Bullous cellulitis as an extraordinary manifestation of a Vibrio cholerae O1 Ogawa infection

@#Vibrio cholerae is a gram-negative bacterium synonymous with its namesake disease, cholera. Thus, gastrointestinal symptoms are the norm and V. cholerae is very rarely associated with skin and soft tissue infections. We describe a case of a 63-year-old Chinese woman with multiple medical comorbidities on corticosteroid therapy who developed fever and a painful swelling on her left leg after being pricked by a branch while gardening. There was no abdominal pain, vomiting or diarrhea. A diagnosis of bullous cellulitis was made clinically, and blood was sent for bacteriological culture. A beta-hemolytic commashaped gram-negative bacillus was isolated from the blood. It was also oxidase-positive and produced an acid/alkaline (A/K) reaction on triple sugar iron agar. It was identified biochemically as Vibrio cholerae. After additional testing, it was found to be of the O1 serogroup and Ogawa serotype. The infection resolved following a 10-day course of high-dose co-trimoxazole therapy.

Expression in Saccharomyces boulardii of recombinant Toxin-coregulated pilus A subunit (TcpA) of Vibrio cholerae O1

Cholera is endemic in over 50 countries with an estimated mortality of 100,000-120,000. Vaccination is considered the complementary key to prevent and control cholera; therefore, alternative vaccine preparations are needed. Toxin Co-regulated Pilus is part of the toxR virulence regulon, which is necessary for colonization in the intestinal mucosa. In order to express Vibrio cholerae TcpA protein in Saccharomyces boulardii, the expression plasmid pYES2 was constructed by inserting tcpA gene isolated from local Vibrio cholerae Eltor Inaba isolates. The new construct was transferred into Saccharomyces boulardii cells and the expression of tcpA gene was induced from the GAL1 promoter by adding galactose to the medium. The SDS-PAGE and Western blot analysis showed the presence of TcpA in yeast. These results showed that Saccharomyces boulardii is a promising host to express Vibrio cholerae toxin TcpA as the first step in attempt to produce an oral Vibrio cholerae vaccine(AU)

El cólera es endémico en más de 50 países. Se estima una mortalidad entre 100.000 - 120.000 debido a esta enfermedad. La vacunación se considera una medida complementaria para prevenir y controlar el cólera, por lo tanto, se necesitan preparaciones vacunales alternativas a las existentes. El Pili corregulado con la toxina, es parte del regulón de virulencia toxR, y es necesario para la colonización en la mucosa intestinal. Para expresar la proteína tcpA de Vibrio cholerae en Saccharomyces boulardii, se construyó el plásmido de expresión pYES2 insertando el gen tcpA obtenido a partir de aislamientos locales de Vibrio cholerae El Tor Inaba. La nueva construcción se transfirió a las células de Saccharomyces boulardii y se indujo la expresión del gen tcpA a partir del promotor GAL1 mediante la adición de galactosa al medio. El análisis mediante SDS-PAGE y Western blot demostró la presencia de TcpA en levaduras. Los resultados demostraron que Saccharomyces boulardii es un hospedero prometedor para expresar el gen tcpA de Vibrio cholerae como el primer paso en el intento de producir una vacuna oral contra Vibrio cholerae(AU)

Comparison of colonization ability of O1 and O139 Vibrio cholerae strains on soft-shelled turtle's surface / 中华预防医学杂志

Objective@#To study the preferred colonization sites of O1 Vibrio cholerae (V.cholerae) and the colonization ability difference for O1 and O139 V. cholerae on soft-shelled turtle's surface.@*Methods@#8 O1 and O139 V. cholerae strains were obtained from branch of diarrheal diseases, Chinese center for disease control and prevention. 63 soft-shelled turtles weighing 150 g and 9 cm in length (diameter of calipash) were selected for use in the study. The preferred colonization sites and proliferation trend were studied by using bioluminescent imaging method. The colonization factors for O1 V. cholerae strains were studied by constructing colonization gene mutant strains (VC1897dmshA, VC1897dgbpA and VC1897dtcpA), performing competition colonization assays and analyzing the competitive indexes. After pairing off O1 and O139 strains respectively to perform 16 competition groups, the colonization difference of these two strains were studied by competition colonization assays.@*Results@#The colonization sites by V. cholerae on soft-shelled turtles surfaces was clustered. More V. cholerae strains colonized on turtle's calipash and carapace on dorsal side and less strain colonized on ventral side. The competition colonization assays showed that colonization ability of O1 serogroup mshA mutant strains were 7.26 times lower than VC1897dlacZ. Besides, the CI value (O139/O1) of 11 out of the 16 competition groups were greater than 2 (between 2.07 and 59.84). Two groups showed values of 1.43 and 0.93 respectively and 3 groups lower than 0.7.@*Conclusion@#The preferred colonization sites for O1 V. cholerae strains on body surface were observed.MSHA was one of the main colonization factors for its colonization. Our study suggested that in general, O139 V. cholerae strains have stronger colonization ability than O1 strains. Besides, strains isolated from soft-shelled turtles tend to have stronger colonization ability than strains isolated from patients.

Susceptibilidad antimicrobiana en aislamientos cubanos de Vibrio cholerae O1 procedentes de muestras clínicas / Antimicrobial susceptibility in Cuban isolates of Vibrio cholerae O1 obtained from clinical samples

Introducción: la re-emergencia de cólera en Haití estableció un nuevo reservorio para el incremento de la séptima pandemia. Esto provocó su diseminación a República Dominicana y a otros países de la región del Caribe, como Cuba y México. Objetivo: estudiar la susceptibilidad antimicrobiana de aislamientos de Vibrio cholerae O1, serotipo Ogawa, biotipo El Tor aisladas de pacientes durante el evento epidemiológico de cólera ocurrido en Cuba entre junio de 2012 y agosto de 2013. Métodos: se realizó el estudio de la susceptibilidad antimicrobiana in vitro en 144 aislamientos de V. cholerae, mediante el método de Bauer-Kirby frente a nueve antimicrobianos: ampicilina, sulfonamida, trimetoprim/sulfametoxazol, cloranfenicol, tetraciclina, doxiciclina, azitromicina, ciprofloxacina y gentamicina, según las normas del Instituto de Estándares de Laboratorio Clínico de los Estados Unidos de América. Resultados: el total de los aislamientos resultaron resistentes al trimetoprim-sulfametoxazol; el 98,7 por ciento lo fue a la sulfonamida y el 90,3 por ciento a la ampicilina. Se obtuvieron valores de sensibilidad intermedia para ciprofloxacina (30,6 por ciento) y cloranfenicol (27,1 por ciento). Se apreciaron niveles de sensibilidad superior al 92 por ciento a los antimicrobianos de primera línea en el tratamiento de la enfermedad (doxiciclina, tetraciclina y azitromicina), así como también a la gentamicina. No se observaron cepas multirresistentes. Conclusiones: los datos aportados por este trabajo demuestran la efectividad in vitro de los antimicrobianos utilizados en el tratamiento la enfermedad diarreica aguda causada por V. cholerae en Cuba(AU)

Introduction: re-emergence of cholera in Haiti created a new reservoir for the increase of the seventh pandemic. This resulted in its spread to the Dominican Republic and other Caribbean countries, such as Cuba and Mexico. Objectives: study the antimicrobial susceptibility of isolates of Vibrio cholerae O1, Ogawa serotype, El Tor biotype, obtained from patients during the cholera epidemiological event occurring in Cuba from June 2012 to August 2013. Methods: a study was conducted of 144 V. cholerae isolates using the Bauer-Kirby method to determine in vitro susceptibility to nine antimicrobials: ampicillin, sulfonamide, trimethoprim/sulfamethoxazole, chloramphenicol, tetracycline, doxycycline, azithromycin, ciprofloxacin and gentamicin, in compliance with standards from the U.S. Clinical and Laboratory Standards Institute. Results: all isolates were resistant to trimethoprim/sulfamethoxazole; 98.7 percent to sulfonamide and 90.3 percent to ampicillin. Intermediate sensitivity values were obtained for ciprofloxacin (30.6 percent) and chloramphenicol (27.1 percent). Sensitivity levels above 92 percent were found for first-line antimicrobials (doxycycline, tetracycline and azithromycin), as well as gentamicin. Multi-drug resistant strains were not found. Conclusions: results reveal the effectiveness in vitro of the antimicrobials used in Cuba to treat acute diarrheal disease caused by V. cholerae(AU)

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.

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.

Significância de anticorpos vibriocidas circulantes em área pós-epidêmica de diarréia, São Bento do Una, Estado de Pernambuco / The importance of circulating vibriocidal antibodies in a pos-epidemic region of diarrhea, São Bento do Una, State of Pernambuco

Verificou-se o nível de anticorpos vibriocidas em 41 indivíduos adultos, sem história passada ou presente de diarréia por Vibrio cholerae O1, residentes no município de São Bento do Una, Pernambuco. Nessa localidade ocorreu no início de 2004 um surto de diarréia, com múltiplos agentes bacterianos envolvidos, incluindo o vibrião colérico. Foi empregado o teste da microtitulação de anticorpos séricos vibriocidas, anti-Ogawa e anti-Inaba, considerando-se como indicativo de infecção por Vibrio cholerae O1, os títulos vibriocidas > 1:640. A freqüência dos reagentes foi de 36 (87,8 por cento) para o sorovar Ogawa, o que evidencia a possível circulação do vibrião colérico, durante e/ou após a epidemia de diarréia.

The levels of vibriocidal antibodies were investigated among 41 adults without any past or present history of diarrhea due to Vibrio cholerae O1 who were living in the municipality of São Bento do Una, Pernambuco. A diarrhea outbreak occurred in this locality at the beginning of 2004, involving multiple bacterial agents, including Vibrio cholerae. The microtitration test was used to investigate the presence of anti-Ogawa and anti-Inaba vibriocidal serum antibodies. Vibriocidal titers e" 1:640 were considered indicative of infection by Vibrio cholerae O1. The frequency of the reagents was 36 (87.8 percent) for the Ogawa serovar, which showed that Vibrio cholerae O1 was possibly circulating during and/or after the diarrhea epidemic.

Detection of viable and viable nonculturable Vibrio cholerae O1 through cultures and immunofluorescence in the Tucumán rivers, Argentina

Vibrio cholerae has been sporadically isolated from rivers in Tucumán, Argentina, since the outbreak in 1991. The aim of this study was to determine the environmental reservoir of the bacterium in these rivers, assessing the presence of Vibrio cholerae non-O1 and O1 (the latter both in its viable culturable and non culturable state) and its relationship to environmental physicochemical variables. 18 water samplings were collected in the Salí River (in Canal Norte and Banda) and the Lules River between 2003 and 2005. Physical-chemical measurements (pH, water temperature, electrical conductivity and dissolved oxygen) were examined. Vibrio cholerae was investigated with conventional culture methods and with Direct Immunofluorescence (DFA-VNC) in order to detect viable non culturable organisms. All isolated microorganisms corresponded to Vibrio cholerae non-O1 and non-O139 (Lules 26 percent, Canal Norte 33 percent and Banda 41 percent). The majority was found during spring and summer and correlated with temperature and pH. Non culturable Vibrio cholerae O1 was detected year round in 38 of the 54 water samples analyzed. Application of the Pearson correlation coefficient revealed that there was no relationship between positive immunofluorescence results and environmental physicochemical parameters. Genes coding for somatic antigen O1 were confirmed in all DFA-VNC-positive samples, whereas the virulence-associated ctxA and tcpA genes were confirmed in 24 samples.

Vibrio cholerae tem sido isolado esporadicamente nos rios da Província de Tucumán, Argentina, desde outubro de 1991. O objetivo deste estudo foi localizar os reservatórios nestes rios, identificar a presença de Vibrio cholerae O1 (em estado cultivável e não cultivável) e relacionar a presença desta bactéria com as variações físico-químicos da água. Foram coletadas dezoito amostras de água do rio Salí (nas localidades de Canal Norte e Banda) e do rio Lules, entre 2003 e 2005. Estas foram submetidas a análises físico-químicos como determinação de pH, temperatura, condutibilidade elétrica e oxigênio dissolvido. A presença de Vibrio cholerae foi verificada por métodos de cultivo convencional e por imunofluorescência direta (DFA-VNC). Todos os microrganismos isolados foram não O1 e não O139 (Lules 26 por cento, Canal Norte 33 por cento e Banda 41 por cento). A maioria foi encontrada na primavera e verão, indicando uma relação com a temperatura e pH. Das 54 amostras analisadas por DFA-VNC, 38 Vibrio cholerae não cultivável, foram detectadas em todas as épocas do ano. As amostras positivas foram confirmadas por PCR para o antígeno somático O1 e para os genes de virulência ctxA e tcpA. Coeficiente de correlação de Pearson revelou que não há relação entre os resultados obtidos por imunofluorescência e a variação dos parâmetros físico-químicos.

Desempeño de los sistemas Cholera-SMART® y Pathogen-Detection-Kit® en el diagnóstico rápido del cólera / Performance of Cholera-SMART® and Pathogen Detection-Kit® in the quick diagnosis of cholera

Objetivos. Comparar el desempeño de dos sistemas rápidos de diagnóstico de cólera con el método de cultivo y proponer una estrategia que permita mejorar la especificidad y la sensibilidad de estos sistemas y disminuir los costos del diagnóstico. Métodos. En el estudio participaron el Centro Nacional de Referencia en Bacteriología (CNRB) del Instituto Costarricense de Investigación y Enseñanza en Nutrición y Salud (INCIENSA) y hospitales de las provincias de Alajuela, Guanacaste y San José, en Costa Rica. Se emplearon 237 muestras de heces para evaluar el desempeño de dos pruebas rápidas para el diagnóstico de Vibrio cholerae O1: Pathogen Detection Kit® (PDK, Intelligent Monitoring Systems, Gainsville, Florida, EUA) y Cholera-SMART® (New Horizons Diagnostics Corp., Columbia, Maryland, EUA), tanto en forma directa (SMART directo y PDK directo) como a partir de cultivos de enriquecimiento de 6 horas (SMART-6 y PDK-6) y de 18 horas (SMART18 y PDK-18) a 37 °C en agua de peptona alcalina. Las muestras diarreicas y semiformadas se cultivaron y se evaluaron con las pruebas rápidas directas; cuando el resultado inicial era negativo se repitieron a las 6 y 18 horas de cultivo. Los hisopados rectales y fecales se evaluaron a partir de cultivos de enriquecimiento de 6 y de 18 horas. Adicionalmente se estudió la sensibilidad analítica de los sistemas rápidos con cultivos puros de 18 a 24 horas de incubación de V. cholerae O1 (cepa SOS-833, CNRB, Costa Rica) y se evaluó la utilidad del análisis microscópico de la motilidad para racionalizar el uso de las técnicas rápidas. Resultados. La sensibilidad, tanto de SMART directo como de PDK directo, fue de 100% en muestras de heces diarreicas y semiformadas y en contenido intestinal de cadáveres. Con estas muestras, el procedimiento SMART directo mostró una especificidad de 100%, mientras que con el PDK directo esta fue de 85,7% a 77,4%, en dependencia del tipo de muestra. Los resultados positivos falsos obtenidos mediante PDK directo resultaron negativos con PDK-6 y PDK-18. Entre los hisopados rectales y fecales de personas con y sin diarrea o que recibieron tratamiento previo con antibióticos se observaron tres resultados negativos falsos con SMART-6 y dos con PDK-6, los cuales resultaron positivos mediante SMART-18 y PDK-18, respectivamente. Ambos sistemas mostraron una concordancia excelente (índice kappa superior a 0,9) en las diferentes modalidades evaluadas. La sensibilidad analítica de ambos sistemas fue de 6 107 ufc/mL de V. cholerae O1, lo que concordó con la observación microscópica de 10 microorganismos o más con motilidad típica de vibriones por campo (aumento de 1000). Las muestras con menos de 10 microorganismos con motilidad típica de vibriones tenían concentraciones entre 6 103 y 6 106 ufc/mL y solo resultaron positivas después de un enriquecimiento de 6­18 horas. Se propone una estrategia para establecer la presencia de Vibrio cholerae O1 en un tiempo inferior al de los métodos convencionales, con valores predictivos positivo y negativo de 100%. Conclusiones. Los sistemas SMART y PDK permiten llegar a un diagnóstico certero de cólera en poco tiempo, no requieren de instrumental complejo ni de personal técnico altamente calificado y funcionan satisfactoriamente en condiciones de campo. Mediante la estrategia propuesta se pueden aumentar la especificidad y la sensibilidad de estos sistemas y se reducen los costos del diagnóstico, lo que permite recomendar su empleo para la vigilancia del cólera en áreas con escasos recursos, donde esta enfermedad constituye un grave problema de salud pública

Objectives. To compare the performance of two rapid systems for the diagnosis of cholera with the culture method, and to propose a strategy for improving the specificity and sensitivity of these systems and reducing the costs involved in making a diagnosis. Methods. The following institutions participated in the study: the National Bacteriology Referral Center (Centro Nacional de Referencia en Bacteriología, CNRB) of the Costa Rican Institute for Research and Teaching in Nutrition and Health (Instituto Costarricense de Investigación y Enseñanza en Nutrición y Salud, INCIENSA) and various hospitals in the provinces of Alajuela, Guanacaste and San José, in Costa Rica. A total of 237 feces samples were used to asses the performance of two tests for the rapid detection of Vibrio cholerae 01: the Pathogen Detection Kit© (PDK, Intelligent Monitoring Systems, Gainesville, Florida, USA) and Cholera-SMART© (New Horizons Diagnostics Corp., Columbia, Maryland, USA), both when applied directly (direct SMART and direct PDK) and when applied to specimens cultured in brothenriched medium for 6 hours (SMART-6 and CPK-6) and for 18 hours (SMART-18 and PDK-18) at 37 °C in alkaline peptone water. Liquid and partially formed stools were cultured and examined by means of the rapid direct test; when the initial result for periods of 6 and 18 hours. Rectal and fecal swabs were obtained from feces cultured in enriched-broth medium for 6 and 18 hours. In addition, we studied the sensitivity of the rapid testing systems by using pure cultures of V. cholerae 01 (strain SOS-833, CNRB, Costa Rica) that were incubated for 18 to 24 hours, and we assessed the usefulness of observing motility under the microscope in order to rationalize the use of rapid methods. Results. The sensitivity of the direct SMART test and of the direct PDK test was 100% when samples obtained from liquid and partially formed stools and from the intestinal contents of dead bodies were used. With these samples, the direct SMART procedure showed a specificity of 100%, whereas the direct PDK procedure showed a specificity that ranged from 85.7% to 77.4%, depending on the type of sample. False positives obtained with the direct PDK method turned out to be negative with PDK-6 and PDK-18. Among the rectal and fecal swabs of persons with and without diarrhea or who had received prior treatment with antibiotics, three results that were negative with the SMART-6 procedure and two that were negative with the PDK-6 procedure turned out to be positive with the SMART-18 and PDK-18 procedures, respectively. Both systems showed excellent concordance (kappa index above 0.9) throughout. Both systems were sensitive to 6 107 colony-forming units per milliliter (cfu/mL), which was concordant with the microscopic observation of 10 microorganisms or more per field with the type of motility that characterizes vibrios (at 1 000 magnification). Samples having fewer than 10 microorganisms with the motility that characterizes vibrios had concentrations between 6 103 and 6 106 cfu/mL and became positive only after incubation in enriched-broth medium for 6 to 18 hours. We propose a strategy for diagnosing the presence of V. cholerae 01 infection in less time than it takes with traditional methods, with positive and negative predictive values of 100%. Conclusions. The SMART and PDK systems make it possible to accurately diagnose cholera quickly, don't require sophisticated equipment or highly qualified technical personnel, and perform satisfactorily in field conditions. Through the proposed strategy, it becomes possible to improve the specificity and sensitivity of these systems and to reduce the cost of making a diagnosis, thus making them suitable for use in cholera surveillance in low-income settings where this disease is a serious public health problem