Vibrio cholera and V. parahaemolyticus coinfection in an oncology patient with gastroenteritis.

Vibrio-related gastroenteritis in the United States is mostly associated with the consumption of raw or improperly cooked seafood. We describe a case of a stage IV lung adenocarcinoma patient who became ill after eating crab while visiting Upstate New York. Molecular testing and culture confirmed a coinfection with V. parahaemolyticus and a nontoxigenic strain V. cholera.

A double-quadratic model for predicting Vibrio species in water environments of Japan.

Vibrio spp. are natural inhabitants of marine and estuarine environments. Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus are the major infectious agents for humans. Their densities are affected by environmental factors such as water temperature and salinity. The detailed contribution of each factor still remains to be elucidated. Here we conducted multi-coastal study in a 21-month period to examine relationships between environmental factors and V. cholerae, V. parahaemolyticus and V. vulnificus densities in sea surface water in eight coastal sites of four prefectures in Japan. Vibrio densities were measured by a most-probable-number with PCR method which is highly sensitive and quantitative (3/100 ml of detection limit). Vibrio densities were analyzed with environmental factors including water temperature, salinity, total dissolved substance, and pH, and their quadratics. A linear regression model suited best for prediction of V. cholerae density. A novel double-quadratic model suited best for the prediction of V. parahaemolyticus and V. vulnificus densities.

A novel adhesive factor contributing to the virulence of Vibrio parahaemolyticus.

Bacterial adhesins play a pivotal role in the tight bacteria-host cells attachment to initiate the downstream processes and bacterial infection of hosts. In this study, we identified a novel adhesin, VpadF in V. parahaemolyticus. Deletion of VpadF in V. parahaemolyticus markedly impaired its attachment and cytotoxicity to epithelial cells, as well as attenuated the virulence in murine model. Biochemical studies revealed that VpadF recognized both fibronectin and fibrinogen. The binding of VpadF to these two host receptors was mainly dependent on the its fifth bacterial immunoglobulin-like group domain and its C-terminal tail. Our finding suggested that VpadF is a major virulence factor of V. parahaemolyticus and a potential good candidate for V. parahaemolyticus infection control for both vaccine development and drug target.

Development of a rapid and simple immunochromatographic assay to identify Vibrio parahaemolyticus.

To rapidly and simply determine whether or not bacterial colonies growing on agar were Vibrio parahaemolyticus, we developed an immunochromatographic assay (VP-ICA) using two different monoclonal antibodies (designated mAb-VP34 and mAb-VP109) against the delta subunit of V. parahaemolyticus-F0F1 ATP synthase. The epitopes recognized by mAb-VP34 and mAb-VP109 were mapped to sequences of eight ((47)LLTSSFSA(54)) and six amino acid residues ((16)FDFAVD(21)), respectively. An amino acid sequence similarity search of the NCBI database using BLASTP showed that both epitopic amino acid sequences were present together only in V. parahaemolyticus. When 124 V. parahaemolyticus strains and 94 strains of 27 other Vibrio species or 35 non-Vibrio species were tested using the VP-ICA, the VP-ICA identified V. parahaemolyticus with 100% accuracy. The VP-ICA rapidly and simply identified the pathogen directly from a single agar colony within 30 min, indicating that VP-ICA will greatly reduce labor and time required to identify V. parahaemolyticus compared with conventional biochemical tests.

Multiple enzymatic profiles of Vibrio parahaemolyticus strains isolated from oysters / Multiple enzymatic profiles of Vibrio parahaemolyticus strains isolated from oysters.

The enzymatic characterization of vibrios has been used as a virulence indicator of sanitary interest. The objective of this study was to determine the enzymatic profile of Vibrio parahaemolyticus strains (n=70) isolated from Crassostrea rhizophorae oysters. The strains were examined for the presence of gelatinase (GEL), caseinase (CAS), elastase (ELAS), phospholipase (PHOS), lipase (LIP), amilase (AML) and DNase. All enzymes, except elastase, were detected in more than 60

of the strains. The most recurrent enzymatic profiles were AML + DNase + PHOS + GEL + LIP (n=16; 22.9

) and AML + CAS + DNase + PHOS + GEL + LIP (n=21; 30

). Considering the fact that exoenzyme production by vibrios is closely related to virulence, one must be aware of the bacteriological risk posed to human health by the consumption of raw or undercooked oysters.

Population structure of clinical and environmental Vibrio parahaemolyticus from the Pacific Northwest coast of the United States.

Vibrio parahaemolyticus is a common marine bacterium and a leading cause of seafood-borne bacterial gastroenteritis worldwide. Although this bacterium has been the subject of much research, the population structure of cold-water populations remains largely undescribed. We present a broad phylogenetic analysis of clinical and environmental V. parahaemolyticus originating largely from the Pacific Northwest coast of the United States. Repetitive extragenic palindromic PCR (REP-PCR) separated 167 isolates into 39 groups and subsequent multilocus sequence typing (MLST) separated a subset of 77 isolates into 24 sequence types. The Pacific Northwest population exhibited a semi-clonal structure attributed to an environmental clade (ST3, N = 17 isolates) clonally related to the pandemic O3:K6 complex and a clinical clade (ST36, N = 20 isolates) genetically related to a regionally endemic O4:K12 complex. Further, the identification of at least five additional clinical sequence types (i.e., ST43, 50, 65, 135 and 417) demonstrates that V. parahaemolyticus gastroenteritis in the Pacific Northwest is polyphyletic in nature. Recombination was evident as a significant source of genetic diversity and in particular, the recA and dtdS alleles showed strong support for frequent recombination. Although pandemic-related illnesses were not documented during the study, the environmental occurrence of the pandemic clone may present a significant threat to human health and warrants continued monitoring. It is evident that V. parahaemolyticus population structure in the Pacific Northwest is semi-clonal and it would appear that multiple sequence types are contributing to the burden of disease in this region.

Multiple enzymatic profiles of Vibrio parahaemolyticus strains isolated from oysters.

The enzymatic characterization of vibrios has been used as a virulence indicator of sanitary interest. The objective of this study was to determine the enzymatic profile of Vibrio parahaemolyticus strains (n=70) isolated from Crassostrea rhizophorae oysters. The strains were examined for the presence of gelatinase (GEL), caseinase (CAS), elastase (ELAS), phospholipase (PHOS), lipase (LIP), amilase (AML) and DNase. All enzymes, except elastase, were detected in more than 60% of the strains. The most recurrent enzymatic profiles were AML + DNase + PHOS + GEL + LIP (n=16; 22.9%) and AML + CAS + DNase + PHOS + GEL + LIP (n=21; 30%). Considering the fact that exoenzyme production by vibrios is closely related to virulence, one must be aware of the bacteriological risk posed to human health by the consumption of raw or undercooked oysters.

Production and characterization of a novel monoclonal antibody against Vibrio parahaemolyticus F0F1 ATP synthase's delta subunit and its application for rapid identification of the pathogen.

We raised monoclonal antibodies (MAbs) against Vibrio parahaemolyticus cell extracts. One of the MAbs, designated MAb-VP34, reacted in enzyme-linked immunosorbent assays (ELISAs) with 140 V. parahaemolyticus strains, regardless of serotype or origin. MAb-VP34 did not detectably react with 96 strains belonging to 27 other Vibrio species (except for Vibrio natriegens) or with 29 non-Vibrio species. These results show that MAb-VP34 is highly specific for V. parahaemolyticus. Western blotting and mass spectrometry analyses revealed that MAb-VP34 recognized V. parahaemolyticus F(0)F(1) ATP synthase's delta subunit. Using MAb-VP34, a rapid and simple immunodot blotting assay (VP-Dot) was developed to determine whether bacterial colonies growing on selective agar, represented V. parahaemolyticus. To evaluate VP-Dot, 20 V. parahaemolyticus strains and 19 non-related strains were tested. The results indicated that VP-Dot is a reliable tool for identification of V. parahaemolyticus colonies. The simple VP-Dot procedure took 40min, indicating that the MAb-VP34 based immunological method will greatly reduce labor, time, and costs required to verify V. parahaemolyticus colonies as compared with the conventional biochemical test.

A polyphasic approach for the differentiation of environmental Vibrio isolates from temperate waters.

Climate change and marine traffic lead to changing species communities in the oceans. Due to increasing seawater temperatures, pathogenic Vibrio species could become significant even in temperate waters. We classified mesophilic Vibrio isolates from the German Bight (North Sea) using a polyphasic approach with special emphasis on Vibrio parahaemolyticus. Matrix-assisted laser desorption/ionization time-of-flight MS was used as a primary screen to classify isolates, 16S rRNA gene and rpoB gene sequencing to identify species. Potential V. parahaemolyticus isolates were screened for regulatory or virulence-related genes (toxR, tlh, tdh, trh). To investigate genomic diversity, we applied repetitive-sequence-based PCRs. Results were evaluated and methods compared using multivariate statistical analysis. Most isolates were classified as V. parahaemolyticus or Vibrio alginolyticus. Reliable differentiation between both species was achieved by rpoB sequencing and toxR detection. Among the fingerprinting methods, ERIC-PCR showed the highest discriminatory power, displaying three separated clusters. These clusters represent the species V. parahaemolyticus, V. alginolyticus and one group in between. The frequent detection of V. parahaemolyticus in the German Bight reveals the urgency for further monitoring. In this context, a polyphasic approach, such as defined in this study, is needed to differentiate populations of V. parahaemolyticus and V. alginolyticus.

Características de Vibrio parahaemolyticus isolados de mexilhões (Perna perna) comercializados em Niterói, Rio de Janeiro / Characteristics of Vibrio parahaemolyticus isolated from mussels (Perna perna) commercialized at Niterói, Rio de Janeiro

O ecossistema marinho é o habitat natural de bactérias como Vibrio parahaemolyticus, um importante patógeno causador de gastrenterite humana associada ao consumo de alimentos marinhos. Na presente investigação, foi avaliada a presença de V. parahaemolyticus a partir de 86 amostras de mexilhões in natura e pré-cozidos. Vibrio parahaemolyticus foi isolado a partir de 11,6 por cento dos mexilhões in natura e pré-cozidos avaliados. Todas as cepas avaliadas demonstraram-se urease positivas e 28,5 por cento Kanagawa positivas sugerindo um potencial patogênico para o homem. Houve a predominância do sorotipo O10:K52 e a identificação da cepa emergente O3:K6. Esses resultados apontam para a relevância epidemiológica de V. parahaemolyticus em casos de gastrenterite humana após consumo de mexilhões sem cozimento adequado (100°C/15min). Além disso, é importante alertar as autoridades de Vigilância Sanitária no Brasil quanto a sua presença na cadeia alimentar e seus riscos para a Saúde Pública.

The marine ecosystem is the natural habitat of bacteria like Vibrio parahaemolyticus, an important pathogen that cause human gastroenteritis associated with seafood consumption. In the present investigation, the presence of V. parahaemolyticus in 86 in natura and precooked mussel samples was evaluated. Vibrio parahaemolyticus was isolated from 11.6 percent of the in natura and precooked mussels. All strains tested were urease-positive and 28.5 percent were Kanagawa-positive, which suggests that they have pathogenic potential for humans. There was predominance of the O10:K52 serotype and the emerging O3:K6 strain was identified. These results show the epidemiological relevance of V. parahaemolyticus in cases of human gastroenteritis following mussel consumption without adequately cooking them (100°C/15min). Moreover, it is important to alert the Brazilian Sanitary Surveillance authorities regarding their presence in the food chain and their public health risks.

Vibrio parahaemolyticus growth under low-iron conditions and survival under high-magnesium conditions.

Since 1996, Vibrio parahaemolyticus serotype O3:K6 and closely related strains have been associated with an increased incidence of V. parahaemolyticus gastroenteritis worldwide, suggesting the emergence of strains with enhanced abilities to cause disease. One hypothesis for the recent emergence of V. parahaemolyticus O3:K6 and related strains is an enhanced capacity for environmental survival relative to other strains, which might result in increased human exposure to these organisms. Therefore, the objective of this study was to test the hypothesis that survival or growth characteristics of clinical V. parahaemolyticus isolates differ from those of nonclinical isolates under different environmental conditions. Twenty-six V. parahaemolyticus isolates selected to represent either clinical or food sources were monitored for either survival following exposure to high magnesium (300 mM) or growth under iron-limited conditions. Isolates in each category (clinical or food) differed widely in survival capabilities following 24 h of exposure to 300 mM Mg2+. Although 4 of 15 clinical isolates grew better at approximately 0.96 microM Fe2+ (iron-limited conditions) than at 50 microM Fe2+ (iron-rich conditions), as an entire group clinical isolates in this study were not more effective at growing under iron-limited conditions than were strains not associated with disease. Within the diverse collection of strains examined in these experiments, neither growth characteristics in low-iron environments nor survival capabilities following exposure to high magnesium concentrations were uniformly different between clinical and nonclinical V. parahaemolyticus isolates. Therefore, neither phenotypic characteristic can be used to reliably differentiate potentially pathogenic V. parahaemolyticus strains.

Revisión y recomendaciones para el manejo de diarrea por Vibrio parahaemolyticus / Review and guidelines for treatment of diarrhea caused by Vibrio parahaemolyticus

A contar del año 1998 se han presentado en Chile tres brotes epidémicos por Vibrio parahaemolyticus, el último de ellos durante el verano del 2005, que afectó a más de 10.000 personas. Los afectados presentaron un cuadro clínico caracterizado por diarrea, náuseas, vómitos, dolor abdominal y fiebre; 6 por ciento de los casos tuvo leucocitos fecales positivos y un paciente falleció. La cepa predominante en los tres brotes ha sido la pandémica O3: K6. El diagnóstico de V. parahaemolyticus se realizó con la confirmación microbiológica de las cepas y tipificación o por asociación epidemiológica. Las cepas fueron susceptibles in vitro a tetraciclina, cefalosporinas de tercera generación, quinolonas y cloranfenicol no observándose susceptibilidad a ampicilina. Todos los casos se asociaron al consumo de mariscos crudos o insuficientemente cocidos. Por la repercusión de este brote, el Ministerio de Salud impulsó la formación de una comisión multidisciplinaria para actualizar los aspectos epidemiológicos, clínicos y microbiológicos, y elaborar una guía de recomendaciones en el manejo de esta infección.

Density-dependent sorting of physiologically different cells of Vibrio parahaemolyticus.

A pure bacterial culture is composed of clonal cells in different physiological states. Separation of those subpopulations is critical for further characterization and for understanding various processes in the cultured cells. We used density-dependent cell sorting with Percoll to separate subpopulations from cultures of a marine bacterium, Vibrio parahaemolyticus. Cells from cultures in the exponential and stationary phases were fractionated according to their buoyant density, and their culturability and ability to maintain culturability under low-temperature and low-nutrient stress (stress resistance) were determined. The buoyant density of the major portion of the cells decreased with culture age. The culturability of stationary-phase cells increased with increasing buoyant density, but that of exponential-phase cells did not. Stress resistance decreased with increasing buoyant density regardless of the growth phase. The results indicate that density-dependent cell sorting is useful for separating subpopulations of different culturabilities and stress resistances. We expect that this method will be a powerful tool for analyzing cells in various physiological states, such as the viable but nonculturable state.

Enumeração e identificação de Vibrio parahaemolyticus em lulas frescas comercializadas no município de Niterói RJ Brasil / Enumeration and identification of Vibrio parahaemolyticus in fresh squids commercialized at Niterói, Rio de Janeiro State, Brazil

Com o objetivo de verificar as condições higiênico-sanitárias do pescado comercializado no município de Niterói, efetuou-se um estudo sobre a enumeração e identificação de Vibrio parahaemolyticus em lulas frescas. Foram utilizadas 50 amostras de lulas frescas, identificadas como pertencentes à espécie Oorytheutis brasiliensís Blainville, 1823. A identificação e enumeração do V. parahaemolyticus foram baseadas nos métodos descritos pela lnternational Commission on Microbiological Specifications for Foods - ICMSF (1983). A classificação de Heiberg (1936) foi utilizada como teste complementar. O V. parahaemolyticus foi identificado em três (6%) amostras, sendo que, em uma o vibrio se desenvolveu em meio com concentração salina a 10%, na prova de halofilismo. O V. parahaemolyticus apresentou os NMPs (Número Mais Provável) com média de 19,3.bactérias/g. As três amostras de V. parahaemolyticus submetidas à classificação de Heiberg apresentaram perfil compatível com o grupo VIl (Sacarose -, Manose + e Arabinose +). Concluiu-se que, mesmo sob comercialização inadequada, com manipulação e resfriamento precários, as amostras analisadas apresentaram o V. parahaemolyticus com frequências e NMPs baixos.

A study on the enumeration and identification of Vibrio parahaemolyticus in freshsquids in Niterói was undertook aiming at checking the hygienic-sanitary conditions of fish commercialized in that City. Fifty fresh squids (Oorytheutis brasiliensis, Blainville, 1823). Methods described by the lnternational Comission on Microbiological Specification for Foods-ICMSF (1983) were used to identify and enumerate V. parahaemoiyticus and Heiberg's classi-fication was a complementai test. v. parahaemoiyticus was identified in three samples (6%); in one of them it grew in an environment with 10% salt concentration during halophilism probe. Most probable number (MPN) average for V. parahaemoiyticus was 19,3 bacteria per gram. The three samples submitted to Heiberg's classification showed a profile compatible with group VIl (sacarose-, manose+, arabinose-). We concluded that, even under inadequate commercialization, manipulation and refrigeration conditions, V. parahaemolyticus frequencies and MPNs are low.

Caracterizaçäo sorológica de amostras de Vibrio Parahaemolyticus isoladas de peixes capturados no litoral brasileiro / Serological characterization of strains of Vibrio parahaemolyticus isolated from fishis caugth off the Brazilian coast

Em 82 peixes de origem marinha e de diferentes espécies, capturados na faixa de litoral compreendida entre Bahia e Rio Grande do Sul, foram detectados 45 ou 54, 8 por cento portadores de Vibrio parahaemolyticus, propiciando o isolamento de 71 culturas. Na caracterizaçäo sorológica (0: K), 13 sorotipos foram identificados (24 ou 33, 7 por cento) e 47 ou 66, 1 por cento das amostras só permitiram o reconhecimento da estrutura "O".os sorotipos mais incidentes foram: 02: k28; 05: K17; 01: 32 e 02: K12; nos grupos sorológicos predominaram 02; 04; 05 e 03. Näo foi evidenciado qualquer vínculo de espécies de peixes com determinados sorotipos ou sorogrupos tampouco, os níveis de isolamento sofreram a influência da sszonalidade.

Ecological relationship between Vibrio parahaemolyticus and agar-digesting vibrios as evidenced by bacteriophage susceptibility patterns.

Twenty bacteriophages active against Vibrio parahaemolyticus and agar-digesting vibrios, isolated from oysters (Crassostrea gigas) and Dungeness crab (Cancer magister) and by induction of a lysogenic agar digester, were tested as to their host range. These phages were specific for V. parahaemolyticus and various agar-digesting vibrios, and interspecies lysis occurred only between these two groups. V. alginolyticus, V. anguillarum and related species, V. cholerae, and a group of marine psychrophilic and psychrotrophic vibrios were not affected. No correlation was observed between the O and K serotypes of V. parahaemolyticus strains and bacteriophage susceptibility patterns, and 7 of 28 strains of V. parahaemolyticus were not lysed by any of the phages. Only two of the phage isolates were capable of lysing all susceptible V. parahaemolyticus strains. No correlation was observed between the inter-and intraspecies genetic relatedness (DNA homologies) of V. parahaemolyticus and agar-digesting vibrios and susceptibility patterns to different bacteriophages. Some of the phages were capable of plaque formation on V. parahaemolyticus as well as on some strains of agar-digesting vibrios that were separated by 70 to 80% differences in their DNA homologies. The possible ecological significance of these vibrio bacteriophages, particularly those having a wide host range, is discussed.