interaction with macrophages and intestinal epithelial cells.

Mycobacterium avium is an environmental microorganism that is adapted to live both in the environment (mainly in water and soil) and in bird, fish and mammal hosts. In humans, M. avium infection is seen in patients with some sort of immunosuppression, such as patients with chronic lung disease, and Acquired Immunodeficiency Syndrome. More recently, other populations were shown to be at risk to develop M. avium disease. For the majority of time, humans acquire M. avium through the intestinal tract where the bacterium comes in contact with and translocates the intestinal mucosa. M. avium possesses a unique manner to interact with the intestinal mucosa, and, following invasion, can enter and survive within macrophages and monocytes. Although in vitro entry seems to be dependent on binding to the complement receptor, this finding has not been observed in vivo where the bacterium appears to enter macrophages by alternative mechanisms. The bacterium appears to trigger little inflammatory response, and is able to adapt itself to different environments in the host.

The defect in the metabolism of erythritol of the Brucella abortus B19 vaccine strain is unrelated with its attenuated virulence in mice.

The role of the defect in erythritol catabolism in the attenuated virulence of Brucella abortus B19 vaccine strain in mice was investigated by means of five different strains: (i) the erythritol sensitive B19 vaccine strain; (ii) a natural erythritol tolerant (NET) mutant obtained spontaneously from B19; (iii) an erythritol resistant derivative from B19 (FJS19) obtained by gene replacement of the deleted ery region; (iv) the erythritol resistant B. abortus 2308 reference virulent strain; and (v) an erythritol sensitive mutant (227 strain) obtained from strain 2308 by transposon insertion in the chromosomal ery region. Besides virulence for mice, erythritol oxidation as well as other phenotypic markers were tested in all the strains. The 2308 and FJS19 strains grew in the presence of erythritol and oxidized the sugar, whereas the B19 and 227 strains did not. The NET strain grew in presence of erythritol but was unable to oxidize it. The B19 vaccine strain and its two erythritol resistant derivatives, NET and FJS19, showed similar residual virulence and splenic time courses in mice. Moreover, the virulent strain 2308 and its erythritol sensitive derivative (227 strain) exhibited similar levels of splenic infection. Altogether, these results demonstrate that the genetic region implicated in erythritol catabolism is not related to the low virulence exhibited by B19 in mice.

Molecular basis of Brucella pathogenicity: an update.

Microorganisms belonging to the genus Brucella can infect humans and many species of animals. Virulence of the brucellae is thought to be essentially due to their capacity to survive and replicate within the phagocytic cells. However, many gaps remain in our understanding of this ability of brucellae to elude the bactericidal effects of host phagocytes, and basic questions remain unanswered. Identification of Brucella gene products which are related to intracellular survival, as well as those which contribute to the induction of protective immunity, is critical to elucidate the molecular mechanisms of the pathogenesis of the organism. The present article summarizes the current status of the research on gene products and other structural or metabolic factors associated with virulence of the brucellae.

Improvement of the Brucella abortus B19 vaccine by its preparation in a glycerol based medium.

The Brucella abortus B19 vaccine strain differs from other Brucella strains in its sensitivity to erythritol. However, erythritol tolerant (Eri(t)) mutants arise from sensitive cultures of B19 at high rate, and may cause persistence and/or abortion when the vaccine is inoculated on adult cattle. Twelve different batches of B19 have been examined for the presence of Eri(t) mutants. All contained Eri(t) variants at a proportion ranging from 10(-4) to 10(-6). In order to eliminate these mutants from the vaccine cultures, we have developed a minimal medium with glycerol as the sole carbon source, named MMG30. Growth of the parental strain B19 (erythritol sensitive) in this medium was fairly good compared with the growth of its Eri(t) derivatives. Culture of the 12 different batches of B19 in liquid MMG30 produced up to a thousandfold decrease in the proportion of Eri(t) mutants present in the vaccine cultures. Use of this medium to grow B19 could represent an easy and considerable improvement of the vaccine, by the reduction of the presence of potentially dangerous Eri(t) mutants.

The Brucella abortus vaccine strain B19 carries a deletion in the erythritol catabolic genes.

Brucella abortus B19, an avirulent strain obtained by spontaneous mutation, is used worldwide as a vaccine for the control of bovine brucellosis. B19 differs from other B. abortus strains in its sensitivity to erythritol. We took advantage of a previously obtained erythritol sensitive Tn5 insertion mutant of B. abortus 2308 to clone the chromosomal region containing erythritol catabolic genes from this representative pathogenic strain and from the vaccine strain B19. Physical mapping with restriction endonucleases and nucleotide sequence determination revealed the existence of a 702 bp long deletion, occurring between two short direct repeats, in the chromosome of B19. This deletion rendered the B19 strain sensitive to erythritol. Two oligonucleotides whose sequences flank this deletion provided an easy method to differentiate B19 from all other B. abortus isolates.

Identification of Brucella abortus B19 vaccine strain by the detection of DNA polymorphism at the ery locus.

As sensitivity to erythritol is the main feature of the Brucella abortus B19 vaccine strain, an attempt to define the genetic basis of this phenotypic trait was made in order to explore its usefulness as a means of molecular identification of strain B19. With this purpose, an erythritol-sensitive derivative of B. abortus strain 2308 was generated by Tn5 transposition methods. Restriction fragments from a cloned 13 kb Tn5-containing EcoRI fragment from the ery locus of strain 2308 were used to probe DNA from B. abortus B19 and other Brucella reference strains. Genetic rearrangement in strain B19 was revealed by the display of restriction fragment length polymorphisms at the ery locus. No pattern differences were detected among all other Brucella strains tested. The polymorphism exhibited by B19 represents the first characterization of this vaccine strain at a molecular level, and can be used to distinguish it from all other Brucella species.