Resistencia antimicrobiana y factores de virulencia en aislados de Vibrio cholerae O1. Cuba, 2012-2015 / Antimicrobial resistance and virulence factors in Vibrio cholerae O1 isolates. Cuba, 2012-2015

Introducción: El cólera es una infección intestinal aguda causada por cepas toxigénicas de Vibrio choleare. La rápida diseminación y emergencia de la multirresistencia que caracteriza a este patógeno, podría interferir en el éxito de la terapia antimicrobiana, por lo que constituye una prioridad monitorear los cambios en los patrones de susceptibilidad, como parte trascendental de la política de control de la resistencia antimicrobiana. Objetivo: Determinar el comportamiento de la resistencia antimicrobiana frente a los antimicrobianos de interés empleados en el tratamiento, la presencia de factores de virulencia enzimáticos y si existe relación entre ambos. Métodos: Se realizó un estudio descriptivo de corte transversal durante julio de 2012 a diciembre de 2015. Se estudiaron 500 aislamientos pertenecientes al cepario del Laboratorio Nacional de Referencia de Enfermedades Diarreicas Agudas del Instituto de Medicina Tropical Pedro Kourí, procedentes de brotes de enfermedades diarreicas agudas de la red nacional de laboratorios de Microbiología de Cuba. Se aplicaron métodos convencionales fenotípicos para determinar el comportamiento de la resistencia antimicrobiana, la presencia de factores enzimáticos y la relación de estos con la resistencia antimicrobiana. Resultados: Los mayores porcentajes de sensibilidad se obtuvieron frente a azitromicina (98 por ciento), doxiciclina (96 por ciento) y ciprofloxacina (93 por ciento) y de resistencia frente a ampicilina (100 por ciento) y trimetoprim-sulfametoxazol (99,4 por ciento). Se encontraron 44 aislados (8,8 por ciento) multirresistente. Todos los aislamientos poseían al menos dos enzimas extracelulares como factores de virulencia, las más frecuentes: gelatinasa (96 por ciento) y lecitinasa (95 por ciento). Conclusiones: Se evidencia una relación directa y proporcional entre la presencia de los factores de virulencia y resistencia antimicrobiana, sinergismo que surgiere mayor patogenicidad de los aislados estudiados procedentes de brotes epidémicos(AU)

Introduction: Cholera is an acute intestinal infection caused by toxigenic strains of Vibrio choleare. The rapid dissemination and emergence of the multiresistance that characterizes this pathogen could interfere with the success of antimicrobial therapy, so it is a priority to monitor changes in susceptibility patterns, as a transcendental part of the resistance control policy antimicrobial. Objective: To determine the behavior of antimicrobial resistance against the antimicrobials of interest used in the treatment, the presence of enzymatic virulence factors and whether there is a relationship between them. Methods: A descriptive cross-sectional study was conducted during July 2012 to December 2015. Where 500 isolates belonging to the cepary of the National Reference Laboratory for Acute Diarrheal Diseases of the Institute of Tropical Medicine Pedro Kourí, from outbreaks of EDA of the national network of Microbiology laboratories in Cuba. Conventional phenotypic methods were applied to determine the behavior of antimicrobial resistance, the presence of enzymatic factors and their relationship with antimicrobial resistance. Results: The highest percentages of sensitivity were obtained against azithromycin (98 percent), doxycycline (96 percent) and ciprofloxacin (93 percent) and resistance to ampicillin (100 percent) and trimethoprim-sulfamethoxazole (99.4 percent). 44 isolated (8.8 percent) multi-resistant were found. All isolates had at least two extracellular enzymes as virulence factors, the most frequent: gelatinase (96 percent) and lecithinase (95 percent). Conclusions: There is a direct and proportional relationship between the presence of virulence factors and antimicrobial resistance, synergism that arises greater pathogenicity of the isolates studied from epidemic outbreaks(AU)

Cepas chilenas de origen clínico de Vibrio cholerae no-O1, no-O139 portan los genes vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF del sistema de secreción T3SS2 presentes en una isla de patogenicidad / Chilean strains of clinical origin of non-O1, non-O139 Vibrio cholerae carry the genes vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF from secretion system T3SS2 present in an island of pathogenicity

Resumen Introducción. Los factores de virulencia de las cepas de Vibrio cholerae no-O1, no-O139 no son claramente conocidos. La cepa de origen septicémico NN1 Vibrio cholerae no-O1, no-O139 fue secuenciada previamente mediante la plataforma Illumina, detectándose en su genoma un fragmento de la isla de patogenicidad VPaI-7 de V. parahaemolyticus. Objetivo: detectar los genes de virulencia vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF en cepas chilenas clínicas de V. cholerae no-O1, no-O139. Material y Métodos: Un total de 9 cepas chilenas de origen clínico de Vibrio cholerae no-O1, no-O139 aisladas entre 2006-2012 fueron analizadas mediante ensayos de reacción de polimerasa en cadena (RPC, en inglés PCR) convencional para los genes de secreción tipo III codificados en dicha isla: vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF. Adicionalmente se determinó la presencia de los genes de virulencia hylA y rtxA. Además, se realizaron ensayos de repetitive element palindromic PCR (REP-PCR) y Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Resultados: la mayoría (6/9) de las cepas chilenas de V. cholerae no-O1, no-O139 contiene todos los genes de secreción tipo III vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF, codificados en una isla de patogenicidad. Además, el total de las cepas (9/9) contiene los genes de virulencia hylA y rtxA. Conclusión: Estos resultados sugieren fuertemente la posibilidad que dichas cepas posean un potencial de virulencia importante en seres humanos.

Backgound: The virulence factors of the Vibrio cholerae non-O1, non-O139 strains are not clearly known. The strain of septicemic origin NN1 Vibrio cholerae non-O1, non-O139 was sequenced previously by the Illumina platform. A fragment of the pathogenicity island VPaI-7 of V. parahaemolyticus was detected in its genome. Aim: To detect the virulence genes vcsN2, vcsC2, vcsV2, vspD, toxR2 y vopF in Chilean strains of V. cholerae non-O1, non-O139. Methods: A total of 9 Chilean strains of clinical origin of Vibrio cholerae non-O1, non-O139 isolated between 2006-2012 were analyzed by conventional PCR assays for type III secretion genes encoded on that island: vcsN2, vcsC2, vcsV2, vspD, toxR2 and vopF. Additionally, the presence of the virulence genes hylA and rtxA was determined. In addition, REP-PCR and ERIC-PCR assays were performed. Results: most (6/9) Chilean V. cholerae non-O1, non-O139 strains contain the type III secretion genes vcsN2, vcsC2, vcsV2, vspD, toxR2 and vopF, encoded in an island of pathogenicity. In addition, all (9/9) the strains contain the virulence genes hylA and rtxA. Conclusion: These results strongly suggest the possibility that those strains possess an important virulence potential in humans.

Bacteriemia por Vibrio cholerae no-O1/no-O139 que porta una región homóloga a la isla de patogenicidad VpaI-7 / Bacteremia caused by Vibrio cholerae non-O1/non-O139 carrying a region homologous to pathogenicity island VpaI-7

Resumen Presentamos un caso de bacteriemia por Vibrio cholerae no-O1/ no-O139 en una mujer de 81 años con un cuadro de dolor abdominal, fiebre, vómitos, diarrea, coluria e ictericia, mientras visitaba una zona rural sin acceso a agua potable. La identificación se realizó por la técnica de espectrometría de masa MALDI-TOF, confirmándose una cepa no toxigénica no-O1/no-139. La caracterización molecular del aislado demostró la ausencia del gen de la toxina del cólera (CTX), y pilus TCP; sin embargo, presentó cinco de los seis genes de virulencia presentes en la isla de patogenicidad homóloga denominada VPaI-7 del V. parahaemolyticus (vcs N2+, vcs C2+, vcs V2+,toxR-, vspD+, T vopF+). Además, el aislado presentó los genes de virulencia hylA y rtxA. Este es el primer caso reportado en Chile de una cepa clínica de V. cholerae no-O1, no-O139 aislada de hemocultivos portador de un segmento homólogo de la isla de patogenicidad denominada VPaI-7 de V. parahaemolyticus, el cual codifica para un sistema de secreción tipo III (TTSS), que probablemente contribuye a su virulencia.

We report a case of V. cholerae non-O1 / non-O139 bacteremia in an 81-year-old woman with abdominal pain, fever, vomiting, liquid stools, choluria and jaundice, while visiting a rural area without access to potable water. The identification was made by the MALDI-TOF mass spectrometry technique and subsequently the non-toxigenic non-O1 / non-139 strain was confirmed in the national reference laboratory. The molecular characterization demonstrated the absence of the cholera toxin gene (CTX), and the TCP pilus, however, presented 5 of 6 virulence genes present in an island of homologous pathogenicity named VPaI-7 of V. parahaemolyticus (vcs N2 +, vcs C2 +, vcs V2 +, toxR-, vspD +, T vopF +) and in addition it was positive for hylAy rtxA virulence genes recognized outside the island. This is the first case reported in Chile of a clinical strain of V. cholerae non-O1, non-O139 isolated from blood culture that carries in its genome a homologous segment of the pathogenicity island named VPaI-7 of V. parahaemolyticus, which codifies for a type III secretion system (TTSS) that probably contributes to his virulence.

Características clinicoepidemiológicas de la enfermedad diarreica aguda por Vibrio cholerae en pacientes de hasta 10 años / Clinical epidemiological characteristics of the acute diarrheal disease due to Vibrio cholerae in patients up to 10 years

Se realizó una investigación observacional, descriptiva y transversal de 95 niños en las edades de 0 a 10 años, con diagnóstico de enfermedad diarreica aguda a causa del Vibrio cholerae, atendidos en el Hospital Infantil Norte Dr Juan de la Cruz Martínez Maceira de Santiago de Cuba, durante el 2016, a fin de caracterizarles según algunas variables clínicas y epidemiológicas. Entre los principales resultados se obtuvo que el grupo etario más afectado fuera el de 0 a 11 meses y el municipio con mayor número de casos el de Santiago de Cuba, los que correspondieron fundamentalmente a las áreas de salud de los policlínicos Frank País García, José Martí Pérez y Josué País García. Asimismo se evidenció que la principal manifestación del proceso infeccioso fue la diarrea líquida y la complicación más frecuente, la deshidratación isotónica moderada. Todos los niños egresaron vivos, lo cual demuestra la eficacia de la atención médica en el territorio suroriental de Cuba

An observational, descriptive and cross-sectional investigation of 95 children aged 0 to 10, with diagnosis of acute diarrheal disease due to Vibrio cholerae, assisted in Dr Juan de la Cruz Martínez Maceira Northern Children Hospital in Santiago de Cuba, was carried out during 2016, in order to characterize them according to some clinical and epidemiological variables. Among the predominant results there were the 0 to 11 months age group as the most affected and the presence of a higher number of cases in Santiago de Cuba municipality, that corresponded mainly to the health areas of Frank País García, José Martí Pérez and Josué País García polyclinics. Also it was evidenced that the main manifestation of the infectious process was the liquid diarrhea and the most frequent complication, the moderate isotonic dehydration. None of the children died, which demonstrates the effectiveness of medical care in the southeastern territory of Cuba

Control de foco de un brote de Cólera. Matanzas 2014 / Control of Focus in front of a Bud of Cholera. Matanzas 2014

Se presenta un estudio de control de foco de un brote de cólera, mediante la técnica observacional del tipo serie de casos, en el mes de julio de 2014 en el Municipio Matanzas, donde se describe, según el método epidemiológico, la relación de los casos detectados con el caso índice; se analiza el problema detectado teniendo en cuenta el enfoque de riesgo del cólera. Se realiza una evolución clínico-epidemiológica de los casos detectados y se analizan las medidas de control de foco aplicadas. Se revisan tanto las historias clínicas individuales en el consultorio del médico de la familia, como las encuestas epidemiológicas y el expediente de control de foco en el departamento de Epidemiología (AU).

A study of control of focus of a bud of cholera is presented, by means of the observational technique of the type series of cases, in the month of July of 2014 in the Municipality Matanzas, where it is described, according to the epidemic method, the relationship of the cases detected with the index case; the detected problem is analyzed keeping in mind the focus of risk of the cholera. He/she is carried out a clinical-epidemic evolution of the detected cases and of the applied measures of focus control. They are revised the clinical histories so much in the clinic of the doctor of the family, as the epidemic surveys and the file of focus control in the department of epidemiology (AU).

Characterization of Vibrio cholerae isolates from 1976 to 2013 in Shandong Province, China

Abstract Cholera continues to be a serious public health issue in developing countries. We analyzed the epidemiological data of cholera from 1976 to 2013 in Shandong Province, an eastern coastal area of China. A total of 250 Vibrio cholerae isolates were selected for PCR analysis of virulence genes and pulsed-field gel electrophoresis (PFGE). The analysis of the virulence genes showed that the positive rates for tcpA and tcpI were the highest among strains from the southwest region, which had the highest incidence rate of cholera. Low positive rates for tcpA, tcpI and ctxAB among isolates from after 2000 may be an influencing factor contributing to the contemporary decline in cholera incidence rates. Spatiotemporal serotype shifts (Ogawa, Inaba, Ogawa, Inaba and O139) generally correlated with the variations in the PFGE patterns (PIV, PIIIc, PIa, PIIIb, PIIIa, PIb, and PII). O1 strains from different years or regions also had similar PFGE patterns, while O139 strains exclusively formed one cluster and differed from all other O1 strains. These data indicate that V. cholerae isolates in Shandong Province have continually undergone spatiotemporal changes. The serotype switching between Ogawa and Inaba originated from indigenous strains, while the emergence of serogroup O139 appeared to be unrelated to endemic V. cholerae O1 strains.

Tipificación molecular de aislados de vibrio cholerae O1 causantes de dos brotes de cólera en Venezuela / Molecular typing of vibrio cholerae O1 isolates causing two outbreaks of cholera in Venezuela

En Venezuela, en junio de 1996, se reportó que los casos de cólera eran causados por V. cholerae O1 serotipo Ogawa. A finales de 1998 se detectó un segundo brote de cólera causado por V. cholerae O1 serotipo Inaba resistente a la ampicilina y el trimetoprim-sulfametoxazol. Para estudiar las relaciones entre las cepas se examinaron veinticinco aislados de Vibrio cholerae O1 obtenidos desde 1996 a 2000 en Venezuela, para determinar la presencia de genes de virulencia y perfiles genómicos. Mediante la reacción en cadena de la polimerasa se determinó la presencia de genes de virulencia. Para determinar el perfil genómico de los aislamientos se utilizó ribotipificación y electroforesis en gel de campo pulsado (PFGE). Todos los aislados resultaron positivos para los genes ctxA, ctxB, zot y ace. El análisis RFLP de los genes RNAr mostró un único patrón de ribotipo V. El análisis de PFGE mostró una similitud de 91,5% independientemente del año o lugar de aislamiento, lo que indica la relación genómica entre los aislados. En conjunto, los datos sugieren que la cepa de V. cholerae O1 resistente a los antibióticos que apareció en 1998 surgió de la cepa epidémica anterior o de otro estrechamente relacionado con el clon anterior, con cambio de serotipo y ganancia de determinantes de resistencia a antibióticos. Es muy importante monitorear continuamente la aparición de la variantes porque mejorará la comprensión de la evolución de nuevos clones de V. cholerae

In Venezuela, cholera reported in June 1996 was caused by V. cholerae O1 serotype Ogawa. Second outbreak of cholera caused by V. cholerae O1 serotype Inaba, resistant to ampicillin and trimethoprim- Sulfamethoxazole, was notify at the end of 1998. Twenty-five isolates of Vibrio cholerae O1 obtained from 1996 to 2000 in Venezuela were examined to study the relationships between strains, presence of virulence genes and genomic profiles. Presence of virulence genes was detected by Polymerase Chain Reaction. Ribotyping and pulsed-field gel electrophoresis (PFGE) were used to determine the genomic profile of isolates. All isolates shown PCR product for ctxA, ctxB, zot and ace genes. RFLP analysis of rRNA gene showed one unique pattern from ribotype V. PFGE analysis revealed a similarity of 91.5%, regardless year or place of isolation, suggesting genomic relatedness among them. Overall, these data suggest that antibiotic resistant V. cholerae O1 El Tor strain that appeared in 1998 emerged from the previous epidemic strain or from another closely related to the previous clone. It is important the continuous monitor the emergence of variants because it will improve our understanding of the evolution of new clones V. cholerae

Plásmido conjugativo portador de integrón clase 1 responsable de la resistencia a los antibióticos en aislados de vibrio cholerae O1 en Venezuela / Conjugative plasmid and a class 1integron responsible for the resistance to antibiotics in vibrio cholerae O1 isolates in Venezuela

Se han descrito aislados de Vibrio cholerae resistentes a una amplia variedad de antibióticos. En Venezuela, durante el brote de cólera ocurrido entre noviembre de 1998 y enero 2000 fueron reportados por primera vez aislados de V. cholerae O1 resistentes a ampicilina, trimetoprim-sulfametoxazole. Usando experimentos de conjugación se determinó la capacidad de transferir los determinantes de resistencia a ampicilina y trimetoprim-sulfametoxazole en 11 aislados. La visualización de plásmidos se realizó utilizando la digestión con nucleasa S1 y electroforesis en campo pulsante. La presencia de integrones de clase 1 fue establecida por PCR y se obtuvo la secuencia de la región variable del integrón. Los determinantes de resistencia fueron transferidos en un plásmido conjugativo de aproximadamente 170 kbp, común a todos los aislados. La resistencia a trimetoprim esta codificada en el gen dfra15, el cual se encuentra en un integrón clase 1 presente en el plásmido. En este estudio, se caracterizó la localización genética de los determinantes que codifican la resistencia a los antibióticos, y al conocer el mecanismo probable de dispersión de los determinantes de resistencia se podrán implementar medidas de control más adecuadas.

Vibrio cholerae has been reported to be resistant to a wide range of antibiotics. V. Cholerae O1 strains resistant to ampicillin, trimethoprim-sulfamethoxazole were isolated for the first time in Venezuela during a cholera outbreak that occurred between November 1998 and January 2000. Using conjugation experiments, the capacity of transfer of the resistance determinants in 11 strains resistant to ampicillin and trimethoprim-sulfamethoxazole was investigated. Plasmid analysis was done by S1 nuclease digestion and pulsed field gel electrophoresis. The presence of class 1 integrons was determined by PCR and the sequence of the gene harbored in the variable region of the integron was obtained. The antibiotic resistance determinants were transferred by a conjugative plasmid of approximately 170 kbp, common to all the isolates. Resistance to trimethoprim is encoded by the dfra15 gene that is harbored by a class 1 integron present in the plasmid. In this study, the genetic location of the determinants that code for resistance to antibiotics was characterized, and knowing the probable mechanism of dispersion of the determinants of resistance, control measures can be implemented most appropriate

DETECTION OF VIRULENCE GENES IN ENVIRONMENTAL STRAINS OF Vibrio cholerae FROM ESTUARIES IN NORTHEASTERN BRAZIL / Detecção de genes de virulência em estirpes de Vibrio cholerae isolados de estuários no Nordeste do Brasil

The objectives of this study were to detect the presence of Vibrio cholerae in tropical estuaries (Northeastern Brazil) and to search for virulence factors in the environmental isolates. Water and sediment samples were inoculated onto a vibrio-selective medium (TCBS), and colonies with morphological resemblance to V. cholerae were isolated. The cultures were identified phenotypically using a dichotomous key based on biochemical characteristics. The total DNA extracted was amplified by PCR to detect ompW and by multiplex PCR to detect the virulence genes ctx, tcp, zot and rfbO1. The results of the phenotypic and genotypic identification were compared. Nine strains of V. cholerae were identified phenotypically, five of which were confirmed by detection of the species-specific gene ompW. The dichotomous key was efficient at differentiating environmental strains of V. cholerae. Strains of V. cholerae were found in all four estuaries, but none possessed virulence genes.

O objetivo deste estudo foi detectar a presença potencial virulência de Vibrio cholerae isolado de estuários do Nordeste do Brasil. Amostras de água e sedimento foram coletadas e inoculadas sobre meio seletivo para víbrios (TCBS) e colônias com características morfológicas de V. cholerae foram isoladas. A identificação fenotípica seguiu chave dicotômica baseada em caraterísticas bioquímicas. Foram empregadas as técnicas de amplificação da polimerase em cadeia (PCR) utilizando o gene ompW e a de multiplex PCR para detecção de genes de virulência (ctx, tcp, zot e rfbO1). Os resultados da identificação das diferentes abordagens foram comparados. Nove cepas de V. cholerae foram identificadas fenotipicamente e cinco confirmadas através da detecção do gene ompW. A chave dicotômica utilizada foi eficiente para a confirmação da espécie. Os quatro estuários analisados apresentaram estirpes de V. cholerae, e nenhuma das cepas isoladas apresentaram genes de virulência.

Cólera, ¿se aproxima una nueva pandemia? / Cholera, is a new pandemy approaching?

Introducción: el cólera es una enfermedad de origen bacteriano que ha causado epidemias, muerte y desolación desde los albores de la humanidad. Aún hoy, con todos los adelantos de las ciencias, sigue siendo una amenaza mortal para los seres humanos, en particular para quienes viven bajo condiciones de pobreza, con poco acceso al agua potable y a un adecuado manejo de excretas; de ahí su interés para la salud pública internacional. Metodología: se realizó una búsqueda virtual de boletines epidemiológicos, reseñas de prensa o comunicados públicos relacionados con el cólera, sobretodo en Colombia, además de una búsqueda de literatura actualizada sobre las características histolopatológicas, clínicas y microbiológicas relevantes para el área de la salud pública. Objetivo: hacer una revisión de la literatura sobre el cólera, con énfasis en la epidemiología y los conceptos básicos de diagnóstico y manejo del evento, con el fin de preparar a la comunidad científica para hacer frente a lo que pudiera ser la nueva pandemia. Desarrollo: se considera que en los últimos 200 años han ocurrido en el mundo entre siete y ocho pandemias de cólera, la última hace alrededor de 20 años. Sin embargo, desde el año 2010, estalló un brote después del terremoto en Haití al parecer con casos provenientes de Nepal que ha expandido una onda epidémica por República Dominicana, Cuba y recientemente México. La propagación de esta epidemia se ha convertido en una preocupación para las Américas, en particular para Colombia cuyo personal de salud, joven y sin la experiencia en las epidemias anteriores, puede no estar preparado para afrontarla. Conclusiones: los avances de la ciencia han sido útiles para monitorear las epidemias, elaborar y probar vacunas, disminuir las tasas de letalidad del 70% a menos del 1%, si recibe manejo adecuado y oportuno. Pero el temor a nuevos brotes y epidemias sigue estando latente, particularmente por epidemias como las ocurridas ...

Background. Cholera is a bacterial disease that has caused epidemics, death and desolation since the dawn of humanity. Nowadays, even with the advances of science, it remains as a deadly threat to humans, particularly for those living under conditions of poverty, with little access to clean water and sanitation, and hence its interest to international public health. It is considered that seven or eight cholera pandemics have already occurred during the last 200 years, the last of them around 20 years ago. However, an outbreak of cholera was reported in 2010, after the earthquake in Haiti since some cases from Nepal, and it has been expanding as an epidemic wave through Dominican Republic, Cuba and recently Mexico. The spread of this epidemic has become a concern for every American country, particularly for Colombia whose health staff, young and inexperienced, might not be prepared to face it. Methods. We did an online research for literary newsletters, press reviews or public statements related to cholera, especially in Colombia, along with a scientific search update on pathological, clinical and microbiological Cholera characteristics relevant to the area of public health. Objective. To review the literature about Cholera, with emphasis on epidemiology and basic concepts of diagnosis and management of the event, in order to prepare the scientific community to deal with what seems to be a new pandemic. Conclusions. Advances in science have been useful for monitoring epidemics, develop and test vaccines, reduce fatality rates of 70% to less than 1% if appropriate and opportune medical management is recieved. But the fear of further outbreaks and epidemics remains latent, particularly epidemics such as those in Haiti, Dominican Republic, Guinea Bissau and South Sudan. It is necessary to raise the performance of preventive measures in communities and assess the readiness of health systems to deal with the eventual arrival of the ...

Loop-mediated isothermal amplification assays for screening of bacterial integrons

BACKGROUND: The occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently. As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection. RESULTS: In this study, three loop-mediated isothermal amplification (LAMP) assays targeting on class 1, 2 and 3 integrons were implemented and evaluated. Optimization of these detection assays were performed, including studing on the reaction temperature, volume, time, sensitivity and specificity (both primers and targets). Application of the established LAMP assays were further verified on a total of 1082 isolates (previously identified to be 397 integron-positive and 685 integron-negative strains). According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 µL, respectively. As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively. Other 685 integron-negative bacteria were negative for the integron-screening LAMP assays, totaling the detection rate and specificity to be 100%. CONCLUSIONS: The intI1-, intI2- and intI3-LAMP assays established in this study were demonstrated to be the valid and rapid detection methodologies for the screening of bacterial integrons.

In vivo antipyretic, antiemetic, in vitro membrane stabilization, antimicrobial, and cytotoxic activities of different extracts from Spilanthes paniculata leaves

BACKGROUND: The study was conducted to evaluate the in vitro antimicrobial activity, cytotoxic, and membrane stabilization activities, and in vivo antiemetic and antipyretic potentials of ethanolic extract, n-hexane and ethyl acetate soluble fractions of Spilanthes paniculata leaves for the first time widely used in the traditional treatments in Bangladesh. RESULTS: In antipyretic activity assay, a significant reduction (P < 0.05) was observed in the temperature in the mice tested. At dose 400 mg/kg-body weight, the n-hexane soluble fraction showed the effect (36.7 ± 0.63°C ) as like as the standard (dose 150 mg/kg-body weight) after 5 h of administration. Extracts showed significant (P < 0.001) potential when tested for the antiemetic activity compared to the standard, metoclopramide. At dose 50 mg/kg-body weight, the standard showed 67.23% inhibition, whereas n-hexane and ethyl acetate soluble fractions showed 37.53% and 24.93% inhibition of emesis respectively at dose 400 mg/kg-body weight. In antimicrobial activity assay, the n-hexane soluble fraction (400 µg/disc) showed salient activity against the tested organisms. It exerts highest activity against Salmonella typhi (16.9 mm zone of inhibition); besides, crude, and ethyl acetate extracts showed resistance to Bacillus cereus and Bacillus subtilis, and Vibrio cholera respectively. All the extracts were tested for lysis of the erythrocytes. At the concentration of 1mg/ml, ethanol extract, and n-hexane and ethyl acetate soluble fractions significantly inhibited hypotonic solution induced lysis of the human red blood cell (HRBC) (27.406 ± 3.57, 46.034 ± 3.251, and 30.72 ± 5.679% respectively); where standard drug acetylsalicylic acid (concentration 0.1 mg/ml) showed 77.276 ± 0.321% inhibition. In case of heat induced HRBC hemolysis, the plant extracts also showed significant activity (34.21 ± 4.72, 21.81 ± 3.08, and 27.62 ± 8.79% inhibition respectively). In the brine shrimp lethality bioassay, the n-hexane fraction showed potent (LC50 value 48.978 µg/ml) activity, whereas ethyl acetate fraction showed mild (LC50 value 216.77 µg/ml) cytotoxic activity. CONCLUSIONS: Our results showed that the n-hexane extract has better effects than the other in all trials. In the context, it can be said that the leaves of S. paniculata possess remarkable pharmacological effects, and justify its folkloric use as antimicrobial, antipyretic, anti-inflammatory, and antiemetic agent. Therefore, further research may be suggested to find possible mode of action of the plant part.

Inhibición de colonización intestinal por Vibrio cholerae con Lactobacillus acidophilus1 en conejos lactantes / Lactobacillus probiotics may prevent Vibrio cholerae colonization in rabbits newborn

Objetivo. Evaluar la capacidad in vitro e in vivo de Lactobacillus acidophilus1, aislado en este estudio, para prevenir enfermedad diarreica causada por Vibrio cholerae 01 OGAWA, en conejos lactantes. Materiales y métodos. Se aisló Lactobacillus acidophilus1 a partir de heces de niños sanos, se seleccionó por su capacidad amilolítica a partir de un grupo de bacterias ácido lácticas (BAL). El mejor sustrato amiláceo para el crecimiento de la BAL fue estandarizado previamente. Con L. acidophilus1 se realizó un ensayo in vitro e in vivo de inhibición antagónica sobre el enteropatógeno y se evaluó la prevención de la colonización por V. cholerae 01 OGAWA en conejos lactantes. Resultados. De acuerdo con el análisis de varianza L. acidophilus1 presentó la mejor capacidad amilolítica respecto a las otras BAL aisladas, p<0.5. Se encontró que un inóculo con una densidad celular de 35x10(6) bacterias/ml en el fermento láctico es capaz de ejercer el mayor efecto antagónico in vitro sobre V. cholerae. Se demostró el efecto probiótico in vivo, ya que los conejos enfrentados con el patógeno y sin recibir probiótico tuvieron una probabilidad de sobrevida menor de 0.25 respecto al grupo de animales retados con el patógeno y simultáneamente alimentados con el probiótico cuya probabilidad de sobrevida fue de 0.95. Conclusiones. L. acidophilus1 se considera un microorganismo probiótico, capaz de sobrevivir a su paso por el tracto gastrointestinal en un modelo animal y prevenir la colonización intestinal por V. cholerae 01 OGAWA en conejos lactantes.

Objective. To assess the capacity in vitro and in vivo of Lactobacillus acidophilus1 isolated in this study, capable of preventing diarrheal disease caused by Vibrio cholerae 01 OGAWA in rabbits newborn. Materials and methods. Lactobacillus acidophilus1 was isolated from faeces of healthy children and was selected from a group of lactic acid bacteria (LAB). The better starchy substrate for the growth of the LAB was previously standardized. Using the L. acidophilus1, there was carried out an in vitro and in vivo assay of antagonistic inhibition on the enteropathogen and it was evaluated the prevention of colonization by V. cholerae 01 Ogawa in rabbits newborn. Results. L. acidophilus1 present the better amylolytic capacity, p<0.5 in the ANDEVA. It was found that an inoculum with a cell density of 35x10(6) bacteria/ml in the lactic ferment is able to exert the greatest antagonistic effect in vitro over V. cholerae. The probiotic effect was demonstrated in vivo, since rabbits exposed to the pathogen without having received probiotic had a lower survival probability of 0.25 regarding the group of animals exposed with the pathogen and simultaneously fed with the probiotic whose probability of survival was of 0.95. Conclusions. L. acidophilus1 is considered a probiotic microorganism capable of surviving when passing through the gastrointestinal tract in an animal model and preventing intestinal colonization by V. cholerae 01 Ogawa in rabbits newborn.

La promoción y prevención de salud es la piedra angular para erradicar el cólera en el primer nivel de atención / Prevention and promotion is the cornerstone in the effort to erradicate cholera in primary care

Introducción:el Cholera morbus ha acompañado a la humanidad desde tiempos inmemoriales. Existen antecedentes en China, India y la Grecia clásica. Sin embargo, sólo se registran datos a partir de 1817 cuando se documenta la primera pandemia surgida en Asia y que se extendió a Turquía y los países árabes. Desde entonces se han sucedió ocho episodios pandémicos de cólera en el mundo. Objetivo: comunicar a la comunidad científica sobre la situación epidemiológica ocurrida en el mes de julio de 2012 en la provincia de Granma, de las medidas de promoción y prevención de salud que se desarrollaron en el primer nivel de atención para el cuadro diarreico causado por el Vibrio cholerae. Estas medidas las implementaron los equipos básicos de salud en el territorio con la participación intersectorial, lo que redunda en la adopción de estilos de vida saludables por parte de la población en general. Conclusión: primaron las actividades de saneamiento ambiental para disminuir la propagación de la enfermedad, complementadas con sugerencias a la comunidad médica sobre la importancia de la vigilancia epidemiológica ante eventuales reemergentes similares.

Introduction.Cholera has accompanied mankind since time immemorial with historical records from China, India and ancient Greece. However, registers are available only since 1817, when the first documented pandemic emerged in Asia and spread to Turkey and Arab countries. Since then, eight episodes of cholera pandemic have occurred in the world. Objective. To inform the scientific community about the epidemiological situation in the province of Granma, Cuba, at the cross-sectional period of July 2012 and the prevention and health promotion measures that were introduced in the first level of care for cases of diarrhea caused by Vibrio cholerae. These measures were implemented in the territory with an intersectoral approach, resulting in the adoption of healthy lifestyles by the general population. Conclusion. Basic sanitation activities were undertaken to slow the spread of the disease, complemented by suggestions to the medical community about the importance of epidemiological surveillance to detect reemergence.

Epidemiologia genômica de vibrio cholerae da epidemia de cólera na América Latina / Genomic Epidemiology of Vibrio cholerae cholera epidemic in Latin America

Vibrio cholerae é o agente causal da cólera, uma infecção ancestral, epidêmica e pandêmica, que é um dos principais problemas de saúde pública no mundo. A humanidade já passou por, pelo menos, seis pandemias de cólera e, atualmente, vive no contexto da sétima pandemia. V. cholerae é classificado em mais de 200 sorogrupos e alguns biotipos. Linhagens de V. cholerae O1 do biótipo clássico e do biotipo El Tor estão relacionadas à sexta (1899–1923) e à sétima (1960 -….) pandemias de cólera, respectivamente. A sétima pandemia chegou à América Latina em 1991 sendo que, inicialmente, sua origem foi relacionada ao Sudoeste Asiático, região endêmica e epidêmica de cólera. Uma segunda hipótese sugere uma origem ambiental local. A partir do início deste século, a epidemia se extinguiu, restando apenas eventuais relatos de casos isolados. Sob a ótica da genética e genômica do V. cholerae, estudamos a linhagem da epidemia da América Latina (AL). Nossos objetivos eram inferir sua origem e identificar marcadores genômicos que possibilitassem seu monitoramento. Utilizamos informações obtidas in vitro e in silico para estabelecer relações genéticas entre isolados de V. cholerae de antes, durante e depois da epidemia que atingiu a América Latina. Analisamos regiões do genoma core e do genoma acessório. Realizamos análises de genômica comparativa com informação de 355 V. cholerae, clínicos e ambientais, de diferentes anos e regiões geográficas. Com base em três genes do genoma core, nossos resultados mostram que a linhagem da epidemia da AL pertence ao clado El Tor, e por sua vez, os genótipos dos principais determinantes de virulência ctxB e tcpA, (genoma acessório), os agrupa com isolados do início da sétima pandemia, incluindo o canônico N16961. As análises genômicas e filogenéticas revelaram a presença de um profago (WASA1) e de uma variante exclusiva da ilha genômica VSP-II. A VSP-II da linhagem da AL é caracterizada por seu gene da integrase e uma inserção de 7 Kb, que contém três genes putativos. Curiosamente, os dois marcadores, que fazem parte do genoma acessório desta linhagem, WASA1 e VSP-II, apresentam relação de similaridade com elementos genéticos identificados em Vibrio vulnificus/Vibrio parahaemolyticus e Vibrio vulnificus/V. cholerae do ambiente, respectivamente. Estes marcadores seriam, portanto, uma fração do mobiloma que evoluiu e se fixou na linhagem de V. cholerae da epidemia da América Latina.

Vibrio cholerae O1 from superficial water of the Tucunduba Stream, Brazilian Amazon

Isolation and genetic characterization of an environmental Vibrio cholerae O1 from the Amazon is reported. This strain lacks two major virulence factors - CTX and TCP - but carries other genes related to virulence. Genetic similarity with epidemic strains is evaluated and the importance of V. cholerae surveillance in the Amazon is emphasized.

Frequência de Escherichia coli e sua sensibilidade aos antimicrobianos em menores de cinco anos hospitalizados por diarreia aguda / The frequency of Escherichia coli and its sensitivity to antimicrobials in children aged under five years admitted to hospital for treatment of acute diarrhea

Determinar a frequência de Escherichia coli diarreiogênica e sua sensibilidade aos antimicrobianos em menores de cinco anos hospitalizados por diarreia aguda. MÉTODOS: estudo prospectivo tipo corte transversal realizado no Instituto de Medicina Integral Prof. Fernando Figueira, entre janeiro de 2010 e fevereiro de 2011. Foram excluídas as crianças com diagnóstico de imunodeficiência ou usando antimicrobianos. Para cada paciente foi feito uma única coleta de swab retal nas primeiras 24 horas de internação. Os patógenos foram identificados na coprocultura e sorotipagem. Os antibiogramas foram obtidos por disco-difusão. RESULTADOS: 140 crianças foram arroladas, em sua maioria provinham de famílias de baixa renda da Região Metropolitana do Recife. Foram isolados 99 microorganismos: 9 (6,4 por cento) E. coli enteropatogênica (EPEC) e 4 (2,9 por cento) E. coli enteroinvasora (EIEC) e 80 (57,1 por cento) outras E.coli não EPEC, não EIEC, 3 (2,1 por cento) Shigella spp e 3 (2,1 por cento) Salmonella spp. O perfil de sensibilidade aos antimicrobianos demonstrou níveis elevados de resistência à ampicilina e sulfametoxazol-trimetoprima. CONCLUSÕES: a baixa frequência de EPEC observada pode estar associada às condições de saneamento básico favoráveis apresentadas pelos pacientes do estudo. A análise local do perfil da sensibilidade da E. coli aos antimicrobianos reforça a recomendação da Organização Mundial de Saúde para o uso racional dessas drogas visando prevenção da resistência bacteriana...

To determine the frequency of diarrheic Escherichia coli and its sensitivity to antimicrobials in children aged under five years admitted to hospital for treatment of acute diarrhea. METHODS: a prospective cross-sectional study was carried out at the Instituto de Medicina Integral Prof. Fernando Figueira, between January 2010 and February 2011. Children were excluded if they had been diagnosed as immunodeficient or were using antimicrobials. A single rectal swab was taken from each patient during the first 24 hours of hospitalization. The pathogens were identified in the coproculture and serotyping. Antibiograms were obtained using disc-diffusion. RESULTS: 140 children were recruited. Most were from low-income families in the Metropolitan Region of Recife. Ninety-nine micro-organisms were isolated: 9 (6.4 percent) enteropathogenic E. coli (EPEC) and 4 (2.9 percent) enteroinvasive E. coli (EIEC) and 80 (57.1 percent) other E.coli that are neither EPEC nor EIEC, 3 (2.1 percent) Shigella spp and 3 (2.1 percent) Salmonella spp. The profile of sensitivity to antimicrobials showed high levels to resistance to ampicillin and sulfametho-xazol-trimetropime. CONCLUSIONS: the low frequency of EPEC found may be associated with basic sanitary conditions among the patients in the study. The local analysis of the profile of sensitivity of E. coli to antimicrobials corroborates the World Health Organization recommendation that these drugs be used prudently to ensure prevention of resistance in bacteria...

Gastroenteritis aguda causada por Vibrio cholerae no-O1, no-O139 que porta una región homologa a la isla de patogenicidad VpaI-7 / Acute gastroenteritis caused by a Vibrio cholerae non-O1, non-O139 strain harboring a genetic region homologous to the VpaI-7 pathogenicity island

Pathogenic Vibrio cholerae isolates, the etiologic agents of cholera, generally express one of two O antigens (O1 or O139). Most environmental isolates are nonpathogenic and are referred to as "non-O1, non-O139". However some V. cholerae non-O1, non-O139 strains are clearly pathogenic and have caused outbreaks or sporadic cases of gastroenteritis and extraintestinal infections in humans. We report a case of acute gastroenteritis by a V. cholerae non-O1, non-O139 harboring a genetic region homologous to a segment of the VpaI-7 V. parahaemolyticus pathogenicity island.

Cepas patogénicas de Vibrio cholerae, el agente causal del cólera, expresan generalmente uno de dos antígenos O (denominados O1 u O139). La mayoría de las cepas ambientales son no patogénicas y corresponden al tipo denominado "no-O1, no-O139". Sin embargo, algunas cepas de este tipo son claramente patogénas y han causado brotes de gastroenteritis e infecciones extra-intestinales en humanos. Se reporta un caso clínico de gastroenteritis aguda causado por una cepa de V. cholerae no-O1, no-O139 que contiene en su genoma una región homóloga a un segmento de la isla de patogenicidad VpaI-7 descrita previamente en V. parahaemolyticus.

Reporte histórico: Primer Aislamiento de Vibrio cholera serogrupo O1 biovar El Tor serovar Inaba durante la epidemia de cólera en el Perú ‑ 1991 / Historical report: first isolation of Vibrio cholera serogroup O1 biovar El Tor serovar Inaba during the cholerae epidemic in Perú ‑ 1991

Hace 20 años apareció una enfermedad diarreica nueva en el Perú y el Laboratorio de Referencia de Enteropatógenos del Instituto Nacional de Salud, cumplió una labor destacada en el aislamiento e identificación rápida y oportuna del Vibrio cholerae. La enfermedad del cólera no se había presentado anteriormente, pero en la última semana de enero de 1991 se detectó un brote epidémico de diarrea aguda con deshidratación intensa y algunos casos de fallecidos. La epidemia afectó, al comienzo, varias localidades del litoral peruano. Equipos de trabajo de la Oficina General de Epidemiología y de los laboratorios del Instituto Nacional de Salud obtuvieron muestras fecales de pacientes con diarrea aguda procedentes de las ciudades de Chancay, Chimbote, Piura y algunos hospitales de Lima. Las muestras colectadas en el medio de transporte de Cary y Blair fueron procesadas en el Laboratorio Nacional de Referencia de Enteropatógenos (LANARE) del Instituto Nacional de Salud. De todas las muestras se aisló e identificó Vibrio cholerae serogrupo O1 biovar El Tor serovar Inaba que mostró ser sensible a la tetraciclina y a otros antibióticos. Esta investigación confirmó el primer brote epidémico de cólera en el Perú.

20 years ago, a new diarrheal disease was introduced in Peru and the Enteropathogens Reference Laboratory of the Instituto Nacional de Salud had an outstanding role in the isolation and rapid and timely identification of Vibrio cholerae. Cholera had not been seen before, but during the last week of January 1991 an outbreak of acute diarrhea was detected, presenting intense dehydration and some deaths. The epidemic affected, in the beginning, many locations of the peruvian coast. Some working teams of the General Office of Epidemiology and of the Instituto Nacional de Salud obtained fecal samples from patients with acute diarrhea coming from the cities of Chancay, Chimbote, Piura and some hospitals in Lima. The collected samples were transported on Cary and Blair media and processed in the National Reference Laboratory of Enteropathogens (LANARE) of the Instituto Nacional de Salud. Vibrio cholerae serogroup 01 biovar El Tor serovar Inaba was isolated from all the samples, it was sensible to tetracyclines and other antibiotics. This research confirmed the first outbreak of cholera in Peru.