Distribution of pathogenic vibrios and Vibrio spp. in the water column and sediment samples from the southern Gulf of Mexico.

This study quantified the distribution of Vibrio spp. by qPCR and pathogenic vibrio species by metagenomics, during 2 oceanographic cruises-XIXIMI-04 and XIXIMI-05 -in the southern Gulf of Mexico (GoMex). A total of 708 samples from various levels of the water column and 22 sediment samples were analyzed, according to a designed net of sampling lines. Sampling was focused on reported water masses with distinctive characteristics, to detect the presence-absence of vibrios. The results indicated that the genus Vibrio was detected along the entire water column and in sediments. Pathogenic vibrios, such as V, campbellii, V. parahaemolyticus, V. vulnificus or V. cholerae were also detected in the water column and in sediments, in both oceanographic cruises. Thus, the ecological conditions of the GoMex permit the growth of Vibrio spp. in deep water environments of the GoMex, despite continuous oil input from natural and anthropogenic sources.

Haitian variant Vibrio cholerae O1 Ogawa caused cholera outbreaks in Odisha.

PURPOSE: Diarrheal disorders particularly cholera cause a significant threat resulting in high morbidity and mortality in the coastal and tribal areas of Odisha. Two sequential diarrheal outbreaks reported in 2016 from Balasore and Rayagada districts of Odisha were investigated to find out the causative organisms, antibiogram profile and molecular analysis of the isolated pathogens. METHOD: Bacteriological analysis and antibiogram profiles of the pathogens were carried out as per the standard procedure followed. The double mismatch amplification mutation (DMAMA) PCR for ctxB gene, sequencing and pulse-field gel electrophoresis (PFGE) were carried out on Vibrio cholerae O1 strains. RESULTS: The rectal swabs and water samples from these districts were positive for V. cholerae O1 Ogawa biotype El Tor. The V. cholerae O1 strains isolated from Balasore district were multidrug resistant to many antibiotics which differed from the isolates of Rayagada district. The DMAMA PCR assay on all clinical and water isolates from these areas and some strains from other districts exhibited ctxB7 allele of V. cholerae O1 which correlates with the sequencing results having different pulsotypes. The Haitian variant of V. cholerae O1 strains which were compared with the V. cholerae O1 strains of 1999 and 2000 exhibited different pulsotypes. CONCLUSION: The present study reports cholera outbreaks due to multidrug resistant ctxB7 allele of V. cholerae O1 from both coastal (Balasore) and tribal (Rayagada) areas of Odisha.

Core and Accessory Genome Analysis of Vibrio mimicus.

Vibrio mimicus is an emerging pathogen, mainly associated with contaminated seafood consumption. However, little is known about its evolution, biodiversity, and pathogenic potential. This study analyzes the pan-, core, and accessory genomes of nine V. mimicus strains. The core genome yielded 2424 genes in chromosome I (ChI) and 822 genes in chromosome II (ChII), with an accessory genome comprising an average of 10.9% of the whole genome for ChI and 29% for ChII. Core genome phylogenetic trees were obtained, and V. mimicus ATCC-33654 strain was the closest to the outgroup in both chromosomes. Additionally, a phylogenetic study of eight conserved genes (ftsZ, gapA, gyrB, topA, rpoA, recA, mreB, and pyrH), including Vibrio cholerae, Vibrio parilis, Vibrio metoecus, and Vibrio caribbenthicus, clearly showed clade differentiation. The main virulence genes found in ChI corresponded with type I secretion proteins, extracellular components, flagellar proteins, and potential regulators, while, in ChII, the main categories were type-I secretion proteins, chemotaxis proteins, and antibiotic resistance proteins. The accessory genome was characterized by the presence of mobile elements and toxin encoding genes in both chromosomes. Based on the genome atlas, it was possible to characterize differential regions between strains. The pan-genome of V. mimicus encompassed 3539 genes for ChI and 2355 genes for ChII. These results give us an insight into the virulence and gene content of V. mimicus, as well as constitute the first approach to its diversity.

Epidemiological and pathogenic characteristics of Haitian variant V. cholerae circulating in India over a decade (2000-2018).

Vibrio cholerae, causative agent of the water-borne disease cholera still threatens a large proportion of world's population. The major biotypes of the pathogen are classical and El Tor. There have been recent reports of variant V. cholerae strains circulating around the world. In the present study, the epidemiological status of V. cholerae strains circulating in the country over a decade was assessed. Also, a comprehensive analysis of the difference in pathogenicity between the different biotypes of V. cholerae strains was evaluated both in-vitro and in-vivo. The amount of CT produced by different biotypes of V. cholerae strains were analyzed by GM1 ELISA and the probable reasons for the difference in toxin production was discussed. MLST analysis grouped the isolates into a single Sequence Type (ST 69) whereas PFGE analysis clustered the isolates into ten different pulsotypes revealing molecular diversity. The circulating strains were identified to produce cholera toxin and CT mRNA intermediate to the classical and prototype El Tor strains. Also, the circulating strains were identified to possess four ToxR binding sequences. In-vivo pathogenicity analysis by rabbit ileal loop fluid accumulation assay revealed the Haitian variant strains to be more hyperemic than the prototype strains.

Deep eutectic solvent-assisted phase separation in chitosan solutions for the production of 3D monoliths and films with tailored porosities.

A facile and greener methodology to obtain pure chitosan-based 3D porous structures in the form of monoliths and films is proposed. It is based on a modified evaporation-induced phase separation process in a chitosan solution precursor. In this approach, a deep eutectic solvent (DES) is used as the nonsolvent system and an ecofriendly, cost effective, simple and versatile alternative for the production of highly structured chitosan materials. The porous heterogeneous structure can be fine-tuned by varying the chitosan content in the precursor solution and chitosan/DES ratio, and enabled the structured polymer to absorb large amounts of water to form hydrogels. This is a versatile and unexplored approach to design porous chitosan with tailored morphology in the absence of crosslinkers, which, based on preliminary studies on V. cholerae biofilm formation, is expected to open new avenues for various applications in biomedical, catalysis, water purification, filtration and other areas where the control of bacterial biofilm formation is critical.

Prevalence and virulence of Vibrio species isolated from raw shrimp from retail markets in Reynosa, Mexico.

In this study, the prevalence of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio mimicus, Vibrio vulnificus and Vibrio spp. in shrimp from retail markets in Reynosa, Mexico was determined. A total of 765 isolates, identified as Vibrio spp. (59·1%), V. cholerae (17·8%), V. mimicus (6·7%) and V. parahaemolyticus (4·6%), were obtained; V. vulnificus was not detected. Most of the strains were isolated from supermarkets (48·1%), followed by street vendors (37·3%) and retail stores (14·6%). Moreover, several virulence genes were identified in V. cholerae: toxR (100%), OmpU (76·5%), hlyA (76·5%), VPI (19·9%) and tcpA (5·1%); in V. mimicus: vmh (100%), wzb (74·5%), pilF (54·9%), VPI (43·1%), OmpU (29·4%) and tdh (9·8%); and in V. parahaemolyticus: toxR (100%), tlh (100%), VP1680 (51·4%) and VPI (11·4%). These results show the low safety of this food and the potential risk to consumers' health, since this product in Mexican cuisine is sometimes served raw or semi-cooked. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the prevalence of pathogenic Vibrio cholerae, Vibrio mimicus and Vibrio parahaemolyticus isolated from shrimp that is commercialized in Reynosa city. This could represent a risk to consumers' health, since outbreaks related to shrimp contaminated with Vibrio have been previously reported. Additionally, shrimp fishing has a major role in Mexico's economy.

Haitian Variant Vibrio cholerae O1 Strains Manifest Higher Virulence in Animal Models.

Vibrio cholerae causes fatal diarrheal disease cholera in humans due to consumption of contaminated water and food. To instigate the disease, the bacterium must evade the host intestinal innate immune system; penetrate the mucus layer of the small intestine, adhere and multiply on the surface of microvilli and produce toxin(s) through the action of virulence associated genes. V. cholerae O1 that has caused a major cholera outbreak in Haiti contained several unique genetic signatures. These novel traits are used to differentiate them from the canonical El Tor strains. Several studies reported the spread of these Haitian variant strains in different parts of the world including Asia and Africa, but there is a paucity of information on the clinical consequence of these genetic changes. To understand the impact of these changes, we undertook a study involving mice and rabbit models to evaluate the pathogenesis. The colonization ability of Haitian variant strain in comparison to canonical El Tor strain was found to be significantly more in both suckling mice and rabbit model. Adult mice also displayed the same results. Besides that, infection patterns of Haitian variant strains showed a completely different picture. Increased mucosal damaging, colonization, and inflammatory changes were observed through hematoxylin-eosin staining and transmission electron microscopy. Fluid accumulation ability was also significantly higher in rabbit model. Our study indicated that these virulence features of the Haitian variant strain may have some association with the severe clinical outcome of the cholera patients in different parts of the world.

Isolation and identification of Vibrio species in the Rio Bravo/Grande and water bodies from Reynosa, Tamaulipas.

The Rio Bravo (Rio Grande) adjoins various states in the Mexican region and has a great importance in water distribution in the northeast Tamaulipas (Mexico). In this work 161 strains were isolated, identified and characterized from the water samples taken from the flow of the Rio Bravo and the two inner canals that cover Reynosa city. The strains were identified as Vibrio cholerae (74·5%), Vibrio spp. (1·2%) and Vibrio mimicus (0·6%). Furthermore, the detected virulence genes in the V. cholerae strains, were the hlyA, ompU, tcpA, toxR genes in 78·3, 62·5, 15·8 and 90·8% respectively. Only the ompU and vmh genes were detected in the V. mimicus strain. These results indicate the presence of multi-toxigenic V. cholerae strains in the Rio Bravo/Grande and in the water bodies from Reynosa city, which could represent a risk for the exposed population. SIGNIFICANCE AND IMPACT OF THE STUDY: Water quality is associated with public health, as it plays an important role in the transmission and epidemiology of pathogens such as Vibrio, since this species have been responsible for human diseases around the world. This study demonstrated the presence of toxigenic Vibrio species in water bodies in Reynosa surroundings, indicating that water bodies may be a source of public health risk.

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

Isolamento de Vibrio spp de camarões comercializados in natura na cidade de São Gonçalo - RJ / Isolation of Vibrio spp from shrimp commercialized in natura in the city of Sao Gonçalo - RJ

Bactérias do gênero Vibrio fazem parte da microbiota de camarões, pois têm capacidade de associar-se à quitina presente no exoesqueleto destes animais e ao zooplancton, que por sua vez são consumidos por estes animais. O gênero contém pelo menos 12 espécies patogênicas, incluindo V. cholerae, responsável por várias pandemias de cólera. A contaminação humana acontece através do consumo de alimentos, principalmente de origem marinha, crus ou mal cozidos. Por se tratar de um tipo de pescado amplamente consumido pela população, este trabalho teve como objetivo investigar a presença de espécies de Vibrio em camarões comercializados in natura na cidade de São Gonçalo-RJ. Os camarões foram adquiridos em duas peixarias da cidade e caracterizados por metodologia convencional e molecular; cento e vinte e nove amostras testaram positivamente para as provas bioquímicas realizadas e, destas, cinquenta e duas testaram positivamente para os testes moleculares. Visando investigar a identidade das espécies de Vibrio, as amostras foram submetidas ao PCR multiplex para 4 espécies (V. cholerae, V. mimicus, V. parahaemolyticus, V. vulnificus). Doze isolados foram identificados como V. parahaemolyticus e 9 como V.cholerae não O1. Dentre os demais isolados, 31 demonstram se tratar de outras espécies de Vibrio spp. O sítio com o maior número de isolados foi a casca, seguida pelo hepatopâncreas/ hemolinfa. A ribotipagem por PCR das 21 cepas demonstrou claramente a separação das cepas de V.parahaemolyticus e V.cholerae. As cepas de V. cholerae e V. parahaemolyticus demonstraram alto índice de resistência a ampicilina (83,33%) e 100% de sensibilidade à nitroflurantoína e tetraciclina. Sete cepas (38,8%) apresentaram perfil de multirresistência a dois antimicrobianos. Nossos resultados demonstram a presença de espécies patogênicas de Vibrio em amostras de pescados amplamente consumidos pela população.

Isolamento de Vibrio spp de camarões comercializados in natura na cidade de São Gonçalo - RJ

Bactérias do gênero Vibrio fazem parte da microbiota de camarões, pois têm capacidade de associar-se à quitina presente no exoesqueleto destes animais e ao zooplancton, que por sua vez são consumidos por estes animais. O gênero contém pelo menos 12 espécies patogênicas, incluindo V. cholerae, responsável por várias pandemias de cólera. A contaminação humana acontece através do consumo de alimentos, principalmente de origem marinha, crus ou mal cozidos. Por se tratar de um tipo de pescado amplamente consumido pela população, este trabalho teve como objetivo investigar a presença de espécies de Vibrio em camarões comercializados in natura na cidade de São Gonçalo-RJ. Os camarões foram adquiridos em duas peixarias da cidade e caracterizados por metodologia convencional e molecular; cento e vinte e nove amostras testaram positivamente para as provas bioquímicas realizadas e, destas, cinquenta e duas testaram positivamente para os testes moleculares. Visando investigar a identidade das espécies de Vibrio, as amostras foram submetidas ao PCR multiplex para 4 espécies (V. cholerae, V. mimicus, V. parahaemolyticus, V. vulnificus). Doze isolados foram identificados como V parahaemolyticus e 9 como V. cholerae não O1. Dentre os demais isolados, 31 demonstram se tratar de outras espécies de Vibrio spp. O Sítio com o maior número de isolados foi a casca, seguida pelo hepatopâncreas/ hemolinfa. A ribotipagem por PCR das 21 cepas demonstrou claramente a separação das cepas de V. parahaemolyticus e V. cholerae. As cepas de V. cholerae e V. parahaemolyticus demonstraram alto índice de resistência a ampicilina (83,33%) e 100% de sensibilidade à nitroflurantoína e tetraciclina. Sete cepas (38,8%) apresentaram perfil de multirresistência a dois antimicrobianos. Nossos resultados demonstram a presença de espécies patogênicas de Vibrio em amostras de pescados amplamente consumidos pela população. (AU)

Vibrio genus is part of the microbiota of shrimps as they have the ability to join the chitin present in the exoskeleton of these animals and to zooplancton, which are consumed by shrimp. The genus contains at least 12 pathogenic species, which includes V. cholerae, responsible for several pandemics of cholera. Human contamination occurs through the consumption of raw or undercooked seafood. Considering it is a type of fish widely consumed by the population, this study aims to investigate the presence of species of Vibrio in shrimps commercialized in São Gonçalo/RJ Shrimps were acquired in two local fishmarkets and characterized by conventional and molecular methods. One hundred and twenty nine isolates tested positive in the biochemical tests. Among them, fifty two have tested positive in molecular tests. In order to investigate the identity of the species of Vibrio, the samples were subjected to the multiplex PCR searching for four species of Vibrio (V. cholerae, V. mimicus, v. parahaemolyticus, V. vulnificus). Twelve isolates were identified as V. parahaemolyticus and 9 as V. cholera non-O1. Thirty one were classified as Vibrio spp. The site with the largest number of isolates was the shell, followed by the hepatopancreas/hemolymph. The PCR ribotyping clearly separate V. parahaemolyticus strains from V. cholerae strains. The strains of V. cholerae and V. parahaemolyticus showed high ampicillin resistance index (83.33%) and 100% sensitivity to nitrofurantoin and tetracycline. Seven strains (38.8%) had profiles of multiresistence to two antimicrobials. Our results demonstrate the presence of pathogenic species of Vibrio in shrimp samples widely consumed by the population.(AU)

Vaccines for viral and bacterial pathogens causing acute gastroenteritis: Part I: Overview, vaccines for enteric viruses and Vibrio cholerae.

Efforts to develop vaccines for prevention of acute diarrhea have been going on for more than 40 y with partial success. The myriad of pathogens, more than 20, that have been identified as a cause of acute diarrhea throughout the years pose a significant challenge for selecting and further developing the most relevant vaccine candidates. Based on pathogen distribution as identified in epidemiological studies performed mostly in low-resource countries, rotavirus, Cryptosporidium, Shigella, diarrheogenic E. coli and V. cholerae are predominant, and thus the main targets for vaccine development and implementation. Vaccination against norovirus is most relevant in middle/high-income countries and possibly in resource-deprived countries, pending a more precise characterization of disease impact. Only a few licensed vaccines are currently available, of which rotavirus vaccines have been the most outstanding in demonstrating a significant impact in a short time period. This is a comprehensive review, divided into 2 articles, of nearly 50 vaccine candidates against the most relevant viral and bacterial pathogens that cause acute gastroenteritis. In order to facilitate reading, sections for each pathogen are organized as follows: i) a discussion of the main epidemiological and pathogenic features; and ii) a discussion of vaccines based on their stage of development, moving from current licensed vaccines to vaccines in advanced stage of development (in phase IIb or III trials) to vaccines in early stages of clinical development (in phase I/II) or preclinical development in animal models. In this first article we discuss rotavirus, norovirus and Vibrio cholerae. In the following article we will discuss Shigella, Salmonella (non-typhoidal), diarrheogenic E. coli (enterotoxigenic and enterohemorragic), and Campylobacter jejuni.

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.

Culture supernatants from V. cholerae O1 ElTor strains isolated from different geographic areas induce cell vacuolation and cytotoxicity / Cepas de V. cholerae O1 biotipo ElTor aisladas de diferente origen geográfico inducen vacuolización celular y citotoxicidad

OBJECTIVE: To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. MATERIAL AND METHODS: Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. RESULTS: All ElTor strains analyzed induced cellular vacuolation. Ribotype 2 strains isolates from the U.S. gulf coast yielded the highest titer of vacuolating activity. Eight of nine strains were haemolytic, while all strains were PCR positive for the hlyA gene. We cloned the hlyA gene from two ElTor strains, a toxigenic (2514-88, ctxAB+) and a non-toxigenic Mexican strain (CM 91-3, ctxAB-). Supernatant from those recombinant E. coli strains induced haemolysis, cell vacuolation and cytotoxicity. RFLP-PCR analysis revealed similarities in the hlyA gene from all strains tested. CONCLUSION: The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.

OBJETIVO: Analizar el efecto vacuolizante de cepas de V. cholerae O1 ElTor aisladas de diferente origen geográfico, incluyendo México. MATERIAL Y MÉTODOS: Se realizaron pruebas de hemolisis, vacuolización y citotoxicidad en células Vero, así como PCR, análisis por RFLP y clonación molecular. RESULTADOS: Todas las cepas indujeron el efecto vacuolizante. Las cepas del ribotipo 2, aisladas de las costas del Golfo en Estados Unidos, presentaron títulos altos de vacuolización. El gen hlyA fue amplificado en las nueve cepas mediante PCR, aunque sólo ocho fueron hemolíticas. Se clonó el gen hlyA de una cepa toxigénica (2514-88, ctxAB+) y de una cepa no toxigénica aislada en México (CM 91-3, ctxAB-). El sobrenadante de las clonas recombinantes indujo hemólisis, efecto vacuolizante y citotoxicidad. El RFLP mostró alta similitud del gen hlyA de las cepas estudiadas. CONCLUSIÓN: El efecto vacuolizante es un fenotipo ampliamente distribuido en cepas epidémicas de V. cholerae O1 biotipo ElTor.

Use of monoclonal antibody for detecting Vibrio cholerae O1 in oysters by means of agglutination test / Detecção de Vibrio cholerae O1 em ostras utilizando anticorpo monoclonal em ensaio de aglutinação

V. cholerae serovar O1 is the etiologic agent responsible for pandemic cholera, and it is considered the most important human pathogenic Vibrio. The symptoms presented by patients affected by this bacterium vary from mild diarrhea to severe disease, and may even lead to death. Among the diverse types of seafood, the oysters pose as an important means for cholera transmission. Traditional methods used to detect V. cholerae are laborious and time-consuming, and there is an urgent need to establish a rapid, sensitive, specific, simple, and low-cost testing . The objective of this trial was, therefore, to assess the performance of a slide agglutination test using latex particles sensitized with specific monoclonal antibodies (MAb) for detecting V. cholerae O1 in experimentally-contaminated oysters. The slide agglutination test based on sensitized latex detected 1.2X102 CFU of bacteria (dilution 1:32). Oyster samples used in the present study, for being experimentally contaminated, were originally free of V. cholerae, but other bacteria were found, such as: Proteus mirabilis, Pseudomonas spp, and other vibrios. The present study demonstrated that the period of time needed to verify the food contamination with V. cholerae is 18 hours, taking in consideration that traditional methods require an average of 7-day period for getting the final results. The produced MAb presented 100% of s

O V. cholerae sorogrupo O1 é o agente etiológico da cólera pandêmica, sendo considerado dentre os víbrios patogênicos ao homem, o mais importante. Os sintomas das infecções por esta bactéria variam de diarréia branda a doença grave podendo até levar a óbito. Dentre os alimentos marinhos, as ostras representam uma das principais vias na transmissão de cólera. Os métodos convencionais para detecção do V. cholerae O1 são laboriosos e demorados havendo, portando, a necessidade de implantar métodos rápidos, sensíveis, específicos, simples e de baixo custo. O objetivo deste estudo foi avaliar a técnica de aglutinação de partículas de látex sensibilizadas com anticorpo monoclonal (AcMo) na detecção de V. cholerae O1 em ostras, contaminadas laboratorialmente. A técnica de aglutinação com látex sensibilizado detectou 1,2x102 UFC da bactéria (diluição 1/32). As amostras de ostras utilizadas para contaminação originalmente não continham V. cholerae, mas outras bactérias foram detectadas, tais como: Proteus mirabilis, Pseudomonas spp. e outros víbrios. O presente estudo demonstrou que a detecção de V. cholerae em alimentos foi reduzida para 18 horas, considerando que pela metodologia convencional a análise é finalizada, em média, em 7 dias. O AcMo produzido apresentou uma sensibilidade e especificidade de 100% para V. cholerae.

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.

Incorporação experimental de Vibrio cholerae O1 não toxigênico em ostras (Crassostrea brasiliana), vivas1 / Experimental incorporation of vibrio cholerae 01 non toxigenic in live oysters (Crassostrea brasiliana)

As ostras são moluscos bivalves que, com freqüência, são consumidas cruas. Podem ser objeto de processos de depuração (descontaminações físicas e químicas), pois este hábito de consumo está associado a eventos de doenças transmitidas por alimentos em vários países. No presente trabalho, foram realizados testes para verificar a possibilidade de contaminação de ostras em laboratório, com o objetivo de estabelecer modelo para avaliações de processos de depuração. As ostras foram mantidas vivas em água do mar previamente ozonizada. Foi procedida a contaminação da água com Vibrio cholerae EI Tor 01 não toxigênico. Foram realizadas determinações analíticas da água e das ostras imediatamente após e depois de 2, 4, 12, 18 e 24 horas da contaminação. Após 2 horas, as ostras apresentaram positividade para o V. cholerae usado no experimento, em até a diluição 107, igual ao número inicial de células viáveis imediatamente após a contaminação. Os níveis de presença da cepa testada manteve-se elevada, tanto na água como nas ostras, durante todo o período de observação (até a diluição 109 na água e 108 nas ostras). Os resultados obtidos indicam que este método é útil, pois permite a manutenção da viabilidade das ostras. A avaliação da eficácia e eficiência de processos tecnológicos de depuração (descontaminação), como o uso de ozônio e de radiação ionizante, pode ser conduzi da, mantendo os parâmetros reais, inclusive com a presença da microbiota autóctone destes moluscos. A comparação das diluições positivas no decorrer do experimento, demonstram que a cepa adaptou-se às condições do experimento, apresentando inclusive multiplicação. Foram realizados três testes independentes (triplicata), para observar possíveis variações na incorporação da cepa. (AU)

Oyster are frequently eaten raw and alive. Because of this, are often Object of depuration process (physical an chemica1 decontamination), since its consumption habit has importante epidemiological implications. It was perfomed essas to verify the possibility of live oyster contamination at aboratoria 1 level: oysters was maintened alive in sea water previously ozonized and then contamined with Vibrio cholerae EI Tor, 01, Ogawa, non toxigenic. Quantitative Analytical determination was realized in sea water and oysters, immediately and 2,4,12,18 and 24h after the contamination. After 2h, oysters presented high numbers of V cholerae, namely positivity in 10. 7 dilution, compatib1e with the initial sea water contamination.The leve1s of the strain used in this experimente was constantly high during the period of observation (positivitytill 10 dilution in water and 10 in oysters).The results obtained indicate that this contamination may be useful for laboratorial observation of etficacy and efficiencyof tecnhological depuration (decontamination) process of oysters, sinceallow measurements at real condictions. Number of viables cells obtained at ditferents periods of time shows that the strain was capable of adaptation, including multiplication, in this experimente. It was realized three independents experiments. (AU)

Análise dos anticorpos vibriocidas e aglutinantes na população urbana do Município do Manacapura/AM (1992/1993) / Vibriocidal and agglutinating antibodies in the urban population in the municipality of Manacapuru/AM (1992-1993)

Foi realizado um estudo sorológico em 1.196 indivíduos residentes na área urbana do município de Manacapuru/AM, visando analisar o perfil dos anticorpos vibriocidas e aglutinantes. Empregou-se o procedimento de amostragem aleatória sistemática na obtenção da amostra populacional. Um ano após, obteve-se uma 2ª amostra de soro de 120 indivíduos (10% da amostra inicial), escolhidos aleatoriamente entre os participantes do inquérito, com o objetivo de avaliar o comportamento dos anticorpos nesse intervalo de tempo. Foram empregados os métodos de microtitulação de anticorpos vibriocidas e de soroaglutinação em tubos. A associação entre os anticorpos estudados foi determinada pelo coeficiente de correlação, calculado com base na distribuição de freqüência dos títulos detectados. A análise dos resultados revelou forte correlação positiva entre os anticorpos (r = 1,0) e queda nos títulos em grande proporção das amostras após um ano.

A serological study was carried out involving 1,196 individuals residents in the urban area of Manacapuru--Amazonas, to evaluate the behavior of vibriocidal and agglutinating antibodies. A systematic random sampling procedure was employed to obtain the sample. A year later a 2nd sample of serum was obtained from 120 individuals selected among the participants of the survey. Vibriocidal antibodies microtitulation and seroagglutination in tubes were employed. The correspondence between the studied antibodies was determined by the correlation coefficient calculated according to the frequency of the titles detected. The analysis of the results revealed positive correlation between the antibodies (r = 1.0) and a decrease in titles in a large proportion of the positive samples one year later.