Development of a multiplex PCR assay for the detection of metallo-beta-lactamase genes in Pseudomonas aeruginosa / Desarrollo de un PCR múltiple para la detección de genes metalo-beta-lactamasa en Pseudomonas aeruginosa

SUMMARY: The appearance of Pseudomonas aeruginosa strains with multi-resistance to antibiotics is a clinical problem of great relevance. The methods for detecting these resistances are laborious and slow, which is a complication when treating patients promptly. In this work, we developed a simple method for simultaneous detection of several carbapenem resistance genes using a multiplex PCR assay. The PCR assay developed, followed by electrophoretic separation of fragments, allows to simultaneously identify the presence of 6 antibiotic resistance genes: bla-VIM (261 bp), bla-IMP (587 bp), bla-SPM (648 bp), bla-GIM-1 (753 bp), bla-NDM-1 (813 bp) and bla-KPC (882 bp). We analyzed 7 clinical isolates of P. aeruginosa obtained in Chile, finding the resistance genes bla-VIM, bla-IMP, bla-SPM, bla-GIM, and bla-NDM in 5 of them. We found a perfect correlation between the detection of various resistance genes by PCR and the results obtained by antibiograms. Interestingly, 2 of the strains possessed 3 different resistance genes simultaneously. Finally, in this work, we found the presence of 3 genes never described before in clinical isolates of P. aeruginosa in Chile (bla-IMP, bla-SPM, and bla-GIM-1). We developed a rapid multiplex PCR test for the simultaneous detection of up to 6 antibiotic resistance genes of the metallo-β-lactamase family in P. aeruginosa.

La aparición de cepas de Pseudomonas aeruginosa con resistencias a diversos antibióticos es un problema clínico de gran relevancia. Los métodos de detección de dichas resistencias son laboriosos y lentos, lo que genera una complicación al momento de tratar a los pacientes oportunamente. En este trabajo desarrollamos un método simple de detección simultánea de varios genes de resistencia a carbapenem, mediante un sistema de PCR múltiple. El ensayo de PCR desarrollado, seguido de una separación electroforética de los amplicones, permite distinguir simultáneamente la presencia de 6 genes de resistencia a antibióticos: bla-VIM (261 pb), bla-IMP (587 pb), bla-SPM (648 pb), bla-GIM-1 (753 pb), bla-NDM-1 (813 pb) y bla-KPC (882 pb). Analizamos 7 aislados clínicos obtenidos en Chile, encontrando en 5 de ellos los genes de resistencia bla-VIM, bla-IMP, bla-SPM, bla-GIM y bla-NDM. Encontramos una perfecta correlación entre la detección de diversos genes de resistencia y los resultados obtenidos mediante antibiogramas. Interesantemente, 2 de las cepas mostraron poseer simultáneamente 3 genes de resistencia distintos. Por último, en este trabajo encontramos la presencia de 3 genes nunca antes descritos en aislados clínicos de P. aeruginosa en Chile (bla-IMP, bla-SPM y bla-GIM-1). Hemos desarrollado un test rápido de PCR múltiple, para la detección simultánea de hasta 6 genes de resistencia a antibióticos de la familia.a de las metallo-b-lactamases en P. aeruginosa.

A rapid preparative-TLC/GC-MS methodology for discriminating between two filmy ferns (Hymenophyllaceae) native from the temperate rain forest of Southern Chile based on their soluble carbohydrates / Una metodología de CCF-preparativa/CG-EM rápida para discriminar entre dos helechos película(Hymenophyllaceae) nativos del bosque templado del sur de Chile basada en sus hidratos de carbono

Filmy ferns are a conspicuous epiphytic component of the temperate rain forest of southern Chile and the more abundant genus is Hymenophyllum represented by 19 species, they are a primitive fern family sharing their limited water lost control (poikilohydricity) with lower non-vascular plants, such as bryophytes and lichens. Because, carbohydrate accumulation is a desiccation tolerance mechanism proposed for desiccation-tolerant vascularized plants, the aim of this investigation was to propose a methodology for soluble carbohydrate analysis in native ferns. Preparative- TLC showed to be a simple and rapid technique for separating Hymenophyllum sugars. GC-MS analysis allowed identifying glucitol (2.0 percent), sucrose (2.0 percent), alpha or beta-melibiose (5.0 percent) and salidroside (4.4 percent) in H. cruentum carbohydrate profile from H. dentatum showed significant differences presenting only two sugars sucrose (1.4 percent) and alpha or beta-melibiose (3.2 percent). Furthermore, salidroside, an antioxidant compound, was identified only in H. cruentum. Our results suggest that H. cruentum, has higher preventive mechanisms than H. dentatum; that would allow delay the effects of desiccation (prevent a rapid dehydration) and thus survive to short periods of drought present in the study area. Due to the presence of salidroside in H. cruentum this species should be more resistant to oxidative stress that H. dentatum.

Los helechos película se pueden encontrar en el bosque templado lluvioso del sur de Chile, siendo el género más abundante Hymenophyllum representado con 19 especies. Son una familia de helechos primitivos que comparten la limitación de control de pérdida de agua (poiquilohidrícos) con plantas no vasculares, tales como briófitas y líquenes. Como el contenido de hidratos de carbono ha sido asociado a la protección de la planta durante la desecación, su análisis es una herramienta importante para dilucidar el mecanismo asociado a este fenómeno. El objetivo de esta investigación fue proponer una metodología fácil y rápida para el análisis de hidratos de carbono en helechos nativos. La CCF-preparativa permitió la separación de hidratos de carbono presentes en plantas del género Hymenophyllum. El análisis por GC-MS logró identificar glucitol (2,0 por ciento), sacarosa (2,0 por ciento), alfa o Beta-melibiosa (5.0 por ciento) y salidrosido (4,4 por ciento) en H. cruentum. El perfil de hidratos de carbono de H. dentatum mostró diferencias significativas, mostrando sólo dos compuestos sacarosa (1,4 por ciento) y alfa o beta- melibiosa (3.2 por ciento). Además, salidrosido, un compuesto antioxidante, sólo fue identificado en H. cruentum. Nuestros resultados sugieren que ante la menor tolerancia a la desecación observada previamente en H. cruentum, esta especie presenta mecanismos que le permitirían retardar los efectos de la desecación (prevención de una rápida pérdida de agua) y así sobrevivir a los periodos cortos de sequía presentes en la zona de estudio. Debido a La presencia de salidrosido en H. cruentum, esta especie debería ser más resistente al estrés oxidativo que H. dentatum.

Applications of recombinant Pichia pastoris in the healthcare industry

Since the 1970s, the establishment and development of the biotech industry has improved exponentially, allowing the commercial production of biopharmaceutical proteins. Nowadays, new recombinant protein production is considered a multibillion-dollar market, in which about 25% of commercial pharmaceuticals are biopharmaceuticals. But to achieve a competitive production process is not an easy task. Any production process has to be highly productive, efficient and economic. Despite that the perfect host is still not discovered, several research groups have chosen Pichia pastoris as expression system for the production of their protein because of its many features. The attempt of this review is to embrace several research lines that have adopted Pichia pastoris as their expression system to produce a protein on an industrial scale in the health care industry.

Recombinants proteins for industrial uses: utilization of Pichia pastoris expression system

The innovation in industrial process with impact in the efficient production is the major challenge for actual industry. A high numerous of enzymes are utilized in at different level of process; the search for new alternatives with better characteristic has become a field of study of great interest, the recombinant protein achievement in a different host system is an alternative widely assessed for production of this. The microorganism Pichia pastoris has been used like a successful expression system in diverse areas, improved the yield and extraction-recovery of the product expressed. The reported of diverse authors in the production of enzymes with different application in industry is varied, in this review the different industry areas and the characteristic of the enzymes produced are detailed.