In silico approach to identify microsatellite candidate biomarkers to differentiate the biovar of Corynebacterium pseudotuberculosis genomes.

Corynebacterium pseudotuberculosis is the causative bacterial agent of the zoonotic disease known as caseous lymphadenitis, and it presents several mechanisms of response to host defenses, including the presence of virulence factors (VFs). The genomes of these bacteria have several polymorphic markers known as microsatellites, or simple sequence repeats (SSRs), that can be used to characterize the genome, to study possible polymorphisms existing among strains, and to verify the effects of such polymorphic markers in coding regions and regions associated with VFs. In this study, several SSRs were identified within coding regions throughout the 54 genomes of this species, revealing possible polymorphisms associated with coding regions that could be used as strain-specific or serotype-specific identifiers of C. pseudotuberculosis. The similarities associated with SSRs amongst the different serum variants of C. pseudotuberculosis, biovars equi and ovis, were also evaluated, and it was possible to identify SSRs located in coding regions responsible for a VF enrolled in pathogenesis known to mediate bacterial adherence (SpaH-type pili virulence factor). Phylogenetic analyses revealed that strains sharing SSR patterns, including the possible polymorphisms identified in the same position of gene-coding regions, were displayed by strains with a common ancestor, corroborating with the Genome Tree Report of the NCBI. Statistical analysis showed that the microsatellite groups belonging to equi and ovis biovars have a significance of 0.006 (p-value) in similarity, thus indicating them as good biomarker candidates for C. pseudotuberculosis.

Impacts of soybean agriculture on the resistome of the Amazonian soil.

The soils of the Amazon are complex environments with different organisms cohabiting in continuous adaptation processes; this changes significantly when these environments are modified for the development of agricultural activities that alter the chemical, macro, and microbiological compositions. The metagenomic variations and the levels of the environmental impact of four different soil samples from the Amazon region were evaluated, emphasizing the resistome. Soil samples from the organic phase from the different forest, pasture, and transgenic soybean monocultures of 2-14 years old were collected in triplicate at each site. The samples were divided into two groups, and one group was pre-treated to obtain genetic material to perform sequencing for metagenomic analysis; another group carried out the chemical characterization of the soil, determining the pH, the content of cations, and heavy metals; these were carried out in addition to identifying with different databases the components of the microbiological communities, functional genes, antibiotic and biocide resistance genes. A greater diversity of antibiotic resistance genes was observed in the forest soil. In contrast, in monoculture soils, a large number of biocide resistance genes were evidenced, highlighting the diversity and abundance of crop soils, which showed better resistance to heavy metals than other compounds, with a possible dominance of resistance to iron due to the presence of the acn gene. For up to 600 different genes for resistance to antibiotics and 256 genes for biocides were identified, most of which were for heavy metals. The most prevalent was resistance to tetracycline, cephalosporin, penam, fluoroquinolone, chloramphenicol, carbapenem, macrolide, and aminoglycoside, providing evidence for the co-selection of these resistance genes in different soils. Furthermore, the influence of vegetation cover on the forest floor was notable as a protective factor against the impact of human contamination. Regarding chemical characterization, the presence of heavy metals, different stress response mechanisms in monoculture soils, and the abundance of mobile genetic elements in crop and pasture soils stand out. The elimination of the forest increases the diversity of genes for resistance to biocides, favoring the selection of genes for resistance to antibiotics in soils.

Stress induced biofilm formation in Propionibacterium acidipropionici and use in propionic acid production.

Propionibacterium acidipropionici produces propionic acid from different sugars and glycerol; the production can be improved by high cell density fermentations using immobilized cells that help to overcome the limitations of the non-productive lag phase and product inhibition. In this study, the use of stress factors to induce P. acidipropionici to form biofilm and its use as an immobilization procedure in fermentations in bioreactors for producing propionic acid was investigated. Citric acid and sodium chloride increased exopolysaccharide production, biofilm forming capacity index and trehalose production. Analysis of the expression of trehalose synthesis-related genes otsA and treY by RT-qPCR showed significantly increased expression of only treY during log phase with citric acid, while FISH analysis showed expression of treY and luxS under the influence of both stress factors. The stress factors were then used for development of microbial biofilms as immobilization procedure on Poraver® and AnoxKaldnes® carriers in recycle batch reactors for propionic acid production from 20 g/L glycerol. Highest productivities of 0.7 and 0.78 g/L/h were obtained in Poraver® reactors, and 0.39 and 0.43 g/L/h in AnoxKaldnes® reactors with citric acid and NaCl, respectively.

Diseño y evaluación de primers in silico del gen E1 del virus de chikungunya para real-time PCR (qPCR) / Design and evaluation of primers in silico of the E1 gene of chukungunya virus for real -time PCR (q PCR)

El diseño de primers es una parte sustancial dentro de los ensayos de PCR, el mismo es aplicado en la caracterización de microorganismos, secuenciación, cuantificación, etc. En los últimos años se ha incrementado herramientas para este fin, se realizaron nuevas variantes, crearon nuevos enfoques, muchas de estas herramientas son de licencia comercial y otros de acceso libre y código abierto permiten al usuario mejorar o modificar según requerimiento el diseño de primers. En este trabajo comparamos las características básicas dentro del diseño de primers, empleando 2 tipos de herramientas web de acceso libre y código abierto: Primer-BLAST y Primer3Plus, se realizó el diseño in silico, análisis de estructuras secundarias y verificación de la especificidad de los primers obtenidos. Los resultados mostraron que el uso de variables adicionales o uso de penaltis mediante el software Primer3Plus afectan en la especificidad, formación de estructuras secundarias de los primers y de los amplicones. Además, se determinó que los parámetros que tienen muchos software por defecto, no aseveran el diseño de primers convenientes y/o específicos, en este sentido es importante realizar el uso de penaltis. Asimismo, se identificó que el uso de Primer3Plus ha permitido disminuir la formación de estructuras secundarias, sin afectar la especificidad de amplificación del marcador seleccionado.

Primer design is a substantial part of PCR assays, which is used in characterization of microorganisms, sequencing, quantification, etc. In recent years, new tools have been added for this purpose, new variants and approaches have been created, many of these tools are commercial and others are free including open source, giving the possibility to improve or modify as required. In this work, we compared the basic characteristics of the primers design, using 2 types of free and open source web tools, PrimerBLAST and Primer3Plus, in this sense the in silico design, analysis of secondary structures and verification of primers designed. The results showed that the use of additional variables or use of penalties using the software Primer3Plus affect in specificity, secondary structures formation in primers and in the products of amplification. In addition, default parameters, that many software have, do not imply that suitable and / or specific primers can be obtained, meaning the importance of the use of penalties. Therefore, the use of Primer3Plus has allowed to decrease the formation of secondary structures without affecting the amplification specificity of the selected marker.

Leishmaniasis in the major endemic region of Plurinational State of Bolivia: Species identification, phylogeography and drug susceptibility implications.

The Plurinational State of Bolivia is one of the Latin American countries with the highest prevalence of leishmaniasis, highlighting the lowlands of the Department of La Paz where about 50% of the total cases were reported. The control of the disease can be seriously compromised by the intrinsic variability of the circulating species that may limit the efficacy of treatment while favoring the emergence of resistance. Fifty-five isolates of Leishmania from cutaneous and mucocutaneous lesions from patients living in different provinces of the Department of La Paz were tested. Molecular characterization of isolates was carried out by 3 classical markers: the rRNA internal transcribed spacer 1 (ITS-1), the heat shock protein 70 (HSP70) and the mitochondrial cytochrome b (Cyt-b). These markers were amplified by PCR and their products digested by the restriction endonuclease enzymes AseI and HaeIII followed by subsequent sequencing of Cyt-b gene and ITS-1 region for subsequent phylogenetic analysis. The combined use of these 3 markers allowed us to assign 36 isolates (65.5%) to the complex Leishmania (Viannia) braziliensis, 4 isolates (7, 27%) to L. (Viannia) lainsoni. and the remaining 15 isolates (23.7%) to a local variant of L. (Leishmania) mexicana. Concerning in vitro drug susceptibility the amastigotes from all isolates where highly sensitive to Fungizone® (mean IC50 between 0.23 and 0.5µg/mL) whereas against Glucantime® the sensitivity was moderate (mean IC50 ranging from 50.84µg/mL for L. (V.) braziliensis to 18.23µg/mL for L. (L.) mexicana. L. (V.) lainsoni was not sensitive to Glucantime®. The susceptibility to miltefosine was highly variable among species isolates, being L. (L.) mexicana the most sensitive, followed by L. (V.) braziliensis and L. (V.) lainsoni (mean IC50 of 8.24µg/mL, 17.85µg/mL and 23.28µg/mL, respectively).

Establecimiento y evaluación de dos métodos de pre tratamiento de muestras de suelo para la extracción de ADN para el estudio de la diversidad bacteriana / Establishment and evaluation of two methods of pre-treatment of soil samples for the extraction of DNA for the study of bacterial diversity

La necesidad de ampliar los conocimientos respecto a los mecanismos bioquímicos y fisiológicos desarrollados por los microorganismos presentes en suelos requiere de una descripción completa de la diversidad microbiana, para lo cual en las últimas décadas se han desarrollado diferentes técnicas moleculares (qPCR, DGGE, T-RFLP, RAPD.) las mismas que requieren una adecuada técnica de extracción de ADN que aseguren el éxito de la descripción de la diversidad microbiana, considerando las características de las muestras de suelos a ser estudiadas. Los protocolos de extracción de ADN generalmente utilizados están basados en la separación de los microorganismos de la matriz antes de la extracción de ADN mediante lisis física o química y por otro lado, la extracción directa del ADN microbiano a partir de muestras de suelo, sin embargo la presencia de sustancias húmicas y fenólicas afectan la calidad del ADN extraído, lo que repercuten en el desarrollo de posteriores estudios moleculares. La finalidad de este estudio fue la de establecer procedimientos de pre tratamientos de 3 tipos de nuestras de suelo (arenoso, arcilloso y francos) para posteriormente describir la riqueza y diversidad bacteriana de las muestras en estudio mediante PCR DGGE. De esta manera se determinó que la adición de CaCO3 en muestras de suelos francos permite la identificación de una mayor diversidad y riqueza bacteriana (10 bandas). Asimismo, la adición de PVPP a suelos arenosos (8 bandas) y arcillosos (3 bandas) también permite obtener las características descritas anteriormente utilizando el método PCR-DGGE. Lo cual indica que los procedimientos de pre tratamiento con CaCO3 y PVPP son específicos para la extracción de ciertas comunidades microbianas.

The knowledge about biochemical and physiological mechanisms by microorganisms in soils are required for a complete description of microbial diversity, lately different molecular techniques have been developed to study this feature (qPCR, DGGE, T- RFLP, RAPD). DNA extraction techniques ensure the description of the microbial diversity success, according the soil samples characteristics. Generally, DNA extraction protocols used for separation of microorganism of matrix soil before DNA extraction by physical and chemical lysis. Other protocol is direct extraction of microbial DNA from soil samples, humic acids and phenolic substances affect the quality of DNA, which affect the development of subsequent molecular studies. The purpose of this study was to establish pretreatment procedures for different kind of soil samples (frank, sandy and clayey) in order to describe richness and bacterial diversity by PCR DGGE. In this sense, we determined the addition of CaCO3 in frank soils samples allows the identification of greater diversity and bacterial richness (10 bands) than the other method. Besides, PVPP pretreatment is no only useful to obtain bacterial diversity in sandy soil (8 bands), but also in clayey soils (3 bands) soils by PCR-DGGE method. This indicates that the pretreatment procedures with CaCO3 and PVPP are specific for soil microbial community's isolation.