Determinación de biopelículas y betalactamasas de espectro extendido en Vibrio cholerae no-O1, no-O139 aislados de pacientes con diarreas en Cuba / Determination of biofilms and extended-spectrum beta-lactamases in Vibrio cholerae non-O1, non-O139 isolates from patients with diarrhea in Cuba

. Vibrio cholerae no-O1, no-O139 es el tercer grupo de bacterias del género Vibrio que con más frecuencia producen diarreas. Sobrevive en los ambientes acuáticos, utilizando la formación de biopelícula como mecanismo de supervivencia que propicia la transmisión de la enfermedad diarreica. Desde 1977 se caracterizan aislados de V. cholerae con resistencia múltiple, y algunos de los mecanismos involucrados incluyen la producción de β-lactamasas de espectro extendido (BLEE). Este trabajo tuvo como objetivo determinar la formación de biopelícula en los aislados cubanos de V. cholerae no-O1, no-O139, causantes de enfermedad diarreica aguda (EDA), y detectar la producción de BLEE en aquellos con resistencia total e intermedia a ampicilina. Se realizó un estudio descriptivo de corte transversal, entre enero 2014 y junio 2015. Se estudiaron 55 aislados caracterizados previamente, que formaban parte del cepario del Laboratorio Nacional de Referencia de EDA del Instituto Pedro Kourí. Para la determinación fenotípica de BLEE se estudiaron 43, de los que ya se conocía su susceptibilidad a ampicilina. El 54,5 por ciento de los aislados resultaron positivos a la formación de biopelícula, y predominaron los clasificados como formadores moderados (46,6 por ciento ) y débiles (36,6 por ciento ). De los 34 resistentes a ampicilina, 26,5 por ciento resultaron positivos a la producción de BLEE. En el caso de los nueve aislados con resistencia intermedia a ampicilina, 44,4 por ciento resultaron positivos. Los resultados del presente estudio contribuyen al conocimiento sobre la capacidad que tienen de persistir en el ambiente y permiten profundizar sobre los mecanismos de resistencia a los antimicrobianos(AU)

Vibrio cholerae non-O1, non-O139 is the third bacterium group from the genus Vibrio most commonly causing diarrhea. It survives in aquatic environments, using the formation of biofilm as a survival mechanism facilitating the transmission of diarrheal disease. Multi-drug resistant V. cholerae isolates have been characterized since the year 1977, and some of the mechanisms involved include the production of extended-spectrum β-lactamases (ESBLs). The purpose of the study was to determine the formation of biofilm in Cuban isolates of V. cholerae non-O1, non-O139 causing acute diarrheal disease (ADD), and detect the production of ESBLs in those with total or intermediate resistance to ampicillin. A descriptive cross-sectional study was conducted from January 2014 to June 2015. The study sample was 55 previously characterized isolates obtained from the strain collection at the ADD National Reference Laboratory of Pedro Kourí Institute. For phenotypic determination of ESBLs, 43 were studied which were known to be susceptible to ampicillin. 54.5 percent of the isolates tested positive for biofilm formation, with a predominance of those classified as moderate (46.6 percent) and weak (36.6 percent) biofilm producers. Of the 34 isolates resistant to ampicillin, 26.5 percent were positive for ESBL production. Of the 9 with intermediate ampicillin resistance, 44.4 % were positive. The results of the present study contribute knowledge about their ability to persist in the environment, and provide insight into antimicrobial resistance mechanisms(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.

Bacteremia por Vibrio cholerae NO-O1/NO-O139 en un paciente cirrótico: Primer reporte de caso en el Perú y revisión de la literatura / Vibrio cholerae NO-O1/NO-O139 bacteremia in a cirrhotic patient: First case report in Peru and literatura review

Vibrio cholerae serogrupo NO-O1/NO-O139 (VCNO) es causa infrecuente de gastroenteritis. Sin embargo, se le asocia a infección extra-intestinal severa en huéspedes inmunocomprometidos, y entre ellas, la bacteremia en pacientes con cirrosis hepática es digna de mención. A continuación, presentamos el caso de una mujer de 58 años, con el diagnóstico de cirrosis hepática de fondo, que desarrolló progresivamente choque séptico, disfunción orgánica múltiple y desenlace fatal al cuarto día de su admisión. Los resultados obtenidos post mortem, de los hemocultivos previamente tomados, aislaron bacilos gram negativos compatibles con Vibrio cholerae. Posteriormente, se identificó el serogrupo NO-O1/NO-O139, a través de aglutinación en placa y PCR negativo para el gen ctxA. El antibiograma mostró susceptibilidad conservada a ampicilina, cloranfenicol, tetraciclina y ciprofloxacino, con resistencia al trimetoprim-sulfametoxazol. El presente caso, descrito en el Hospital Nacional Dos de Mayo, es hasta la fecha, el primer reporte de bacteremia VCNO en el Perú.

Non-O1, non-O139 Vibrio cholerae (NOVC) strains are an uncommon cause of gastroenteritis. However, they have been recently associated with severe extraintestinal infections in immunocompromised hosts. Among them, bacteremia in cirrhotic patients is noteworthy. We present the case of a 58-year-old woman with cirrhosis that developed septic shock, multiple organ failure and died four days after admission. Blood cultures yielded Gram-negative rods identified as Vibrio cholerae. Further serogrouping by slide agglutination and a negative PCR for ctxA gen confirmed the strain to be NOVC. Antimicrobial susceptibility testing showed sensitivity to ampicillin, chloramphenicol, tetracycline and ciprofloxacin; and resistance to trimethoprim-sulfamethoxazole. To the best of our knowledge, this is first report in Peru, described in the Hospital Nacional Dos de Mayo, of NOVC bacteremia.

Bacteriemia por Vibrio cholerae no-O1, no-O139 en un paciente en hemodiálisis crónica / Non-O1, non-O139 Vibrio cholerae bacteremia in a chronic hemodialysis patient

Vibrio cholerae no-O1, no-O139 es un agente poco frecuente como causal de bacteriemias y no hay informes que documenten su presencia en pacientes en hemodiálisis crónica. Se describe el caso de una paciente en hemodiálisis crónica que presentó un cuadro de sepsis, por lo cual inició un tratamiento con vancomicina y ceftacidima. Al cabo de seis horas y media de incubación en el sistema BACT/ALERT de hemocultivo, se evidenció la presencia de bacilos curvos gram negativos, posteriormente identificados como Vibrio cholerae mediante pruebas bioquímicas convencionales y el uso de los kits API 20 NE y VITEK 2. La evaluación del serogrupo y de la presencia de factores de patogenicidad, realizada en el laboratorio de referencia, determinó que el microorganismo hallado pertenecía al serogrupo no-O1, no-O139. No se detectó la toxina de cólera, tampoco el factor de colonización ni la toxina termoestable. El aislamiento presentó sensibilidad frente a ampicilina, trimetoprima-sulfametoxazol, ciprofloxacina, tetraciclina, ceftacidima y cefotaxima por el método de difusión con discos y por VITEK 2. La paciente cumplió 14 días de tratamiento con ceftacidima endovenosa, con evolución favorable.

Non-O1, and non-O139 Vibrio cholerae is an infrequent cause of bacteremia. There are no reports of such bacteremia in chronic hemodialysis patients. This work describes the case of a chronic hemodialysis patient that had an episode of septicemia associated with dialysis. Blood cultures were obtained and treatment was begun with vancomycin and ceftazidime. After 6.5 hours of incubation in the Bact/Alert system there is evidence of gram-negative curved bacilli that were identified as Vibrio cholerae by conventional biochemical tests, API 20 NE and the VITEK 2 system. This microorganism was sent to the reference laboratory for evaluation of serogroup and virulence factors and was identified as belonging to the non-O1 and non-O139 serogroup. The cholera toxin, colonization factor and heat-stable toxin were not detected. The isolate was susceptible to ampicillin, trimethoprim-sulfamethoxazole, ciprofloxacin, tetracycline, ceftazidime and cefotaxime by the disk diffusion method and the VITEK 2 system. The patient received intravenous ceftazidime for a 14 day- period and had a favorable outcome.

A Case of Liver Abscess and Bacteremia Caused by Vibrio cholerae Non-O1 / 대한소화기학회지

Vibrio cholerae non-O1 have caused several well-studied food-borne outbreaks of gastroenteritis and also have been responsible for sporadic cases of otitis media, wound infection, and bacteremia. Few cases of liver abscess caused by Vibrio cholerae non-O1 have been reported. A 73-year-old man with underlying diabetes mellitus was admitted with nausea, vomiting, dyspepsia and febrile sensation. We identified Vibrio cholerae non-O1 in his blood cultures and multiple hepatic microabscess on abdominal computed tomography. He was treated with systemic antibiotics and fluid therapy, but died due to septic shock on sixth day. We report here, a case of liver abscess with bacteremia due to Vibrio cholerae non-O1 in a patient with diabetes mellitus.

Caracterización de aislamientos de Vibrio cholerae no-O1, no-O139 asociados a cuadros de diarrea / Characterization of Vibrio cholerae non-O1 and non-O139 isolates associated with diarrhea

La infección por Vibrio cholerae, el agente causal del cólera, se trasmite al hombre por ingestión de agua y alimentos contaminados. Aunque son los serogrupos O1 y O139 los que habitualmente se asocian al cólera epidémico, los aislamientos de otros serogrupos también son causales de gastroenteritis e infecciones extra-intestinales. Durante el período 2003-2005, se investigó la presencia de V. cholerae en la materia fecal de niños con diarrea atendidos en el Hospital del Niño Jesús, Tucumán. Se recuperaron 34 aislamientos de V. cholerae no-O1, no-O139. Se determinaron sus perfiles de virulencia por PCR, la sensibilidad a los antimicrobianos y la diversidad genética por electroforesis en campo pulsado. Se obtuvieron ocho perfiles de virulencia, aunque ningún aislamiento fue positivo para la toxina colérica ni para la toxina termoestable. Cuatro aislamientos fueron positivos para el sistema de secreción de tipo tres. El 17,6% de los aislamientos fueron resistentes o de sensibilidad intermedia a ampicilina y el 5,9% fueron resistentes a trimetoprima-sulfametoxazol. Los aislamientos resultaron muy diversos: se hallaron 27 patrones distintos en 29 aislamientos tipificables por electroforesis en campo pulsado. A pesar de su baja incidencia, V. cholerae continúa siendo un agente causal de diarrea en niños, los que se ven afectados por una amplia variedad de cepas circulantes.

Vibrio cholerae, etiologic agent of cholera, is transmitted to humans by ingestion of contaminated food or water. Even though serogroups O1 and O139 are the ones usually associated to epidemic cholera, isolates from other serogroups also cause gastroenteritis and extraintestinal infections. During the period 2003-2005, presence of V. cholerae in stools was investigated in children with diarrhea that seaked assistance at the Niño Jesús Hospital in Tucumán. Thirty four isolates of V. cholerae non-O1, non-O139 were recovered. We characterized the isolates studying its virulence factors by PCR, antimicrobial susceptibility patterns and genetic diversity by pulsed-field gel electrophoresis. Eight virulence patterns were obtained although no isolate was positive for the cholera toxin or the thermostable toxin. Four isolates were positive for the type three secretion system. The 17.6% of the isolates were resistant or intermediate to ampicillin and 5.9% were resistant to trimethoprim-sulfamethoxazole. By SfiI-PFGE, all isolates were genetically very diverse, as 27 different patterns were identified in 29 typeable isolates by pulsed-field gel electrophoresis. Although it has a low incidence, V. cholerae continues to be a causative agent of diarrhea in children, who are affected by a variety of circulating strains of V. cholerae non-O1, non-O139.

Vibrio cholerae non-O1,non-O139 Isolated from Pleural Effusion Following Total Gastrectomy

We isolated non-O1, non-O139 Vibrio cholerae from pleural effusion in a patient with recurred advanced gastric caner after total gastrectomy. We also recovered the organism from the patient's stool culture. The patient did not experience gastrointestinal symptoms such as diarrhea except heartburn and epigastric discomfort from stomach cancer before admission. The suspected route of infection is directly from the gastrointestinal tract through the previous surgical wounds. After antibiotic treatment, no more V. cholerae was isolated and the patient was well discharged from the hospital. This is the first report of V. cholerae infection associated with pleural effusion in a long-term latent carrier of the organism.

A cólera no Estado do Rio Grande do Norte--Brasil--perfil sorológico e de sensibilidade do Vibrio cholerae a diferentes antimicrobianos / Cholera in Rio Grande do Norte State--Brazil: sorology and sensitivity of Vibrio cholerae to different antimicrobials

PURPOSE: The emergence of multiple resistance to antimicrobials in Vibrio cholerae isolated in the state of Ceará, Brazil, alerted researchers in this area to the sensitivity to antimicrobials of strains isolated in Rio Grande do Norte (RN), Brazil. METHODS: One hundred and four strains of V. cholerae of human origin, isolated by Laboratório Central de Saúde Pública Dr. Almino Fernandes, were serologically typified and evaluated for in vitro sensitivity to eight antibiotics belonging to different groups (polymyxine, tetracycline, chloramphenicol, nitrofurantoin, sulphazotrin, pefloxacine, erythromycine, ampicillin). The strains were collected from patients suspected of contracting choleric diarrhea in the year 1999, in Natal/RN/Brazil. RESULTS: From the sample total, 100 were identified as V. cholerae, serogroup O:1, biotype El Tor, with 99 (95.3%) belonging to serovar Ogawa and only 1 (0.9%) to serovar Inaba. The 4 remaining were characterized as non O:1 V. cholerae, with 3 (2.9%) biochemically identified as Heiberg type I and 1 (0.9%) as type II. All the V. cholerae serogroup O:1 strains were sensitive to tetracycline, chloramphenicol, sulphazotrin, pefloxacine, erythromycine and resistant to polymyxine. In relation to nitrofurantoin, only 1 was sensitive. Only 1 was resistant to ampicillin. The non O:1 V. cholerae strains were resistant to polymyxine. CONCLUSIONS: The results showed sensitivity in 100% of the V. cholerae serogroup O:1 strains to tetracycline, an elective drug in the treatment of cholera, and an absence of multiple resistant strains in our environment. An interesting finding was the frequency of serovar Ogawa in 1999, considering the greater incidence of serovar Inaba in other years of cholera outbreaks in RN.

Virulence factors of non-O1 non-O139 Vibrio cholerae isolated in Córdoba, Argentina / Factores de virulencia de Vibrio cholerae no-O1 no-O139 aislados en Córdoba, Argentina

V. cholerae non-O1 non-O139 serogroups isolated from clinical and environmental sources in Córdoba, Argentina, were analyzed for the presence and expression of virulence genes. Most of the strains studied contained the genes toxR and hlyA, but lacked ctxA, zot, ace, tcpA and stn. The culture supernatants were tested for hemolytic and cytotoxic activity. The enterotoxic potential of the strains was studied in a rabbit ileal loop assay and their genetic profiles were compared by PFGE. The environmental strains varied in their virulence phenotype and showed no-clonal relationships. The clinical strains were highly enterotoxic, hemolytic, proteolytic and showed indistinguishable PFGE profiles, although they differed in their cytotoxic activity. This is the first description, using cell culture and “in vivo” studies, of the virulence properties of non-O1 non-O139 V. cholerae from Argentina.

En este trabajo se analizó la presencia y expresión de genes de virulencia en V. cholerae no-O1 no-O139 de origen clínico y ambiental, aislados en Córdoba, Argentina. La mayoría de las cepas estudiadas contiene los genes toxR y hlyA, pero no ctxA, zot, ace, tcpA y stn. Se analizó la actividad hemolítica y citotóxica de estas cepas en los sobrenadantes de cultivo, así como su potencial enterotóxico en ensayos de asa ileal ligada de conejo. Además, los aislamientos fueron comparados por sus perfiles genéticos en PFGE. Las cepas del medio ambiente mostraron variación en su fenotipo de virulencia y no mostraron relación clonal. Las cepas clínicas fueron muy enterotóxicas, hemolíticas, proteolíticas y mostraron perfiles indistinguibles de PFGE, aunque mostraron diferencias en su actividad citotóxica. En este trabajo se describen por primera vez, utilizando ensayos de cultivo celular e “in vivo”, propiedades de virulencia de V. cholerae no-O1 no-O139 aislados en Argentina.