Draft genome sequence of Mycobacterium brumae ATCC 51384

Here, we report the draft genome sequence of Mycobacterium brumae type strain ATCC 51384. This is the first draft genome sequence of M. brumae, a nonpathogenic, rapidly growing, nonchromogenic mycobacterium, with immunotherapeutic capacities

Mice with Pulmonary Tuberculosis Treated with Mycobacterium vaccae Develop Strikingly Enhanced Recall Gamma Interferon Responses to M. vaccae Cell Wall Skeleton

Whole heat-killed Mycobacterium vaccae is used as an immunotherapeutic agent in tuberculosis (TB), but the compound(s) that triggers its immunostimulatory ability is not known. Here, we show that among different subcellular fractions, the cell wall skeleton induced a prominent expression of gamma interferon in splenocytes from both non-TB and TB M. vaccae-treated mice

Trehalose polyphleates, external cell wall lipids in mycobacterium abscessus, are associated with the formation of clumps with cording morphology, which have been associated with virulence

Mycobacterium abscessus is a reemerging pathogen that causes pulmonary diseases similar to tuberculosis, which is caused by Mycobacterium tuberculosis. When grown in agar medium, M. abscessus strains generate rough (R) or smooth colonies (S). R morphotypes are more virulent than S morphotypes. In searching for the virulence factors responsible for this difference, R morphotypes have been found to form large aggregates (clumps) that, after being phagocytozed, result in macrophage death. Furthermore, the aggregates released to the extracellular space by damaged macrophages grow, forming unphagocytosable structures that resemble cords. In contrast, bacilli of the S morphotype, which do not form aggregates, do not damage macrophages after phagocytosis and do not form cords. Cording has also been related to the virulence of M. tuberculosis. In this species, the presence of mycolic acids and surface-exposed cell wall lipids has been correlated with the formation of cords. The objective of this work was to study the roles of the surface-exposed cell wall lipids and mycolic acids in the formation of cords in M. abscessus. A comparative study of the pattern and structure of mycolic acids was performed on R (cording) and S (non-cording) morphotypes derived from the same parent strains, and no differences were observed between morphotypes. Furthermore, cords formed by R morphotypes were disrupted with petroleum ether (PE), and the extracted lipids were analyzed by thin layer chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry. Substantial amounts of trehalose polyphleates (TPP) were recovered as major lipids from PE extracts, and images obtained by transmission electron microscopy suggested that these lipids are localized to the external surfaces of cords and R bacilli. The structure of M. abscessus TPP was revealed to be similar to those previously described in Mycobacterium smegmatis. Although the exact role of TPP is unknown, our results demonstrated that TPP are not toxic by themselves and have a function in the formation of clumps and cords in M. abscessus, thus playing an important role in the pathogenesis of this species

Desenvolupament de problemes virtuals i interactius per a l'aprenentatge de la microbiologia

El web Microbiologia Interactiva promou l'aprenentatge de la Microbiologia a través de la resolució de problemes teòrics i pràctics. El material s'ha dissenyat per ser utilitzat a les classes presencials i també com a eina d'autoaprenentatge. La innovació més destacada és la integració dels aspectes teòrics i dels pràctics, gràcies al fet que, a més de continguts teòrics, el web inclou un laboratori virtual de Microbiologia on es poden realitzar diverses tècniques bàsiques de Microbiologia obtenint una simulació de resultats. El web va adreçat a estudiants que entren en contacte per primera vegada amb el món invisible dels microorganismes, és a dir a estudiants de primer o segon curs d'una titulació universitària que inclogui aquesta matèria. La implementació d'un prototip d'aquesta nova eina s'efectua de forma pilot durant el curs acadèmic 2007-2008 a les assignatures de Microbiologia (titulació de Biologia) i de Microbiologia I (titulació de Biotecnologia). L'avaluació que s'ha fet en aquest primer semestre mostra una molt bona acceptació per part dels estudiants així com del professorat que l'ha implementat. En aquesta avaluació s'han identificat determinades disfuncions del web, que ja han estat corregides en la versió final, la qual s'ha simplificat i conté diferents recursos d'ajuda. Aquesta eina, que es considera única, servirà de base per al desenvolupament de futurs webs que tractin de l'aprenentatge de la Microbiologia a un nivell més avançat.La web Microbiología Interactiva promueve el aprendizaje de la Microbiología a través de la resolución de problemas teóricos y/o prácticos. El material se ha diseñado para ser utilizado en las clases presenciales y también como herramienta de autoaprendizaje. La innovación más destacada es la integración de los aspectos teóricos y prácticos, gracias a que, además de contenidos teóricos, la web incluye un laboratorio virtual de microbiología donde se pueden realizar diversas técnicas básicas de Microbiología obteniendo una simulación de resultados. La web se dirige a estudiantes que entran en contacto por primera vez con el mundo invisible de los microorganismos, es decir, a estudiantes de primer o segundo curso de una titulación universitaria que incluya esta materia. La implementación de un prototipo de esta nueva herramienta se ha efectuado durante el curso académico 2007-2008 en las asignaturas de Microbiología (titulación de Biología) y de Microbiología I (titulación de Biotecnología). La evaluación que se ha hecho en este primer semestre muestra una muy buena aceptación por parte delos estudiantes, así como del profesorado que lo ha implementado. En esta evaluación se han identificado algunas disfunciones de la web, que ya se han corregido en la versión final, que se ha simplificado y contiene diversos recursos de ayuda. Esta herramienta, que se considera única, servirá de base para el desarrollo de futuras webs que se ocupen del aprendizaje de la Microbiología a un nivel más avanzado.The Interactive Microbiology website promotes the learning of microbiology by resolving theoretical and/or practical problems. The material was designed to be used in the classroom, as well as for independent learning. The most noteworthy innovation is the integration of the theoretical and practical aspects, thanks to the fact that in addition to theoretical contents the website also includes a virtual microbiology laboratory where students can practice a variety of basic microbiology techniques with a simulation of the results. The website is targeted to students who are entering into contact with the invisible world of micro-organisms for the first time, that is, to students in their first or second year of the university degree programme that covers this material. A prototype of this new tool was implemented as a pilot during academic year2007-08 in the Microbiology course (Biology degree programme) and the Microbiology I course (Biotechnology degree programme). The evaluations from this first term show a high degree of acceptance by both the students and the professors who have implemented it. This evaluation also identified certain dysfunctions of the website which have already been corrected in the final version, which was also simplified and contains a variety of help resources. This tool, which is considered unique, will serve as the foundation for the development of future websites dealing with learning microbiology at a more advanced level

Mycobacteria clumping increase their capacity to damage macrophages

The rough morphotypes of non-tuberculous mycobacteria have been associated with the most severe illnesses in humans. This idea is consistent with the fact that Mycobacterium tuberculosis presents a stable rough morphotype. Unlike smooth morphotypes, the bacilli of rough morphotypes grow close together, leaving no spaces among them and forming large aggregates (clumps). Currently, the initial interaction of macrophages with clumps remains unclear. Thus, we infected J774 macrophages with bacterial suspensions of rough morphotypes of M. abscessus containing clumps and suspensions of smooth morphotypes, primarily containing isolated bacilli. Using confocal laser scanning microscopy and electron microscopy, we observed clumps of at least five rough-morphotype bacilli inside the phagocytic vesicles of macrophages at 3 h post-infection. These clumps grew within the phagocytic vesicles, killing 100% of the macrophages at 72 h post-infection, whereas the proliferation of macrophages infected with smooth morphotypes remained unaltered at 96 h post-infection. Thus, macrophages phagocytose large clumps, exceeding the bactericidal capacities of these cells. Furthermore, proinflammatory cytokines and granuloma-like structures were only produced by macrophages infected with rough morphotypes. Thus, the present study provides a foundation for further studies that consider mycobacterial clumps as virulence factors

Prevalence and concentration of non-tuberculous mycobacteria in cooling towers by means of quantitative PCR: a prospective study

Postprint (published version

Mycobacterial surface characters remodeled by growth conditions drive different tumor-infiltrating cells and systemic IFN-gamma/IL-17 release in bladder cancer treatment

The mechanism of action of intravesical Mycobacterium bovis BCG immunotherapy treatment for bladder cancer is not completely known, leading to misinterpretation of BCG-unresponsive patients, who have scarce further therapeutic options. BCG is grown under diverse culture conditions worldwide, which can impact the antitumor effect of BCG strains and could be a key parameter of treatment success. Here, BCG and the nonpathogenic Mycobacterium brumae were grown in four culture media currently used by research laboratories and BCG manufacturers: Sauton-A60, -G15 and -G60 and Middlebrook 7H10, and used as therapies in the orthotopic murine BC model. Our data reveal that each mycobacterium requires specific culture conditions to induce an effective antitumor response. since higher survival rates of tumor-bearing mice were achieved using M. brumae-A60 and BCG-G15 than the rest of the treatments. M. brumae-A60 was the most efficacious among all tested treatments in terms of mouse survival, cytotoxic activity of splenocytes against tumor cells, higher systemic production of IL-17 and IFN-gamma, and bladder infiltration of selected immune cells such as ILCs and CD4(TEM). BCG-G15 triggered an antitumor activity based on a massive infiltration of immune cells, mainly CD3(+) (CD4(+) and CD8(+)) T cells, together with high systemic IFN-gamma release. Finally, a reduced variety of lipids was strikingly observed in the outermost layer of M. brumae-A60 and BCG-G15 compared to the rest of the cultures, suggesting an influence on the antitumor immune response triggered. These findings contribute to understand how mycobacteria create an adequate niche to help the host subvert immunosuppressive tumor actions

Mycolicibacterium brumae is a safe and non-toxic immunomodulatory agent for cancer treatment

Intravesical Mycobacterium bovis Bacillus Calmette-Guérin (BCG) immunotherapy remains the gold-standard treatment for non-muscle-invasive bladder cancer patients, even though half of the patients develop adverse events to this therapy. On exploring BCG-alternative therapies, Mycolicibacterium brumae, a nontuberculous mycobacterium, has shown outstanding anti-tumor and immunomodulatory capabilities. As no infections due to M. brumae in humans, animals, or plants have been described, the safety and/or toxicity of this mycobacterium have not been previously addressed. In the present study, an analysis was made of M. brumae- and BCG-intravenously-infected severe combined immunodeficient (SCID) mice, M. brumae-intravesically-treated BALB/c mice, and intrahemacoelic-infected-Galleria mellonella larvae. Organs from infected mice and the hemolymph from larvae were processed to count bacterial burden. Blood samples from mice were also taken, and a wide range of hematological and biochemical parameters were analyzed. Finally, histopathological alterations in mouse tissues were evaluated. Our results demonstrate the safety and non-toxic profile of M. brumae. Di erences were observed in the biochemical, hematological and histopathological analysis between M. brumae and BCG-infected mice, as well as survival curves rates and colony forming units (CFU) counts in both animal models. M. brumae constitutes a safe therapeutic biological agent, overcoming the safety and toxicity disadvantages presented by BCG in both mice and G. mellonella animal models

Mycobacteria emulsified in olive oil-in-water trigger a robust immune response in bladder cancer treatment

The hydrophobic composition of mycobacterial cell walls leads to the formation of clumps when attempting to resuspend mycobacteria in aqueous solutions. Such aggregation may interfere in the mycobacteria-host cells interaction and, consequently, influence their antitumor effect. To improve the immunotherapeutic activity of Mycobacterium brumae, we designed different emulsions and demonstrated their efficacy. The best formulation was initially selected based on homogeneity and stability. Both olive oil (OO)- and mineral oil-in-water emulsions better preserved the mycobacteria viability and provided higher disaggregation rates compared to the others. But, among both emulsions, the OO emulsion increased the mycobacteria capacity to induce cytokines' production in bladder tumor cell cultures. The OO-mycobacteria emulsion properties: less hydrophobic, lower pH, more neutralized zeta potential, and increased affinity to fibronectin than non-emulsified mycobacteria, indicated favorable conditions for reaching the bladder epithelium in vivo. Finally, intravesical OO-M. brumae-treated mice showed a significantly higher systemic immune response, together with a trend toward increased tumor-bearing mouse survival rates compared to the rest of the treated mice. The physicochemical characteristics and the induction of a robust immune response in vitro and in vivo highlight the potential of the OO emulsion as a good delivery vehicle for the mycobacterial treatment of bladder cancer

Dissemination of <i>Mycobacterium tuberculosis</i> is associated to a <i>SIGLEC1</i> null variant that limits antigen exchange via trafficking extracellular vesicles

The identification of individuals with null alleles enables studying how the loss of gene function affects infection. We previously described a non‐functional variant in SIGLEC1, which encodes the myeloid‐cell receptor Siglec‐1/CD169 implicated in HIV‐1 cell‐to‐cell transmission. Here we report a significant association between the SIGLEC1 null variant and extrapulmonary dissemination of Mycobacterium tuberculosis (Mtb) in two clinical cohorts comprising 6,256 individuals. Local spread of bacteria within the lung is apparent in Mtb‐infected Siglec‐1 knockout mice which, despite having similar bacterial load, developed more extensive lesions compared to wild type mice. We find that Siglec‐1 is necessary to induce antigen presentation through extracellular vesicle uptake. We postulate that lack of Siglec‐1 delays the onset of protective immunity against Mtb by limiting antigen exchange via extracellular vesicles, allowing for an early local spread of mycobacteria that increases the risk for extrapulmonary dissemination