Characterization of microevolution events in Mycobacterium tuberculosis strains involved in recent transmission clusters.

Under certain circumstances, it is possible to identify clonal variants of Mycobacterium tuberculosis infecting a single patient, probably as a result of subtle genetic rearrangements in part of the bacillary population. We systematically searched for these microevolution events in a different context, namely, recent transmission chains. We studied the clustered cases identified using a population-based universal molecular epidemiology strategy over a 5-year period. Clonal variants of the reference strain defining the cluster were found in 9 (12%) of the 74 clusters identified after the genotyping of 612 M. tuberculosis isolates by IS6110 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive units-variable-number tandem repeat typing. Clusters with microevolution events were epidemiologically supported and involved 4 to 9 cases diagnosed over a 1- to 5-year period. The IS6110 insertion sites from 16 representative isolates of reference and microevolved variants were mapped by ligation-mediated PCR in order to characterize the genetic background involved in microevolution. Both intragenic and intergenic IS6110 locations resulted from these microevolution events. Among those cases of IS6110 locations in intergenic regions which could have an effect on the regulation of adjacent genes, we identified the overexpression of cytochrome P450 in one microevolved variant using quantitative real-time reverse transcription-PCR. Our results help to define the frequency with which microevolution can be expected in M. tuberculosis transmission chains. They provide a snapshot of the genetic background of these subtle rearrangements and identify an event in which IS6110-mediated microevolution in an isogenic background has functional consequences.

Characterization of Mycobacterium tuberculosis Beijing isolates from the Mediterranean area

BACKGROUND. The Beijing lineage of Mycobacterium tuberculosis is causing concern due to its global distribution and its involvement in severe outbreaks. Studies focused on this lineage are mainly restricted to geographical settings where its prevalence is high, whereas those in other areas are scarce. In this study, we analyze Beijing isolates in the Mediterranean area, where this lineage is not prevalent and is mainly associated with immigrant cases. RESULTS. Only 1% (N = 26) of the isolates from two population-based studies in Spain corresponded to Beijing strains, most of which were pan-susceptible and from Peruvian and Ecuadorian patients. Restriction fragment length polymorphism typing with the insertion sequence IS6110 identified three small clusters (2-3 cases). Mycobacterial interspersed repetitive unit-variable number tandem repeat typing (MIRU-15) offered low discriminatory power, requiring the introduction of five additional loci. A selection of the Beijing isolates identified in the Spanish sample, together with a sample of Beijing strains from Italy, to broaden the analysis context in the Mediterranean area, were assayed in an infection model with THP-1 cells. A wide range of intracellular growth rates was observed with only two isolates showing an increased intracellular replication, in both cases associated with contained production of TNF-alpha. No correlation was observed between virulence and the Beijing phylogenetic group, clustered/orphan status, or resistance. The Beijing strain responsible for extensive spread on Gran Canaria Island was also identified in Madrid, but did not lead to secondary cases and did not show high infectivity in the infection model. CONCLUSIONS. The Beijing lineage in our area is a non-homogeneous family, with only certain highly virulent representatives. The specific characterization of Beijing isolates in different settings could help us to accurately identify the virulent representatives before making general assumptions about this lineage.

Amplicon DNA melting analysis for the simultaneous detection of Brucella spp and Mycobacterium tuberculosis complex. Potential use in rapid differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis.

Some sites of extrapulmonary tuberculosis and focal complications of brucellosis are very difficult to differentiate clinically, radiologically, and even histopathologically. Conventional microbiological methods for the diagnosis of extrapulmonary tuberculosis and complicated brucellosis not only lack adequate sensitivity, they are also time consuming, which could lead to an unfavourable prognosis. The aim of this work was to develop a multiplex real-time PCR assay based on SYBR Green I to simultaneously detect Brucella spp and Mycobacterium tuberculosis complex and evaluate the efficacy of the technique with different candidate genes. The IS711, bcsp31 and omp2a genes were used for the identification of Brucella spp and the IS6110, senX3-regX3 and cfp31 genes were targeted for the detection of the M. tuberculosis complex. As a result of the different combinations of primers, nine different reactions were evaluated. A test was defined as positive only when the gene combinations were capable of co-amplifying both pathogens in a single reaction tube and showed distinguishable melting temperatures for each microorganism. According to the melting analysis, only three combinations of amplicons (senX3-regX3+bcsp31, senX3-regX3+IS711 and IS6110+IS711) were visible. Detection limits of senX3-regX3+bcsp31 and senX3-regX3+IS711 were of 2 and 3 genome equivalents for M. tuberculosis complex and Brucella while for IS6110+IS711 they were of 200 and 300 genome equivalents, respectively. The three assays correctly identified all the samples, showing negative results for the control patients. The presence of multicopy elements and GC content were the components most influencing the efficiency of the test; this should be taken into account when designing a multiplex-based SYBR Green I assay. In conclusion, multiplex real time PCR assays based on the targets senX3-regX3+bcsp31 and senX3-regX3+IS711 using SYBR Green I are highly sensitive and reproducible. This may therefore be a practical approach for the rapid differential diagnosis between extrapulmonary tuberculosis and complicated brucellosis.

Tesis electrónicas de la Universidad de Los Andes: adaptación y uso de la Plataforma Tede

Uno de los proyectos de gran envergadura de la Universidad de Los Andes (ULA) es la creación de la Biblioteca Digital de Tesis Electrónicas. Su consecución garantiza elevados niveles de interacción entre los actores internos y externos de la ULA a través de la consulta y publicación de la producción científica e intelectual de la Institución. En este artículo se presenta, en particular, la experiencia en la adaptación de la plataforma TEDE para la publicación de tesis electrónicas. La adaptación de la plataforma TEDE se llevó a cabo en tres etapas, a saber: a) Instalación, configuración y prueba de funcionalidad de la plataforma, b) Configuración y pruebas para el caso ULA, y c) Adaptación de la plataforma a la ULA. El resultado de esta adaptación fue una plataforma única que cumple con los requerimientos básicos establecidos para la publicación de tesis electrónicas en la ULA y que se ajusta perfectamente a cualquier institución académica de Venezuela. Muestra de esta experiencia ULA es su implantación en otras universidades nacionales como la Universidad del Zulia (LUZ), la Universidad Simón Bolívar (USB) y la Universidad Nacional Experimental de los Llanos Ezequiel Zamora (UNELLEZ).

Fluorescent quantitative PCR of Mycobacterium tuberculosis for differentiating intestinal tuberculosis from Crohn's disease

Intestinal tuberculosis (ITB) and Crohn's disease (CD) are granulomatous disorders with similar clinical manifestations and pathological features that are often difficult to differentiate. This study evaluated the value of fluorescent quantitative polymerase chain reaction (FQ-PCR) for Mycobacterium tuberculosis (MTB) in fecal samples and biopsy specimens to differentiate ITB from CD. From June 2010 to March 2013, 86 consecutive patients (38 females and 48 males, median age 31.3 years) with provisional diagnoses of ITB and CD were recruited for the study. The patients' clinical, endoscopic, and histological features were monitored until the final definite diagnoses were made. DNA was extracted from 250 mg fecal samples and biopsy tissues from each patient. The extracted DNA was amplified using FQ-PCR for the specific MTB sequence. A total of 29 ITB cases and 36 CD cases were included in the analysis. Perianal disease and longitudinal ulcers were significantly more common in the CD patients (P<0.05), whereas night sweats, ascites, and circumferential ulcers were significantly more common in the ITB patients (P<0.05). Fecal FQ-PCR for MTB was positive in 24 (82.8%) ITB patients and 3 (8.3%) CD patients. Tissue PCR was positive for MTB in 16 (55.2%) ITB patients and 2 (5.6%) CD patients. Compared with tissue FQ-PCR, fecal FQ-PCR was more sensitive (X2=5.16, P=0.02). We conclude that FQ-PCR for MTB on fecal and tissue samples is a valuable assay for differentiating ITB from CD, and fecal FQ-PCR has greater sensitivity for ITB than tissue FQ-PCR.

Identificación de genes Vinculados con la Condición de resistencia a Fármacos de Primera Línea en Mycobacterium tuberculosis.

Tesis (Maestría en Ciencias con Acentuación en Microbiología) UANL, 2011.

Análisis de la resupuesta humoral contra la proteína Hspx de mycobacterium tuberculosis como biomarcador de tuberculosis latente.

Tesis (Maestría en Ciencias con Acentuación en Inmunobiología) UANL, 2012.

Aportaciones al mecanismo de acción del ácido mesodihidroguaiarético sobre mycobacterium tuberculosis H37Rv.

Tesis (Maestría en Ciencias con Orientación en Farmacia) UANL, 2013.

Secuenciación del genoma de una clona de mycobacterium tuberculosis resistente a todos los medicamentos antituberculosis de primera línea.

Tesis (Maestría en Ciencias con orientación en Biología Molecular e Ingeniería Genética) UANL, 2014.

Análisis de los perfiles de expresión genética de cepas multifármacorresistentes de mycobacterium tuberculosis expuestas a nuevos compuestos contra tuberculosis pulmonar.

Tesis (Doctorado en Ciencias con Especialidad en Microbiología) UANL, 2012.

Polimorfismo de longitud de fragmentos amplificados como método de genotipificación de aislamientos clínicos de mycobacterium tuberculosis.

Tesis (Doctor en Ciencias con Especialidad en Biotecnología) UANL, 2009.

Obtención de nuevos compuestos con actividad contra mycobacterium tuberculosis a partir de leucophyllum frutescens.

Tesis (Doctor en Ciencias con Especialidad en Química Biomédica) UANL, 2011.

Cambio social y trayectorias académicas, un análisis longitudinal : acceso, éxito y abandono del alumnado de las carreras de ciclo largo de la Universidad de Murcia.

Analizar el proceso de cambio producido en el alumnado de la Universidad de Murcia desde 1980 hasta la actualidad. Conocer las distintas estrategias del alumnado universitario en función de su origen social determinado por el nivel educativo y situación profesional de sus padres. También se intenta responder a cuestiones críticas sobre la evolución reciente de la Enseñanza Superior en la Universidad de Murcia, tanto en sus dimensiones globales (oferta y demanda de plazas, tasas de participación, funcionamiento, éxito, retención y deserción), como en las más detalladas.. Alumnos matriculados en las carreras de ciclo largo en la Universidad de Murcia entre los años 1980 y 1999.. El estudio se desarrolla en 2 fases : en una primera, se realiza un análisis descriptivo temporal de las características básicas del alumnado universitario para cada titulación (edad, sexo, actividad laboral, origen familiar educativo y ocupacional, y modalidades de acceso). Esto permite comparar la evolución en el tiempo de los perfiles correspondientes a la matrícula universitaria. La segunda fase comprende un análisis diferencial de los alumnos que concluyen con éxito sus estudios y aquellos que, por diversas razones, 'abandonan'. De nuevo, los perfiles pertinentes establecidos, permiten discriminar los factores asociados al éxito o al abandono, en sus diferentes tipos. . Análisis descriptivo temporal de las características básicas del alumnado universitario para cada titulación de segundo ciclo. Análisis multivariante. Técnicas de regresión logística. Índice de consumo de tiempo para el éxito (ICTE). Coeficiente de correlación de Pearson. Correlación Bilateral Paramétrica Bivariada. Razón de suertes o doble razón. Paquetes informáticos no especificados. Es destacable por su magnitud, el fenómeno del enorme abandono que se observa en la enseñaza superior de segundo ciclo. No se puede afirmar, que todos aquellos que abandonan este nivel formativo renuncien a proseguir sus estudios, pero sí es cierto que la realidad de la institución nos muestra que un porcentaje muy alto de los matriculados no continúan en las carreras de ciclo largo, y muy especialmente, en aquellas carreras con altos contenidos prácticos, y en consecuencia altamente costosas para la institución. Otra de las conclusiones a las que se ha llegado se refiere al cambio socioprofesional y los niveles de estudios de las madres de los alumnos inscritos en la Universidad, que no son más que el reflejo de lo acaecido en la sociedad. También se ha observado que la transición sostenida de una universidad eminentemente masculina a una donde la presencia femenina es predominante, se ha debido al significativo incremento de la matrícula durante la década de los 80 y parte de los 90, pero sobre todo gracias a la notable presión de la mujer en el sistema académico universitario..

Thiol and Sulfenic Acid Oxidation of AhpE, the One-Cysteine Peroxiredoxin from Mycobacterium tuberculosis: Kinetics, Acidity Constants, and Conformational Dynamics

Drug resistance and virulence of Mycobacterium tuberculosis are partially related to the pathogen`s antioxidant systems. Peroxide detoxification in this bacterium is achieved by the heme-containing catalase peroxidase and different two-cysteine peroxiredoxins. M. tuberculosis genome also codifies for a putative one-cysteine peroxiredoxin, alkyl hydroperoxide reductase E (MtAhpE). Its expression was previously demonstrated at a transcriptional level, and the crystallographic structure of the recombinant protein was resolved under reduced and oxidized states. Herein, we report that the conformation of MtAhpE changed depending on its single cysteine redox state, as reflected by different tryptophan fluorescence properties and changes in quaternary structure. Dynamics of fluorescence changes, complemented by competition kinetic assays, were used to perform protein functional studies. MtAhE reduced peroxynitrite 2 orders of magnitude faster than hydrogen peroxide (1.9 x 10(7) M(-1) s(-1) vs 8.2 x 10(4) M(-1) s(-1) at pH 7.4 and 25 degrees C, respectively). The latter also caused cysteine overoxidation to sulfinic acid, but at much slower rate constant (40 M(-1) s(-1)). The pK(a) of the thiol in the reduced enzyme was 5.2, more than one unit lower than that of the sulfenic acid in the oxidized enzyme. The pH profile of hydrogen peroxide-mediated thiol and sulfenic acid oxidations indicated thiolate and sulfenate as the reacting species. The formation of sulfenic acid as well as the catalytic peroxidase activity of MtAhpE was demonstrated using the artificial reducing substrate thionitrobenzoate. Taken together, our results indicate that MtAhpE is a relevant component in the antioxidant repertoire of M. tuberculosis probably involved in peroxide and specially peroxynitrite detoxification.