Estandarización del análisis multi-locus de número variable de repeticiones en tándem automatizado para tipificación de Staphylococcus aureus Resistentes a Meticilina causantes de infecciones invasivas en población pediátrica paraguaya / Standardization of automated multi-locus variable number tandem repeats analysis for Methicillin-resistant Staphylococcus aureus typing isolated from invasive infections in paraguayan pediatric population

Introducción: Staphylococcus aureus es considerado uno de los patógenos humanos más importantes a nivel mundial y sus niveles de resistencia a meticilina han aumentado incluso en cepas aisladas de personas sin factores de riesgo nosocomial, por lo que la tipificación genética de los clones circulantes es fundamental para comprender los patrones de diseminación. Objetivo: Obtener la tipificación de SARM que causaron infecciones invasivas a niños mediante el empleo de la técnica de análisis multi-locus de número variable de repeticiones en tándem (MLVA) automatizada. Materiales y Métodos: Estudio observacional, descriptivo de corte transverso. Resultados: Se analizaron 25 cepas SARM que representan más de 700 aislamientos de S. aureus colectados en los años 2010, 2012 y 2013 de 4 hospitales de referencia nacional. La automatización de la técnica MLVA incluyó la tipificación del 88% (22/25) de los aislamientos en estudio, resultando 3 perfiles diferentes, cada uno asociado a un "spa tipo" distinto, siendo el perfil 1-t019 el predominante (86%), seguido por el perfil 3-t002 (9%), arrojando 100% de concordancia con el método MLVA manual, así como una alta concordancia con el método estándar de oro, PFGE. Conclusiones: La inclusión de un método de análisis de fragmentos automatizado permitió llevar a cabo la caracterización de aislamientos mejorando el tiempo de respuesta y manteniendo una alta sensibilidad en comparación con el método manual.

Introduction: Staphylococcus aureusis considered one of the most critical human pathogens worldwide, and its levels of methicillin resistance have increased even in strains isolated from people without nosocomial risk factors. Therefore the genetic typing of circulating clones is essential to understand dissemination patterns. Objective: Obtain the MRSA typing that caused invasive infections in children by using the automated multi-locus variable number of tandem repeats (MLVA) analysis technique. Materials and methods: Observational, descriptive, cross-sectional. Results: 25 strains MRSA representing more than 700S. aureusisolates collected in 2010, 2012, and 2013 from 4 national reference hospitals were analyzed. The MLVA automation included the typing of 88% (22/25) isolates, resulting in 3 different profiles, each one associated with a different spa type, being the 1-t019 the predominant (86%), followed by the 3-t002 profile (9%), yielding 100% concordance with the MLVA manual, as well as high concordance with the standard gold method, PFGE. Conclusions: The inclusion of an automated fragment analysis method led to the characterization of isolates, improving response time, and maintaining high sensitivity compared to the manual process.

Distribution of Locus of Adhesion and Autoaggregation and hes Gene in STEC Strains from Countries of Latin America.

Shiga toxin-producing Escherichia coli (STEC) are zoonotic food pathogens associated with foodborne diarrheal illness, hemorrhagic colitis, and complications such as hemolytic uremic syndrome (HUS). The ability to adhere to epithelial cells is an important virulence trait, and pathogenicity islands (PAIs) play an important role on it. Some STEC carrying a PAI named locus of enterocyte effacement (LEE-positive) have been frequently associated to HUS; however, STEC that do not carry LEE (LEE-negative) have also been associated with this outcome. The burden of disease caused by LEE-negative STEC has increased recently in several countries like Argentina, Chile, and Paraguay. A new PAI -the Locus of Adhesion and Autoagregation (LAA)-has been associated to severe disease in humans. In this study, we aimed to analyze the distribution of LAA and its possible predictor, the gene hes, in LEE-negative STEC strains isolated from Chile and Paraguay from different sources. The presence of the different LAA modules and hes were detected by PCR. LAA was found in 41.6% and 41.0% of strains isolated from Chile and Paraguay, respectively. Strains were isolated from diverse origins and belonged to several serogroups including O91, O103, and O113. The hes gene was detected in 50% of the isolates from Paraguay and Chile. Therefore, the detection of LAA and hes in STEC could complement current genetic evaluation schemes, allowing to classify LEE negative STEC strains as LAA-positive or LAA-negative STEC strains.

Caracterización molecular de aislamientos de Escherichia coli productores de toxina Shiga obtenidos en 2 establecimientos ganaderos del Paraguay Caracterización molecular de aislamientos de STEC de ganado bovino paraguayo / Molecular characterization of Shiga toxin producing Escherichia coli isolated from 2 livestock establishments of Paraguay

Resumen Escherichia coli productora de toxina Shiga (STEC) es un patógeno de importancia alimentaria en los humanos, el bovino es su principal reservorio. El objetivo de este estudio fue determinar la portación de STEC en bovinos del Paraguay y analizar el perfil de virulencia y los serotipos de los aislados reunidos. Se estudiaron 197 muestras de hisopado rectal de bovinos y un promedio de 5 a 50 colonias por bovino positivo a genes stx 1 /stx 2. Se amplificaron por PCR los genes stx 1, stx 2, saa, ehxA y eae. El 84,8% de los bovinos resultaron portadores de STEC. Los perfiles de virulencia predominantes fueron stx 2 y stx 2 /saa/ehxA. La serotipificación se realizó por reacciones de aglutinación en 60 aislamientos seleccionados, se encontró un aislamiento del serogrupo O103, capaz de producir infecciones en humanos. Este trabajo muestra los primeros datos de portación de STEC de ganado bovino paraguayo y señala la necesidad de efectuar otros estudios con mayor cobertura territorial, para lograr una visión completa de este fenómeno.

Abstract Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen in humans, with cattle being the main reservoir. The objective of this study was to determine the carrying of STEC in Paraguayan bovines and to analyze the virulence profile and serotypes of these isolates. A total of 197 samples of bovine fecal samples and an average of 5 to 50 colonies from stx 1 /stx 2 positive samples were studied. The stx 1 , stx 2 , saa, ehxA and eae genes were amplified by PCR. 84.8% of the cattle were carriers of STEC. The predominant virulence profiles were stx 2 and stx 2 /saa/ehxA. The serotyping was performed by agglutination reactions for 60 selected isolates, resulting in isolation of serogroup O103, which could produce infections in humans. This work shows the first data of STEC carriers in Paraguayan cattle, and indicates the need for other studies with greater territorial coverage for a complete vision of this phenomenon.

[Molecular characterization of Shiga toxin producing Escherichia coli isolated from 2 livestock establishments of Paraguay]. / Caracterización molecular de aislamientos de Escherichia coli productores de toxina Shiga obtenidos en 2 establecimientos ganaderos del Paraguay.

Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen in humans, with cattle being the main reservoir. The objective of this study was to determine the carrying of STEC in Paraguayan bovines and to analyze the virulence profile and serotypes of these isolates. A total of 197 samples of bovine fecal samples and an average of 5 to 50 colonies from stx1/stx2 positive samples were studied. The stx1, stx2, saa, ehxA and eae genes were amplified by PCR. 84.8% of the cattle were carriers of STEC. The predominant virulence profiles were stx2 and stx2/saa/ehxA. The serotyping was performed by agglutination reactions for 60 selected isolates, resulting in isolation of serogroup O103, which could produce infections in humans. This work shows the first data of STEC carriers in Paraguayan cattle, and indicates the need for other studies with greater territorial coverage for a complete vision of this phenomenon.

Estandarización de una técnica de PCR múltiple para la detección de los serogrupos O157, O104 y "big six" de Escherichia coli productora de la toxina Shiga (STEC) / Standardization of a multiplex PCR technique for the detection of Shiga toxin-producing Escherichia coli (STEC) serogroups O157, O104 and "big six"

Los serogrupos O26, O45, O103, O104, O111, O121, O145 y O157 de STEC se relacionan con un elevado número de casos de SUH a nivel mundial, por lo que están incluidos dentro de las categorías de mayor riesgo para los humanos, según los criterios de autoridades alimentarias de Estados Unidos y Europa. El método convencional de identificación de antígenos O y H se realiza por aglutinación con antisueros de conejo. Este método además de ser muy costoso y laborioso, no se encuentra disponible en el país para empleo masivo. En este contexto, el objetivo de este estudio observacional descriptivo de corte transverso ha sido la estandarización de una técnica de PCR múltiple para la detección de estos 8 serogrupos, a fin de contar con un sistema de detección eficiente, sensible y con potencial de aplicación en la industria alimentaria. Se estandarizaron reacciones de PCR empleando como controles positivos cepas E. coli de referencia correspondientes a la totalidad de los serogrupos citados. Se obtuvieron productos de tamaños esperados para cada serogrupo, no se observaron amplificaciones cruzadas o falsos positivos. Esta técnica estandarizada podría representar una herramienta rápida y menos costosa que la técnica serológica, con la capacidad de ser aplicada a diferentes matrices, permitiendo la detección de estos serogrupos en aislados STEC de ganado en pie, fuentes de agua de consumo, alimentos e incluso en aislamientos clínicos asociados a enfermedades humanas(AU)

STEC serogroups O26, O45, O103, O104, O111, O121, O145, and O157, are related to a high number of cases of HUS worldwide, so they are included in the categories of greatest risk for humans, according to the food administration criteria of the United States and Europe. The conventional method of identifying antigens O and H is carried out by agglutination with rabbit antisera. This method is very expensive and laborious and is not available in the country for massive-scale use. In this context, the objective of this cross-sectional descriptive observational study has been the standardization of a multiplex PCR technique for the detection of these 8 serogroups, in order to have an efficient and sensitive detection system with the potential for application in the food industry. PCR reactions were standardized using as positive controls reference E. coli strains to correspond to all the mentioned serogroups. Products of expected sizes were obtained for each serogroup; no cross-amplification or false positives were observed. This standardized technique could represent a quick and less expensive tool than the serological technique, with the possibility to be applied to different kind of samples, allowing the detection of these serogroups in STEC isolates of live cattle, sources of drinking water, food and even in clinical isolates associated with human diseases(AU)

Portación de fimH en aislados de Escherichia coli productor de Toxina Shiga provenientes de ganado bovino, Departamento Cordillera, Paraguay / Portability of fimH in isolates of Shiga toxin-producing Escherichia coli from cattle, Department of Cordillera, Paraguay

Ciertas cepas de Escherichia coli productoras de toxina Shiga (STEC) tienen la capacidad de formar biofilm en alimentos y otras superficies, aumentando su potencial como fuente de contaminación. El gen fimH se ha asociado a la capacidad de formación de biofilm en E.coli. Este estudio observacional descriptivo de corte transverso se realizó con el objetivo de describir la portación de fimH en aislados STEC provenientes de muestras de materia fecal de ganado bovino del Departamento de Cordillera en el 2016. Los aislados de STEC se obtuvieron por cultivos, extracción de ADN y amplificación por PCR de genes stx1 y stx2. El gen fimH se detectó por PCR convencional. Un total de 1006 aislamientos de E. coli se sometieron a extracción de ADN y amplificación por PCR para genes stx1 y stx2. De éstos, 269 se identificaron como STEC, en los que la detección del gen fimH se realizó por PCR convencional. Un producto de PCR representativo se sometió a secuenciación del gen fimH y mostró 100% de homología con secuencias de la Base de Datos de GenBank. De 269 aislamientos STEC, 129 aislamientos (48%) resultaron ser portadores del gen fimH y por tanto con potencial de formar biofilm. Esta frecuencia elevada representa un riesgo de persistencia de estos patógenos en elementos y superficies de trabajo de sitios de expendio y manipulación de productos cárnicos. Este trabajo contribuye como una herramienta esencial para seguir con la línea de investigación, obteniendo datos de suma importancia que ayuden a describir la situación de riesgo de contaminación alimentaria en que se encuentra el país(AU)

Synthesis and Docking of Novel 3-Indolylpropyl Derivatives as New Polypharmacological Agents Displaying Affinity for 5-HT1A R/SERT.

A series of novel 3-indolylpropyl derivatives was synthesized and evaluated for their binding affinities at the serotonin-1A receptor subtype (5-HT1A R) and the 5-HT transporter (SERT). Compounds 11b and 14b exhibited the highest affinities at the 5-HT1A R (Ki = 43 and 56 nM), whereas compounds 11c and 14a were the most potent analogs at the SERT (Ki = 34 and 17 nM). On the other hand, compounds 14b and 11d showed potent activity at both targets, displaying a profile that makes them promising leads for the search for novel potent ligands with a dual mechanism of action. Molecular docking studies in all the compounds unveiled relevant drug-target interactions, which allowed rationalizing the observed affinities.

Prevalence of Escherichia coli adhesion-related genes in neonatal calf diarrhea in Uruguay.

INTRODUCTION: Neonatal calf diarrhea (NCD), one of the most important diseases of neonatal dairy and beef calves in Uruguay, has become relevant in association with intensive systems. This disease generates substantial economic losses every year worldwide as a result of increased morbidity and mortality. Escherichia coli, one of the pathogens associated with NCD, can express several fimbrial and afimbrial adhesins. The objective of this study was to assess the presence of clpG, f5, f17A, f17G(II), and f17G(I) genes that encode three important adhesins expressed in diarrheagenic E. coli: F5, F17 and CS31A, isolated from feces of calves in Uruguay. METHODOLOGY: Feces of 86 (70 diarrheic and 16 healthy) calves, from 15 animal facilities in Uruguay, were collected between 2012 and 2013. Biochemical and molecular identification were performed to finally obtain 298 E. coli isolates. Partial amplification of adhesion-related genes was performed by polymerase chain reaction. RESULTS: The most prevalent gene was f17A (31.2%), followed by f17G(II), clpG, f17G(I) and f5 (25.8%, 17.5%, 3.7% and 0.7%, respectively). All genes were present in diarrheic and healthy animals except f5 and f17G(I); these genes were present only in affected calves, although in low numbers. CONCLUSIONS: This is the first report of the presence of F5, F17, and CS31A genes in E. coli strains from NCD cases in Uruguay. Prevalence values of the genes, except f5, were in accordance with regional findings. It is expected that further characterization of locally transmitted strains will contribute to control a problem of regional and international magnitude.

Similarities between the Binding Sites of SB-206553 at Serotonin Type 2 and Alpha7 Acetylcholine Nicotinic Receptors: Rationale for Its Polypharmacological Profile.

Evidence from systems biology indicates that promiscuous drugs, i.e. those that act simultaneously at various protein targets, are clinically better in terms of efficacy, than those that act in a more selective fashion. This has generated a new trend in drug development called polypharmacology. However, the rational design of promiscuous compounds is a difficult task, particularly when the drugs are aimed to act at receptors with diverse structure, function and endogenous ligand. In the present work, using docking and molecular dynamics methodologies, we established the most probable binding sites of SB-206553, a drug originally described as a competitive antagonist of serotonin type 2B/2C metabotropic receptors (5-HT2B/2CRs) and more recently as a positive allosteric modulator of the ionotropic α7 nicotinic acetylcholine receptor (nAChR). To this end, we employed the crystal structures of the 5-HT2BR and acetylcholine binding protein as templates to build homology models of the 5-HT2CR and α7 nAChR, respectively. Then, using a statistical algorithm, the similarity between these binding sites was determined. Our analysis showed that the most plausible binding sites for SB-206553 at 5-HT2Rs and α7 nAChR are remarkably similar, both in size and chemical nature of the amino acid residues lining these pockets, thus providing a rationale to explain its affinity towards both receptor types. Finally, using a computational tool for multiple binding site alignment, we determined a consensus binding site, which should be useful for the rational design of novel compounds acting simultaneously at these two types of highly different protein targets.