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O consumo de pescado tem crescido, seja pela maior consciência dos consumidores, seja por estratégias de marketing dos governos. Nesse contexto, destaca-se a culinária japonesa, cuja base principal é o pescado. Desta forma, objetivou-se avaliar os sashimis e vegetais Comercializados em restaurantes do Recife através do Número Mais Provável (NMP) de Vibrio parahaemolyticus e coliformes totais e termotolerantes, pesquisa de Vibrio spp. e Escherichia coli. Analisaram-se 30 amostras coletadas em cinco estabelecimentos. Não se observou diferença significativa nos índices de contaminação dos restaurantes pesquisados. Verificou-se presença de Escherichia coli em duas amostras de sashimi e em oito amostras de vegetais. Identificaram-se Vibrio damsela, V. fluvialis, V. furnissii, V. hollisae e V. harveyi, não sendo isolado V. parahaemolyticus. Conclui-se que alguns restaurantes necessitam implantar as Boas Práticas de Fabricação (BPF) para que os sashimis e vegetais comercializados não veiculem microrganismos, diminuindo a incidência das doenças transmitidas por alimentos.

O consumo de pescado tem crescido, seja pela maior consciência dosconsumidores, seja por estratégias de marketing dos governos. Nesse contexto, destaca-se a culinária japonesa, cuja base principal é o pescado. Desta forma, objetivou-se avaliar os sashimis e vegetais comercializados em restaurantes do Recife através do Número MaisProvável (NMP) de Vibrio parahaemolyticus e coliformes totais e termotolerantes, pesquisa de Vibrio spp. e Escherichia coli. Analisaram-se 30 amostras coletadas em cinco estabelecimentos.Não se observou diferença significativa nos índices de contaminação dos restaurantes pesquisados. Verificou-se presença de Escherichia coli em duas amostras de sashimi e em oito amostras de vegetais. Identificaram-se Vibrio damsela, V. fluvialis, V. furnissii, V. hollisae e V. harveyi, não sendo isolado V. parahaemolyticus. Conclui-se que alguns restaurantes necessitam implantar as Boas Práticas de Fabricação (BPF) para que os sashimis e vegetaiscomercializados não veiculem microrganismos, diminuindo a incidência das doenças transmitidas por alimentos.The fish consumption has grown, either for a higher sense of the consumers or for governments marketing strategies. In this context, the Japanese culinary is distinguished, whose main basis is fish. Thus, this study aims to evaluate the sashimi and vegetablescommercialized in restaurants of Recife through Most likely Number (NMP) of Vibrio parahaemolyticus, plus total and termotolerantes coliforms, research of Vibrio spp. and Escherichia coli. 30 samples collected in five establishments were analyzed. Significant difference in the indexes of contamination of the searched restaurants was not observed. Presence of Escherichia coli was verified in two samples of sashimi and eight vegetable samples. damsela, V. fluvialis, V. furnissii, V. hollisae and V. harveyi were identified, not being isolated V. parahaemolyticus. One conclusion was that some restaurants need to implement Good Fabrication Practices (BPF), so that the commercialized sashimi and vegetables do not propagate microorganisms, minimizing the incidence of the illnesses transmitted by food.

Coral reefs perform a major role in regulating marine biodiversity and serve as hotspot for highly dynamic and diverse microbiomes as holobionts. Corals around Ishigaki, however, are at risk due to tremendous stressors including elevation of seawater temperature, eutrophication and so on. However, no information is currently available on how Vibrio diversity fluctuates spatially and temporally due to environmental determinants in Ishigaki coral reef ecosystems. The aim of this study is to elucidate spatiotemporal Vibrio diversity dynamic at both community and population levels and to assess the environmental drivers correlated to Vibrio abundance and diversity. The Vibrio community identified based on pyrH gene phylogeny of 685 isolates from seawater directly connecting to Ishigaki coral holobionts consisted of 22 known and 12 potential novel Vibrionaceae species. The most prominent species were V. hyugaensis, V. owensii and V. harveyi followed by V. maritimus/V. variabillis, V. campbellii, V. coralliilyticus, and Photobacterium rosenbergii. The Vibrio community fluctuations, assessed by PCoA with UniFrac distance and clustering with Euclidiean distance were varied less not only by year but also by site. Interestingly, significant positive correlation was observed between rising seawater temperature and the abundance of V. campbellii (r = 0.62; P < 0.05) whereas the opposite was observed for V. owensii (r = -0.58; P < 0.05) and the C6 group of V. hyugaensis (r = -0.62; P < 0.05). AdaptML-based microhabitat differentiation revealed that V. harveyi, V. campbellii, P. rosenbergii, and V. coralliilyticus populations were less-ecologically distinctive whereas V. astriarenae and V. ishigakensis were ecologically diverse. This knowledge could be important clue for the future actions of coral conservation. [ABSTRACT FROM AUTHOR]

Marine environmental conditions are naturally changing throughout the year, affecting life cycles of hosts as well as parasites. In particular, water temperature is positively correlated with the development of many parasites and pathogenic bacteria, increasing the risk of infection and diseases during summer. Interannual temperature fluctuations are likely to alter host–parasite interactions, which may result in profound impacts on sensitive ecosystems. In this context we investigated the parasite and bacterial Vibrionaceae communities of four common small fish species (three-spined stickleback Gasterosteus aculeatus , Atlantic herring Clupea harengus , European sprat Sprattus sprattus and lesser sand eel Ammodytes tobianus ) in the Northern Wadden Sea over a period of two years. Overall, we found significantly increased relative diversities of infectious species at higher temperature differentials. On the taxon-specific level some macroparasite species (trematodes, nematodes) showed a shift in infection peaks that followed the water temperatures of preceding months, whereas other parasite groups showed no effects of temperature differentials on infection parameters. Our results show that even subtle changes in seasonal temperatures may shift and modify the phenology of parasites as well as opportunistic pathogens that can have far reaching consequences for sensitive ecosystems. [ABSTRACT FROM AUTHOR]

Gyrodactylus anguillae and Vibrio vulnificus are pathogens of critical significance in anguilliculture. Both agents were involved in mortalities affecting earthen pond cultured European eel, Anguilla anguilla, in Egypt during the period extended from June to September 2014. Moribund eels showed excessive slimy mucus and haemorrhages on the external body surface. All investigated eels were parasitized with the monogenean, Gyrodactylus anguillae. Majority of specimens 93.3% were concomitantly found to be infected with V. vulnificus. No other parasitic or bacterial infections were noticed in the investigated fish samples. V. vulnificus was detected in Gyrodactylus anguillae homogenates collected from diseased fish. Bacterial isolates were confirmed by polymerase chain reaction (PCR). G. anguillae were demonstrated in skin and gills histopathological sections. Varieties of circulatory, proliferative as well as degenerative changes were noticed in haemopoietic tissues. It was concluded that G. anguillae presumably enhanced eels vulnerability to V. vulnificus infections through providing portals of entry as well as acting as mechanical vectors. [ABSTRACT FROM AUTHOR]

The Vibrio genus is widespread in marine and brackish environments, and several species are human and animal pathogens of global importance. Vibrios adapt rapidly to many environmental stresses, so that brackish environments can be both a suitable niche and a possible reservoir for them. To test the occurrence of culturable vibrios and their possible correlation with environmental factors in a temperate brackish environment, a 1-year sampling study was performed in three brackish ponds located along the Central Thyrrenian coast in the Macchiatonda Nature Reserve (Santa Marinella, district of Rome, Italy). Molecular methods were used to detect Vibrio cholerae, V. parahaemolyticus, and V. vulnificus pathogenicity-associated genes among the Vibrio isolates. Out of 130 Vibrio isolates identified by sequencing a recA fragment, 70 harbored virulence-associated genes including ctx, ace, tcpA, tdh, trh, vvhA, vllY, and toxRS, so confirming the spread of virulence determinants across the environmental isolates. Ecological analysis showed that, although the water temperature is known to be a strong predictor of abundance and distribution of vibrios, its influence accounts for 27 % of the observed variance in the Macchiatonda samples, increasing to 40 % when combined with salinity. [ABSTRACT FROM AUTHOR]

The Red Sea has a unique geography and ecosystem and its shores are very rich in mangrove, macro-algae and coral reefs. Different sources of pollution are affecting the Red Sea shores and waters which impacts biological life including microbial life. We assessed the effects of industrialization, along the Egyptian Red Sea coast in eight coastal sites and two lakes, on microbial life. The bacterial community in sediment samples was analyzed using bacterial 16S rDNApyrosequencing of V6-V4 hypervariable regions. Taxonomical assignment of 131,402 significant reads to major bacterial taxa revealed five main bacterial phyla dominating the sampled Red Sea sites. This includes Proteobacteria (68%), Firmicutes (13%), Fusobacteria (12%), Bacteriodetes (6%) and Spirochetes (0.03%). Further analysis revealed distinct bacterial consortium formed mainly of: 1) marine Vibrio’s- suggesting a Marine Vibrio phenomenon 2) potential human pathogens and 3) oil-degrading bacteria. We discuss a distinct microbial consortium in Solar Lake West near Taba/Eilat and Saline Lake in Ras Muhammad; revealing the highest abundance of human pathogens versus no pathogens, respectively. Our results draw attention to the affects of industrialization on the Red Sea, and suggest further analysis to overcome hazardous affects on the impacted sites.

To date 142 species have been described in the Vibrionaceae family of bacteria, classified into seven genera; Aliivibrio, Echinomonas, Enterovibrio, Grimontia, Photobacterium, Salinivibrio and Vibrio. As vibrios are widespread in marine environments and show versatile metabolisms and ecologies, these bacteria are recognized as one of the most diverse and important marine heterotrophic bacterial groups for elucidating the correlation between genome evolution and ecological adaptation. However, on the basis of 16S rRNA gene phylogeny, we could not find any robust monophyletic lineages in any of the known genera. We needed further attempts to reconstruct their evolutionary history based on multilocus sequence analysis (MLSA) and/or genome wide taxonomy of all the recognized species groups. In our previous report in 2007, we conducted the first broad multilocus sequence analysis (MLSA) to infer the evolutionary history of vibrios using nine housekeeping genes (the 16S rRNA gene, gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA), and we proposed 14 distinct clades in 58 species of Vibrionaceae. Due to the difficulty of designing universal primers that can amplify the genes for MLSA in every Vibrionaceae species, some clades had yet to be defined. In this study, we present a better picture of an updated molecular phylogeny for 86 described vibrio species and 10 genome sequenced Vibrionaceae strains, using 8 housekeeping gene sequences. This new study places special emphasis on 1) eight newly identified clades (Damselae, Mediterranei, Pectenicida, Phosphoreum, Profundum, Porteresiae, Rosenbergii, and Rumoiensis); 2) clades amended since the 2007 proposal with recently described new species; 3) orphan clades of genomospecies F6 and F10; 4) phylogenetic positions defined in 3 genome-sequenced strains (N418, EX25, and EJY3); and 5) description of V. tritonius sp. nov., which is a member of the Porteresiae clade.

Background: The genus Vibrio is clinically significant and major pathogenic Vibrio species causing human Vibrio infections are V. cholerae, V. parahaemolyticus, V. vulnificus, V. alginolyticus and V. mimicus. In this study, we screened for novel genetic markers using comparative genomics and developed a Vibrio multiplex PCR for the reliable diagnosis of the Vibrio genus and the associated major pathogenic Vibrio species. Methods: A total of 30 Vibrio genome sequences were subjected to comparative genomics, and specific genes of the Vibrio genus and five major pathogenic Vibrio species were screened. The designed primer sets from the screened genes were evaluated by single PCR using DNAs from various Vibrio spp. and other non-Vibrio bacterial strains. A sextuplet multiplex PCR using six primer sets was developed to enable detection of the Vibrio genus and five pathogenic Vibrio species. Results: The designed primer sets from the screened genes yielded specific diagnostic results for target the Vibrio genus and Vibrio species. The specificity of the developed multiplex PCR was confirmed with various Vibrio and non-Vibrio strains. This Vibrio multiplex PCR was evaluated using 117 Vibrio strains isolated from the south seashore areas in Korea and Vibrio isolates were identified as Vibrio spp., V. parahaemolyticus, V. vulnificus and V. alginolyticus, demonstrating the specificity and discriminative ability of the assay towards Vibrio species. Conclusions: This novel multiplex PCR method could provide reliable and informative identification of the Vibrio genus and major pathogenic Vibrio species in the food safety industry and in early clinical treatment, thereby protecting humans against Vibrio infection. [ABSTRACT FROM AUTHOR]

Rapid growth of shrimp farming industry is affected by the recurrence of diverse diseases, among which vibriosis is predominant. Eco-friendly disease management strategy by the application of antagonistic probiotics is widely accepted. In the present study, culture conditions of antagonistic probiotics, Pseudomonas MCCB 102 and 103, were optimized to enhance their biomass production and antagonistic activity against the shrimp pathogen V. harveyi MCCB 111. Primarily, one-dimensional screening was carried out to fix the optimum range of sodium chloride concentration, pH and temperature. The second step optimization was done using a full-factorial central composite design of response surface methodology. As per the model, 12.9 g/L sodium chloride and pH 6.5 for Pseudomonas MCCB 102, and 5 g/L sodium chloride and pH 7 for Pseudomonas MCCB 103 were found to be ideal to maximize antagonistic activity. However, optimum temperature was the same (25 °C) for both isolates. Finally, the models were experimentally validated for enhanced biomass production and antagonistic activity. The optima for biomass and antagonistic activity were more or less the same, suggesting the possible influence of biomass on antagonistic activity. [ABSTRACT FROM AUTHOR]

Plating environmental samples on vibrio-selective chromogenic media is a commonly used technique that allows one to quickly estimate concentrations of putative vibrio pathogens or to isolate them for further study. Although this approach is convenient, its usefulness depends directly on how well the procedure selects against false positives. We tested whether a chromogenic medium, CHROMagar Vibrio (CaV), used alone (single-plating) or in combination (double-plating) with a traditional medium thiosulfate-citrate-bile-salts (TCBS), could improve the discrimination among three pathogenic vibrio species ( Vibrio cholerae , Vibrio parahaemolyticus , and Vibrio vulnificus ) and thereby decrease the number of false-positive colonies that must be screened by molecular methods. Assays were conducted on water samples from two estuarine environments (one subtropical, one tropical) in a variety of seasonal conditions. The results of the double-plating method were confirmed by PCR and 16S rRNA sequencing. Our data indicate that there is no significant difference in the false-positive rate between CaV and TCBS when using a single-plating technique, but determining color changes on the two media sequentially (double-plating) reduced the rate of false positive identification in most cases. The improvement achieved was about two-fold on average, but varied greatly (from 0- to 5-fold) and depended on the sampling time and location. The double-plating method was most effective for V. vulnificus in warm months, when overall V. vulnificus abundance is high (false positive rates as low as 2%, n = 178). Similar results were obtained for V. cholerae (minimum false positive rate of 16%, n = 146). In contrast, the false positive rate for V. parahaemolyticus was always high (minimum of 59%, n = 109). Sequence analysis of false-positive isolates indicated that the majority of confounding isolates are from the Vibrionaceae family, however, members of distantly related bacterial groups were also able to grow on vibrio-selective media, even when using the double-plating method. In conclusion, the double-plating assay is a simple means to increase the efficiency of identifying pathogenic vibrios in aquatic environments and to reduce the number of molecular assays required for identity confirmation. However, the high spatial and temporal variability in the performance of the media mean that molecular approaches are still essential to obtain the most accurate vibrio abundance estimates from environmental samples. [ABSTRACT FROM AUTHOR]

Abstract: Seafood can pose a public health concern to consumers. It is often consumed raw and may be contaminated with several foodborne pathogens. In order to guarantee the safety of seafood, real-time polymerase chain reaction (PCR) protocols may be used as these enable results to be provided within 24 h. The first goal of our work was to develop real-time PCR protocols enabling the detection of six foodborne pathogens that may be present in seafood products (Campylobacter jejuni, Campylobacter coli, enterohemorrhagic Escherichia coli, Salmonella spp., Vibrio parahaemolyticus, and Vibrio vulnificus). The corresponding gene targets were: 50S/VS1, rfbE, ttr, tlh, and vvp. A multiplex PCR was also developed to detect the virulence genes of V. parahaemolyticus: tdh and trh. A total of 420 bacterial strains belonging to four different genera/strains were used in this study. Sensitivity and specificity were always 100%, except in the case of Salmonella spp., where three strains were not detected by our PCR protocols. The second objective of our work was to assess the detection limit of our real-time PCR protocols on artificially contaminated seafood products (raw shrimps, cooked shrimps, and raw mussels), purchased in public stores. Six different levels of contamination were assayed in four replicates for each matrix. The real-time PCR protocols enabled a better level of detection than the ISO methods, except for Salmonella in raw shrimps and for V. vulnificus in shrimps (raw and cooked). The estimated level of detection was between 1 and 47 cfu/25 g sample for the ISO norms and between 1 and 315 cfu/25 g sample for the real-time PCR protocols tailored in our work. The real-time PCRs developed in our work allowed for good selectivity, sensitivity, and specificity. The sensitivity on seafood products was estimated at a level of 100%, except for Salmonella (97%). In the spiking assays, the levels of detection were lower with the real-time PCR protocol than those obtained with the ISO method. This was not the case for V. vulnificus in raw and cooked shrimps and for Salmonella in raw shrimps. These real-time PCR protocols appear to be good alternative methods for surveillance of seafood products to ensure the absence of foodborne pathogens. One additional conclusion is that laboratories have to use enrichment media that are compatible with those recommended by ISO standards. This may facilitate the isolation of the pathogen if the real-time PCR protocol gives a suspect positive signal during the first step of the seafood analysis. [Copyright &y& Elsevier]

The Red Sea possesses a unique geography, and its shores are rich in mangrove, macro-algal and coral reef ecosystems. Various sources of pollution affect Red Sea biota, including microbial life. We assessed the effects of industrialization on microbes along the Egyptian Red Sea coast at eight coastal sites and two lakes. The bacterial communities of sediment samples were analyzed using bacterial 16S rDNA pyrosequencing of V6-V4 hypervariable regions. The taxonomic assignment of 131,402 significant reads to major bacterial taxa revealed five main bacterial phyla dominating the sampled sites: Proteobacteria (68%), Firmicutes (13%), Fusobacteria (12%), Bacteriodetes (6%), and Spirochetes (0.03%). Further analysis revealed distinct bacterial consortia that primarily included (1) marine Vibrio spp.--suggesting a "marine Vibrio phenomenon"; (2) potential human pathogens; and (3) oil-degrading bacteria. We discuss two divergent microbial consortia that were sampled from Solar Lake West near Taba/Eilat and Saline Lake in Ras Muhammad; these consortia contained the highest abundance of human pathogens and no pathogens, respectively. Our results draw attention to the effects of industrialization on the Red Sea and suggest the need for further analysis to overcome the hazardous effects observed at the impacted sites. [ABSTRACT FROM AUTHOR]

Microorganisms play an indispensable role in the ecological functioning of marine environment. Some species are sensitive while others are insensitive for a specific pollutant. The aim of this work is a preliminary study of the quantitative and qualitative distribution of cultivable vibrios in sediments and water samples characterized by different toxicity levels. For 1 year, in three suitably selected sampling stations of Mar Piccolo in Taranto (Ionian Sea, Italy), we have evaluated the toxicity level by Microtox® system, vibrios, total, and fecal coliform densities. The results of the Microtox® tests showed sediments characterized by an elevated level of toxicity, while the interstitial water of the same sites always showed biostimulatory phenomenon. The quantitative results show that vibrios and coliforms are more abundant in water than in sediment samples. The most often isolated strains were: Vibrio alginolyticus, Vibrio mediterranei, Vibrio metschinkovii, and Vibrio splendidus II. This work is the first example of study on the distribution of Vibrio species related to toxicity evaluation conducted by the Microtox® bioassay. The results show the different distribution of Vibrionaceae in two environmental matrices analyzed and characterized by different levels of toxicity. [ABSTRACT FROM AUTHOR]

Analysis of conditions of formation of microplanktonic microbial communities (MC) was carried out for the Pechora Sea using the data of winter 2002-2005. The patterns of distribution of different MC groups in different water types are discussed. Regular variations of the indices of abundance in a gradient of physicochemical variables were revealed. The estimated average bacterial numbers for under-ice conditions were 234 × 10 cells/mL, with the average cell volume of 0.58 μm and total cell area ∼550 mm/L. The estimated total microbial biomass in the 0-25 m water horizon of the Pechora Sea is 187 × 10 g wet biomass. [ABSTRACT FROM AUTHOR]

Serum resistance is recognized as one of the most important pathogenic traits of bacterial pathogens, and no control measure is available. Based on our previous discovery that pathogenic Escherichia coli represses glycine, serine, and threonine metabolism to confer serum resistance and that the reactivation of this pathway by exogenous glycine could restore serum sensitivity, we further investigate the mechanism underlying the action of glycine in Vibrio alginolyticus . Thus, V. alginolyticus is treated with glycine, and the proteomic change is profiled with tandem mass tag-based quantitative proteomics. Compared to the control group, glycine treatment influences the expression of a total of 291 proteins. Among them, a trap-type mannitol/chloroaromatic compound transport system with periplasmic component, encoded by N646&#95;0992, is the most significantly increased protein. In combination with the pathway enrichment analysis showing the altered fructose and mannitol metabolism, mannitol has emerged as a possible metabolite in enhancing the serum killing activity. To demonstrate this, exogenous mannitol reduces bacterial viability. This synergistic effect is further confirmed in a V. alginolyticus - Danio rerio infection model. Furthermore, the mechanism underlying mannitol-enabled serum killing is dependent on glycolysis and the pyruvate cycle that increases the deposition of complement components C3b and C5b-9 on the bacterial surface, whereas inhibiting glycolysis or the pyruvate cycle significantly weakened the synergistic effects and complement deposition. These data together suggest that mannitol is a potent metabolite in reversing the serum resistance of V. alginolyticus and has promising use in aquaculture., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kou, Wu, Chen and Peng.)

Una práctica mundial en granjas de cultivo acuícola es el uso rutinario de antimicrobianos, incluyendo compuestos de valor en terapéutica humana; sin embargo, todavía prevalecen las vibriosis atribuibles fundamentalmente a Vibrio harveyi luminiscentes. Se determinó la resistencia frente a 12 antimicrobianos y al agente vibriostático, con el método en disco de difusión en agar, de miembros del género Vibrio aislados de lisas criollas silvestres (Mugil curema) y de camarones peneidos (Litopenaeus schmitti, L. vannamei y L. stylirostris), silvestres y cultivados, así como del agua y sedimento de su entorno. Se tomaron muestras del riñón, hepatopáncreas e intestino de los animales, y también de macerados de larvas de camarones. En general los ejemplares se observaron sanos a su llegada al laboratorio, con excepción de los provenientes de los 3 primeros muestreos realizados en una camaronera en 1996, donde un grupo de larvas de cultivo presentó problemas sanitarios. Fueron aislados 629 vibrios, de los cuales el mayor grupo, claramente identificado, estuvo formado por bacterias de la especie V. harveyi. Se detectó que el 93% de los vibrios presentaron resistencia múltiple, la cual fue elevada (R ³ 50%) a la eritromicina, estreptomicina, kanamicina, novobiocina, penicilina G, polimixina B, tetraciclina y triple sulfa. No se encontró diferencia estadística significativa (P £ 0,05) entre la especie V. harveyi y el resto de las especies de Vibrio ubicadas en un segundo grupo, considerando en conjunto los ambientes silvestre y de cultivo. Sin embargo, el nivel de resistencia de las cepas de esta especie se hizo significativamente mayor (P £ 0,05), al analizar en forma individual las de V. harveyi aisladas de las larvas enfermas. Aquí las cepas fueron 100% resistentes en un rango comprendido entre 7 y 10 de los antimicrobianos probados. El alto nivel de resistencia de las bacterias recuperadas de animales cultivados, puede ser una consecuencia del amplio uso de antimicrobianos. En lo referente a los animales silvestres, la alta resistencia detectada podría involucrar la transferencia de factores R mediante plásmidos.

De 3.250 fezes diarréicas, recebidas para diagnóstico microbiológico em laboratório clínico particular, no Recife, Brasil, isolaram-se 55 (1,7%) linhagens de Vibrio. O estudo foi realizado entre maio de 1989 e maio de 1991. Para o isolamento de Vibrio, os espécimes fecais foram enriquecidos em água peptonada alcalina suplementada com 2% de NaCl e subcultivados em ágar tiosulfato-citrato-sais biliares-sacarose (TCBS). Das espécies isoladas, V. parahaemolyticus foi a mais freqüente (24 cepas), seguida de V. furnissii (15 cepas), V. cholerae não 01 (6 cepas), V. alginolyticus (4 cepas), V. fluvialis (2 cepas) e Vibrio sp. (1 cepa). Do ponto de vista custo-benefício, a baixa taxa de isolamento de Vibrio levanta dúvidas acerca da utilidade do TCBS na rotina enterobacteriológica dos laboratórios clínicosOf 3250 diarrheal stools received for microbiologic diagnosis at a private clinical laboratory in Recife, Brazil, strains of Vibrio were isolated from 55 (1.7%). The study was carried out from May 1989 through May 1991. For recovering Vibrio, fecal samples were enriched in alkaline peptone water supplemented with 2% NaCl and subcultured on thiosulfate-citrate-bile salts-sucrose agar (TCBS). Of the recovered species, V. parahaemolyticus was most commonly found (24 strains), followed by V. furnissii (15 strains), V. cholerae non-01 (6 strains), V. alginolyticus (4 strains), V. fluvialis (2 strains), and Vibrio sp. (1 strain). The low isolation rate of Vibrio raises doubts about the cost-effectiveness of the use of TCBS in the routine enterobacteriologic workup of clinical laboratories

This study aimed to detect the presence of quorum-sensing (QS) signalling molecules and evaluate the in vivo virulence gene expression in shrimp challenged with acute hepatopancreatic necrosis disease (AHPND) positive Vibrio isolates. Three types of QS signal molecules, namely N-acyl-homoserine lactone (AHL), Autoinducer-2 (AI-2) and Cholerae autoinducer-1-like (CAI-1) molecules were detected in AHPND causing isolates using Agrobacterium tumefaciens KYC55, Vibrio campbellii JMH597 and V. campbellii JAF375 biosensors respectively. The presence of QS genes in all the 12 AHPND positive isolates was further justified by conducting molecular screening of the QS-related genes, luxR and luxS. Challenged groups demonstrated a significant (P < 0.05) increase in the expression levels of the QS master regulator gene luxR compared with the unchallenged group. The expression of pirA -, pirB -, toxR - and luxR genes peaked at 36 h in shrimp challenged with AHPND positive Vibrio parahaemolyticus BpShHep31 and V. harveyi BpShHep24. However, compared to the group challenged with V. parahaemolyticus BpShHep31, the V. harveyi BpShHep24 challenged group demonstrated 45.1-, 126.0-, 19.7- and 10.7- fold higher expression of pirA , pirB , toxR and luxR respectively. • The autoinducer-1 (AI-1) and autoinducer-2 (AI-2) are the major quorum sensing signals produced by the AHPND isolates. • Expression level of pirA -, pirB -, luxR- and toxR- regulated virulence genes increased proportionally with high mortality rates in the shrimp challenged with AHPND positive isolates. • Up-regulation of the production capacity of pirA, pirB, toxR and luxR were observed at 36 h. [ABSTRACT FROM AUTHOR]

Background: The emergence, resurgence and spread of human food-borne pathogenic Vibrios are one of the major contributors to disease burden and mortality particularly in developing countries with disputable sanitary conditions. Previous research on pathogenic Vibrio cholerae and Vibrio parahaemolitycus derived from clinical samples has proposed links between acquisition of virulence and multiple drug resistance traits and intercellular transmissibility of mobile genetic elements in the environment. To date, very few information is available on environmental Vibrio isolates. In this study, we characterized eleven Vibrio strains bearing the SXT/R391-like integrative and conjugative elements (ICEs) derived from aquatic products and environment in the Yangtze River Estuary, China. Results: The eleven Vibrio strains were isolated in 2010 to 2011, and taxonomically identified, which included six Vibrio cholerae, three Vibrio parahaemolyticus, one Vibrio alginolyticus and one Vibrio natriegens. Most of the strains displayed strong resistance phenotypes to ampicillin, mercury and chromium. The majority of their ICEs, which belong to S and R exclusion system groups, contain ICEs-chromosome junction sequences and highly conserved core-genes required for ICE transfer. However, comparative sequence analysis uncovered interesting diversity in their mosaic accessory gene structures, which carry many novel genes that have not been described in any known ICEs to date. In addition, antibiotic resistance was transmitted by ICEVchChn6 and ICEVpaChn1 from V. cholerae, V. parahaemolyticus to E. coli MG1655 via conjugation, respectively. Our data also revealed that the ICEs characterized in this study are phylogenetically distant from most of the SXT/R391 ICEs reported previously, which may represent a novel cluster likely shaped by the ecological environment in the Yangtze River Estuary, China. Conclusions: This study constitutes the first investigation of ICEs-positive Vibrio spp. in the Yangze River Estuary, China. The newly identified ICEs were characterized with mosaic accessory gene structures and many novel genes. The results demonstrated self-transmissibility of antibiotic resistance mediated by two of the ICEs from V. cholerae, V. parahaemolyticus to E. coli via conjugation, respectively. Our results also revealed that the ICEs examined in this study may represent a novel cluster in the SXT/R391 family. [ABSTRACT FROM AUTHOR]

The molecular basis of antagonistic activity of a number of probiotic bacteria was analyzed using plasmid curing method. Earlier, six bacteria having potential probiotic activity under laboratory conditions were screened from 96 isolates which were obtained from shrimp rearing environment from south-west coastal region of Bangladesh. Among the isolated probionts, five were subjected to plasmid curing method and three were found to harbor plasmids, as was revealed in an agarose gel electrophoresis. Curing of these plasmids demonstrated that the bacteria lost its antagonistic activity, indicating the activity could be plasmid-borne. The antagonistic activities of the rest two isolates however, were not plasmid-oriented as no plasmid was recovered from them. [ABSTRACT FROM AUTHOR]

Microbial safety of recreational water is one of the major human public health issues in developing countries. Three water bodies, the Tbilisi Sea, Kumisi and Lisi lakes, in the South Caucasus region near Tbilisi, Georgia, were monitored in 2006-2009 to determine microbiological quality using standard methods. Microbial pollution indicators were determined in parallel with phytoplankton abundance and measurement of a number of physical--chemical parameters. Kumisi Lake, a brackish water body in an active agricultural area, appeared to be the most polluted, whereas the Tbilisi Sea, a freshwater reservoir was the least polluted. High values for fecal indicators in all three lakes in summer and early autumn were revealed. In our study, total enterococci counts (TEC) appeared to be a better indicator than either fecal or total coliform counts for the evaluation of fresh and brackish microbial water quality. We found significant correlation between total Vibrio counts and TEC for all three water bodies. Prevalence of somatic coliphages and V. cholerae-specific phages as additional water pollution indicator significantly correlated with abundance of the host bacteria. Particular phytoplankton groups in the lakes responded to the changes of fecal indicators; however, no correlation was observed between dominant zooplankton taxonomic groups and microbial parameters. [ABSTRACT FROM AUTHOR]

Some bacteria produce a satellite RNA-DNA complex termed msDNA, multicopy single-stranded DNA. In this report, msDNA from Vibrio parahaemolyticus, a cause of acute gastroenteritis, was identified and named msDNA-Vp96. The retron element containing the ret gene, encoding the reverse transcriptase (RT) that is responsible for msDNA production, was cloned and characterized. Comparison of msDNA-Vp96 and msDNA-Vc95, from Vibrio cholerae, showed a high level of sequence similarity. We exchanged the two ret genes to examine whether msDNA was produced by the RT from different sources. We found that RT-Vp96 of V. parahaemolyticus was able to synthesize msDNA-Vc95 of V. cholerae and vice versa. To the best of our knowledge, this is the first report that RT from different bacterial species can synthesize msDNA. [ABSTRACT FROM AUTHOR]

The present study was aimed to investigate the occurrence of multiple antibiotic resistance, the role of plasmids and their relationship with the multiple antibiotic resistance in 3 0 Vibrios isolated from selected seafoods of Kerala, India. The isolated Vibrios were screened for plasmid DNA and were tested for transformation and conjugation efficiencies. Antibiotic resistance studies revealed that the levels of Multiple Antibiotic Resistance of Vibrio strains to various antibiotics differed considerably and were found to be varied in the expression of their resistance pattern. All studied Vibrio strains were found to be resistant to antibiotics; amoxycillin, ampicillin and carbeniciliin. 87 % were resistant to rifampicin; 74% to cefuroxime; 67 to streptomycin; 53% to norfloxacin and ciprofloxacin and 47% to furazolidone and nalidixic acid. The nine strains isolated from crustaceans and from molluscans have been found to harbor 1-3 plasmids with size varies from 5.98 kb to 19.36 kb. The average transformation efficiency is about 5x10-8 and the conjugation efficiency is varied from 2.1x 10-3 to 10-9. The study of antibiotic resistance pattern could be useful to test the extent of drug resistance in seafoods and help to devise a nationwide antibiotic policy. [ABSTRACT FROM AUTHOR]

Vibrios are halophilic bacteria that are ubiquitous in marine environments. Their occurrence in tropical lakes has rarely been investigated. In this study, the predominance and diversity of spp. was investigated over a 12-month period in a coastal lagoon, Songkhla Lake, in southern Thailand. Water samples were collected at 2 stations in the estuary near Yor Island in Songkhla Lake. The predominant vibrios were detected by a culture-based method, using thiosulfate-citrate-bile salt-sucrose agar and CHROMagar Vibrio. The diversity of Vibrio spp. was evaluated using denaturant density gradient electrophoresis (DGGE). The highest numbers of total vibrios and in both areas were observed during the summer. There was no significant correlation between the numbers of vibrios, including V. parahaemolyticus, and either the water temperature or plankton density. Variations in Vibrio species were observed with changes in salinity. Vibrio parahaemolyticus and non-O1/non-O139 were detected during the rainy season when the salinity dropped to nearly 0 parts per thousand. In both areas, was the most prominent species detected by the culture method, whereas Vibrio parahaemolyticus was detected by DGGE, every month. Other Vibrio spp. of potential public health concern were also detected by the culture method; they included , , and . [ABSTRACT FROM AUTHOR]

The use of chemotherapeutic agents has been questioned for disease management in shrimp aquaculture due to the emergence of drug-resistant bacteria and enduring residual effects in the environments. Instead, microbial interventions in sustainable aquaculture have been proposed, and among them, the use of probiotics as an alternative to antibiotics has gained considerable attention. Earlier, we short-listed nineteen isolates out of ninety six bacteria, isolated from shrimp ghers of coastal areas of Bangladesh that exhibited antagonistic potential against pathogenic vibrios, and non-hemolytic property, presumably indicating their harmless behavior in mammalian system at least in vitro. This prompted us to study their cytotoxic effect in vivo using brine shrimp Artemia franciscana. Six isolates were found having higher lethal values compared with others. Four of them, identified as Pseudomonas and Bacillus spp. exhibited the lowest inhibition against Artemia and were considered safe. Further, in a co-culture experiment it was evident that the isolated probiotic bacteria were shown to outcompete the growth of pathogenic Vibrio harveyi due to their antagonistic properties. The probionts can now therefore be tested in culture environment of Bangladesh in order to understand the effectiveness of microbial control of bacterial diseases in shrimp aquaculture. [ABSTRACT FROM AUTHOR]

The present study was aimed to detect the plasmid profile, the role of plasmid associated multiple antibiotic resistance of Vibrios isolated from coastal waters of Kerala. The isolated plasmids from antibiotic resistant Vibrios were tested for the presence of integrons using polymerase chain reaction (PCR) to elucidate the presence of plasmid borne integron, a key element in horizontal gene transfer. 100 isolates of Vibrios from water samples of shrimp farms and coastal landing sites were tested for the antibiogram profile to 22 antibiotics and the presence of the plasmids. Antibiotic resistance studies revealed that 78% were expressing multiple antibiotic resistance (MAR), defined as the isolates having resistant to more than three resistance determinants. The levels of resistance exhibited by isolates to specific antibiotics vary between 94 % and 6%. In the plasmid profiling test, only 17 isolates (21%) harbored plasmid DNA which ranged in size from 1.4 to 25 kb, separating the isolates into various plasmid profiles. Interestingly it was observed from the plasmid profiling that 11 strains among them had a single plasmid, 4 strains were with two plasmids and one each of having three and four plasmids. The role of native plasmids in antibiotic resistance in Vibrios was confirmed by isolation and transformation of the plasmids in E.coli DH5a, followed by antibiotic resistance assay. Both the plasmids and the associated antibiotic resistance of selected isolates could be transferred to a recipient by transformation and conjugation. The study demonstrates the plasmid-mediated drug resistance as a contributor to the antibiotic resistance in Vibrios of the aquatic system and their ability to disseminate the resistance by means of transformation, conjugation and by horizontal gene transfer element like integrons. Antibiotic resistance marker and its location were confirmed from the resistant plasmids from Vibrio isolates by using curing protocols. As in many developing countries, antimicrobial resistance epidemiology is still in the infancy stage in the locality of the study. To our knowledge, there are no reports available on the plasmid mediated multiple antibiotic resistance in Vibrio isolates from coastal waters of Kerala and our study on plasmid profiling will provide a current baseline profile of plasmid mediated resistance of Vibrios from coastal waters in Kerala and thereby will provide a significant insight about Indian scenario. [ABSTRACT FROM AUTHOR]

Vibrios are highly motile, facultatively anaerobic bacteria that are ubiquitous in aquatic environments and part of the normal intestinal microflora of healthy fish, but some species can cause vibriosis. The adherence of vibrios to host fish intestines is a significant event not only for their survival and growth, but also in terms of pathogenicity. However, the molecular mechanism underlying the adhesion of vibrios to the intestinal tract of fish is not fully understood. We report here the identification of intestinal glycosphingolipid (GSL) receptors to which pathogenic vibrios attach in typical mariculture fish. Thin-layer chromatography overlay assays using five species of S-labeled vibrios and intestinal glycosphingolipids of seven species of mariculture fish revealed that all of the fish tested possessed GM3 (NeuAcα2-3Galβ1-4Glcβ1-1′Cer) and/or GM4 (NeuAcα2-3Galβ1-1′Cer) as major acidic intestinal GSLs and that all of the vibrios tested specifically adhered to GM3 and/or GM4. Our results demonstrate that these GSLs were able to function as a receptor for the various vibrios tested. Analysis of the relationship between sugar structure and receptor activity for vibrios revealed that 'NeuAcα2-3Galβ1-' is required at the non-reducing end of glycosphingolipids for the bacteria to attach. [ABSTRACT FROM AUTHOR]

Abstract: From 2006 to 2008, microbial water quality was monitored along the Georgian coast of the Black Sea. Temperature, pH, salinity, and dissolved oxygen were measured, along with a variety of aquatic microbial parameters, including heterotrophic plate count (HPC), total culturable bacterial count (TCBC), and chlorophyll a (Chl-a) concentration. Total and fecal coliforms and total enterococci counts were recorded as indicators of fecal pollution. Vibrio bacteria, and Escherichia coli- and Vibrio-specific bacteriophages were isolated and enumerated to determine their relationships to standard marine pollution indicators. Persistent microbial pollution was observed, particularly in the summer months, with a higher rate of contamination in estuaries. Microbial indicators generally showed seasonal dependence, suggesting that temperature may influence bacterial dynamics in this environment. No correlation was apparent between fecal pollution indicators and physical–chemical and aquatic microbial parameters, although there were significant relationships amongst the indicators themselves, as well as with the prevalence of Vibrio bacteria and phage. [Copyright &y& Elsevier]

Wetland dynamics are probably linked to cholera endemicity in South Asia. We focus on links between Vibrio cholerae abundance, chitin content and suspended particle load in size fractions of suspended particulate matter (SPM) along the salinity gradient of Sunderban mangrove waters. SPM decreased downstream, while salinity increased from 0.2 to 4. Particulate organic carbon (90 ± 25 μM) and nitrogen (9.1 ± 3.3 μM) highly correlated with SPM and turbidity, suggesting a significant contribution of fine particles to organic matter. Total chitin ranged 1-2 mg/l and decreased downstream. The distribution among size fractions of SPM, chitin and V. cholerae O1 (the bacterial serogroup mainly associated with cholera epidemics) was similar, with ~98% of the total in the fraction <20 μm. In comparison, the number of V. cholerae O1 attached to zooplankton and microplankton size classes >20 μm was almost negligible, in contrast to usual assumptions. Thus, microdetritus, nanoplankton and fungal cells in size classes <20 μm represent a chitinaceous substrate on which V. cholerae can grow and survive. Total bacteria, cultivable vibrios and V. cholera O1 increased 5-10 times downstream, together with salinity and nitrite concentration. Overall, nitrate and silicate concentrations were relatively constant (>22 μM N and 100 μM Si). However, nitrite increased ~9 times in the outer sector, reaching ~1.2 μM N, probably as a result of increased abundance of nitrate-reducing vibrios. A characterization of Vibrio habitats that takes account of the presence of nitrate-reducing bacteria could improve the understanding of both mangrove nitrogen cycling and cholera seasonality. [ABSTRACT FROM AUTHOR]

Abstract: Cholera has been a public health burden in Ghana since the early 1970s. Between 1999 and 2005, a total of 25,636 cases and 620 deaths were officially reported to the WHO. In one of the worst affected urban cities, fecal contamination of surface water is extremely high, and the disease is reported to be prevalent among inhabitants living in close proximity to surface water bodies. Surface runoff from dump sites is a major source of fecal and bacterial contamination of rivers and streams in the study area. This study aims to determine (a) the impacts of surface water contamination on cholera infection and (b) detect and map arbitrary shaped clusters of cholera. A Geographic Information System (GIS) based spatial analysis is used to delineate potential reservoirs of the cholera vibrios; possibly contaminated by surface runoff from open space refuse dumps. Statistical modeling using OLS model reveals a significant negative association between (a) cholera prevalence and proximity to all the potential cholera reservoirs (R 2 =0.18, p <0.001) and (b) cholera prevalence and proximity to upstream potential cholera reservoirs (R 2 =0.25, p <0.001). The inclusion of spatial autoregressive coefficients in the OLS model reveals the dependency of the spatial distribution of cholera prevalence on the spatial neighbors of the communities. A flexible scan statistic identifies a most likely cluster with a higher relative risk (RR=2.04, p <0.01) compared with the cluster detected by circular scan statistic (RR=1.60, p <0.01). We conclude that surface water pollution through runoff from waste dump sites play a significant role in cholera infection. [Copyright &y& Elsevier]

Aims: The aim of this study was to characterise and identify vibrios isolated from the haemolymph of apparently healthy adult spider crabs ( Maja brachydactyla) wild-caught in the Spanish localities of Galician coast and in the Canary Islands and also from captive animals held at IRTA’s facilities in the Ebro Delta of Catalonia, north-west Spanish Mediterranean coast. Methods and Results: A total of 277 bacterial isolates were obtained, and of these, 171 were characterised with rep-PCR, resulting electrophoretic bands were analysed and clusters formed. Identification of representative strains of each cluster was made by sequencing the 16S rRNA. Samples from animals caught in Galicia and captive at IRTA (around 15–18°C) rendered mostly species belonging to the Splendidus clade (72·2 and 76·6% respectively), commonly found in cold waters (below 20°C). Higher species diversity was found in the haemolymph of the captive animals. In the warmer Canary Islands waters (around 21°C), the diversity of vibrios is dominated by three clades, Harveyi ( Vibrio core group, 39·3%), Orientalis (23·2%) and Splendidus (21·4%) with a species diversity that equals that of the colder captive animals. Conclusions: Differences in the vibrios populations were found in the haemolymph extracted from animals collected from the three localities. Potential new species were found, and their description is under way. Significance and Impact of Study: As with other invertebrates, spider crabs also contain a diverse population of vibrios. These findings should help researchers to diagnose when a crab is infected. [ABSTRACT FROM AUTHOR]

Abstract: Vibrios are Gram-negative rod-shaped bacteria that are widespread in the coastal and estuarine environments. Some species, e.g. Vibrio anguillarum and V. tapetis, comprise serious pathogens of aquatic vertebrates or invertebrates. Other groups, including Grimontia (=Vibrio) hollisae, Photobacterium (=Vibrio) damselae subsp. damselae, V. alginolyticus, V. harveyi (=V. carchariae), V. cholerae, V. fluvialis, V. furnissii, V. metschnikovii, V. mimicus, V. parahaemolyticus and V. vulnificus, may cause disease in both aquatic animals and humans. The human outbreaks, although low in number, typically involve wound infections and gastro-intestinal disease often with watery diarrhoea. In a minority of cases, for example V. vulnificus, there is good evidence to actually associate human infections with diseased animals. In other cases, the link is certainly feasible but hard evidence is mostly lacking. [Copyright &y& Elsevier]

Abstract: Bivalves are filter-feeders that can accumulate large numbers of bacteria, in particular Vibrio species; these can persist within bivalve tissues largely depending on their sensitivity to the hemolymph bactericidal activity. In this work, functional parameters of the hemolymph of Mytilus galloprovincialis were evaluated in response to in vivo challenge with different bacteria (Gram(−) Vibrio anguillarum and V. splendidus, Gram(+) Micrococcus lysodeikticus). Mussels were injected with heat-killed bacteria or PBS-NaCl (controls) and hemolymph sampled from 3 to 48 h post-injection (p.i.). In hemocytes, all bacteria induced significant lysosomal membrane destabilisation (LMS) from 3 h p.i. with V. splendidus >V. anguillarum >M. lysodeikticus. LMS showed recovery for both M. lysodeikticus and V. anguillarum, whereas a further time-dependent decrease was observed for V. splendidus. Bacterial challenge also induced a rapid (from 3 h p.i.) and significant increase in serum lysozyme activity; the effect was persistent with M. lysodeikticus and transient for the two Vibrio species. In order to evaluate whether in vivo challenge may affect the subsequent capacity of hemolymph to kill bacteria, the bactericidal activity was tested in an in vitro assay towards E. coli. At 48 h. p.i. hemolymph samples from V. anguillarum-injected mussels showed a significant increase in E. coli killing (+35% with respect to controls); a smaller effect was observed with V. splendidus-injected mussels (+16%), whereas M. lysodeikticus was ineffective. Moreover, hemolymph from V. anguillarum-injected mussels showed an in vitro bactericidal activity towards V. anguillarum 2-folds higher than that of controls. Changes in total hemocyte counts (THC) and in hemocyte populations were evaluated by Flow cytometry at 6 and 48 h p.i., indicating a decrease in THC followed by recovery with all bacteria. Moreover, at 6 h p.i. a general decrease in the percentage of granulocytes was observed (V. splendidus >V. anguillarum >M. lysodeikticus), followed by complete and partial recovery with M. lysodeikticus and V. anguillarum, respectively, but not with V. splendidus. The results demonstrate the existence of differential functional immune responses in M. galloprovincialis to different bacteria. [Copyright &y& Elsevier]

To determine the bacteriological quality of fishery products in the different stages from commercialization, monthly samples were taken during March 2004 and February 2006 from a Costa Rica marketing chain. Microbiological analyses were made to determine total coliforms (CT), faecal coliforms (CF), Escherichia coil (EC), aerobic total count (RTA), Salmonella sp., Staphylococcus aureus, Vibrio cholerae and Vibrio parahaemolyticus. Three body zones were analyzed: skin, belly and muscle. There were differences in the amount of CT between parts: skin had the highest counts, 11% of samples were identified as E. coli, and 2.5% of total counts were higher than the legal limit. Only 1.3% of the samples were S. aureus-positive. Salmonella sp., V cholerae and V. parahaemolyticus were absent. [ABSTRACT FROM AUTHOR]

Abstract: Taxonomic characterization was performed on the putative N2-fixing microbiota associated with the coral species Mussismilia hispida, and with its sympatric species Palythoa caribaeorum, P. variabilis, and Zoanthus solanderi, off the coast of São Sebastião (São Paulo State, Brazil). The 95 isolates belonged to the Gammaproteobacteria according to the 16S rDNA gene sequences. In order to identify the isolates unambiguously, pyrH gene sequencing was carried out. The majority of the isolates (n=76) fell within the Vibrio core group, with the highest gene sequence similarity being towards Vibrio harveyi and Vibrio alginolyticus. Nineteen representative isolates belonging to V. harveyi (n=7), V. alginolyticus (n=8), V. campbellii (n=3), and V. parahaemolyticus (n=1) were capable of growing six successive times in nitrogen-free medium and some of them showed strong nitrogenase activity by means of the acetylene reduction assay (ARA). It was concluded that nitrogen fixation is a common phenotypic trait among Vibrio species of the core group. The fact that different Vibrio species can fix N2 might explain why they are so abundant in the mucus of different coral species. [Copyright &y& Elsevier]

Abstract: The aim of the present study was to characterize and identify vibrios isolated from cultured clams in Galicia (NW Spain). A total of 759 isolates were obtained, phenotypically characterized, grouped and assigned to the genus Vibrio. Subsequently, the genomic diversity of 145 representative strains was analyzed by means of amplified fragment length polymorphism (AFLP), which revealed a high genetic diversity amongst these isolates. Only 57 out of 145 strains could be identified to the species level, and they were distributed in 13 AFLP clusters. V. cyclitrophicus, V. splendidus and V. alginolyticus were the most abundantly represented species. Eighty-eight isolates remained unidentified, 59 were distributed over 16 clusters, while 29 were unclustered. Sequencing of the 16S rRNA and two house-keeping genes (rpoA and recA) from representative strains belonging to eight unidentified clusters with the highest number of isolates confirmed their assignation to the Vibrionaceae family, and some of these probably represent new species within the genus. The present study confirmed that the phenotypic characterization of vibrios is not sufficient to identify them at the species level. A wide diversity of vibrios was found in cultured clams from all four geographic locations analyzed. In total, more than 12 Vibrio species and at least three potential new species in this genus were identified. [Copyright &y& Elsevier]