Genomic epidemiology of Vibrio parahaemolyticus from acute diarrheal patients in Shenzhen City from 2013 to 2021 / 中华预防医学杂志

Objective: To characterize the prevalence and genomic epidemiology of Vibrio parahaemolyticus from acute diarrheal patients in Shenzhen City from 2013 to 2021. Methods: Based on the Shenzhen Infectious Diarrhea Surveillance System, acute diarrheal patients were actively monitored in sentinel hospitals from 2013 to 2021. Whole-genome sequencing (WGS) of Vibrio parahaemolyticus isolates was performed, and the genomic population structure, serotypes, virulence genes and multilocus sequence typing were analyzed. Outbreak clusters from 2019 to 2021 were explored based on single-nucleotide polymorphism analysis. Results: A total of 48 623 acute diarrhea cases were monitored in 15 sentinel hospitals from 2013 to 2021, and 1 135 Vibrio parahaemolyticus strains were isolated, with a positive isolation rate of 2.3%. Qualified whole-genome sequencing data of 852 isolates were obtained. Eighty-nine serotypes, 21 known ST types and 5 new ST types were identified by sequence analysis, and 93.2% of strains were detected with toxin profile of tdh+trh-. 8 clonal groups (CGs) were captured, with CG3 as the absolute predominance, followed by CG189. The CG3 group was dominated by O3:K6 serotype and ST3 sequence type, while CG189 group was mainly O4:KUT, O4:K8 serotypes and ST189a and ST189 type. A total of 13 clusters were identified, containing 154 cases. About 30 outbreak clusters with 29 outbreak clusters caused by CG3 strains from 2019 to 2021. Conclusion: Vibrio parahaemolyticus is a major pathogen of acute infectious diarrhea in Shenzhen City, with diverse population structures. CG3 and CG189 have been prevalent and predominant in Shenzhen City for a long time. Scattered outbreaks and persistent sources of contamination ignored by traditional methods could be captured by WGS analysis. Tracing the source of epidemic clone groups and taking precise prevention and control measures are expected to significantly reduce the burden of diarrhea diseases caused by Vibrio parahaemolyticus infection in Shenzhen City.

ToxR represses the synthesis of c-di-GMP in Vibrio parahaemolyticus / 生物工程学报

Vibrio parahaemolyticus, the main pathogen causing seafood related food poisoning worldwide, has strong biofilm formation ability. ToxR is a membrane binding regulatory protein, which has regulatory effect on biofilm formation of V. parahaemolyticus, but the specific mechanism has not been reported. c-di-GMP is an important second messenger in bacteria and is involved in regulating a variety of bacterial behaviors including biofilm formation. In this study, we investigated the regulation of ToxR on c-di-GMP metabolism in V. parahaemolyticus. Intracellular c-di-GMP in the wild type (WT) and toxR mutant (ΔtoxR) strains were extracted by ultrasonication, and the concentrations of c-di-GMP were then determined by enzyme linked immunosorbent assay (ELISA). Three c-di-GMP metabolism-related genes scrA, scrG and vpa0198 were selected as the target genes. Quantitative real-time PCR (q-PCR) was employed to calculate the transcriptional variation of each target gene between WT and ΔtoxR strains. The regulatory DNA region of each target gene was cloned into the pHR309 plasmid harboring a promoterless lacZ gene. The recombinant plasmid was subsequently transferred into WT and ΔtoxR strains to detect the β-galactosidase activity in the cellular extracts. The recombinant lacZ plasmid containing each of the target gene was also transferred into E. coli 100λpir strain harboring the pBAD33 plasmid or the recombinant pBAD33-toxR to test whether ToxR could regulate the expression of the target gene in a heterologous host. The regulatory DNA region of each target gene was amplified by PCR, and the over-expressed His-ToxR was purified. The electrophoretic mobility shift assay (EMSA) was applied to verify whether His-ToxR directly bound to the target promoter region. ELISA results showed that the intracellular c-di-GMP level significantly enhanced in ΔtoxR strain relative to that in WT strain, suggesting that ToxR inhibited the production of c-di-GMP in V. parahaemolyticus. qPCR results showed that the mRNA levels of scrA, scrG and vpa0198 significantly increased in ΔtoxR strain relative to those in WT strain, suggesting that ToxR repressed the transcription of scrA, scrG and vpa0198. lacZ fusion assay showed that ToxR was able to repress the promoter activities of scrA, scrG and vpa0198 in both V. parahaemolyticus and E. coli 100λpir. EMSA results showed that His-ToxR was able to bind to the regulatory DNA regions of scrA and scrG, but not to the regulatory DNA region of vpa0198. In conclusion, ToxR inhibited the production of c-di-GMP in V. parahaemolyticus via directly regulating the transcription of enzyme genes associated with c-di-GMP metabolism, which would be beneficial for V. parahaemolyticus to precisely control bacterial behaviors including biofilm formation.

Epidemiological and etiological characteristics of Vibrio parahaemolyticus strains causing foodborne disease outbreaks in Guangdong Province from 2017 to 2020 / 中华预防医学杂志

Objective: To study the epidemiological and pathogenic characteristics of Vibrio parahaemolyticus isolated from outbreaks cases in Guangdong Province, 2017-2020. Methods: Epidemiological characteristics of 87 outbreak events caused by Vibrio parahaemolyticus were analyzed. Strains were serotyped, and then analyzed by pulsed-field gel electrophoresis (PFGE). Results: The food-borne disease outbreak caused by Vibrio parahaemolyticus was found in 16 cities. 44.8% (39/87) and 37.9% (33/87) of the outbreaks occurred in hotels, restaurants and school canteens, respectively. Improper food processing and storage (40.2%, 35/87) and cross contamination caused by indiscriminate raw and cooked food (25.3%, 22/87) were the main causes of food-borne disease outbreaks of Vibrio parahaemolyticus. The main serotypes of patient derived strains were O3:K6 (87.5%) and O4:KUT (22.5%). The similarity value between O3:K6 type isolates was 65.5%-100.0%, and the PFGE pattern similarity value of O4:KUT type isolates was 66.5%-100.0%. Conclusion: Outbreaks caused by Vibrio parahaemolyticus are widely distributed in Guangdong province. It is necessary to strengthen the publicity and education on the correct handling of food in hotels, restaurants, schools, and unit canteens. O3:K6 and O4:KUT serotypes are the main serotypes of the outbreak. There is genetic diversity among the epidemic strains.

Visual Detection of Vibrio parahaemolyticus using Combined CRISPR/Cas12a and Recombinase Polymerase Amplification / 生物医学与环境科学（英文）

Objective@#To establish an ultra-sensitive, ultra-fast, visible detection method for Vibrio parahaemolyticus (VP) .@*Methods@#We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) combined with recombinase polymerase amplification and visual detection (CRISPR/Cas12a-VD).@*Results@#CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10 -18 M (single molecule detection) within 30 min without cross-reactivity against other bacteria. When detecting pure cultures of VP, the consistency of results reached 100% compared with real-time PCR. The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10 2 CFU/g.@*Conclusion@#The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods, such as real-time PCR, and has great potential for preventing the spread of pathogens.

Diversidad molecular de variantes patogénicas de Vibrio parahaemolyticus en el Perú / Molecular diversity in pathogenic variants of vibrio parahaemolyticus in Peru

RESUMEN Con el objetivo de determinar la diversidad de variantes patogénicas de Vibrio parahaemolyticus en el Perú durante el periodo 1995-2017, se analizaron 102 genomas peruanos (97 clínicos y 5 ambientales) empleando el esquema de tipificación multilocus y BLASTn para la búsqueda de genes de virulencia. Se identificaron 15 tipos de secuencia diferentes, encontrándose que el genotipo ST3, perteneciente al clon pandémico, fue el más abundante, con 52% (n=53); seguido por el ST120, con 23,5% (n=24); y el complejo clonal CC345, con 11,8% (n=12). Un total de 89 cepas analizadas presentaron genes que codifican la isla de patogenicidad VpaI-7 (87,3%), mientras que 96 presentaron el gen tdh (94,1%), y 6, el trh (5,9%). Durante el periodo evaluado, se resalta la predominancia del ST3, causante de un importante brote en el pasado del Perú, además de otros genotipos patógenos que representan un riesgo latente en salud pública asociado al consumo de alimentos marinos.

ABSTRACT During the period from 1995 to 2017, in order to determine the diversity of Vibrio parahaemolyticus pathogenic variants in Peru, 102 Peruvian genomes (97 from a hospital setting and 5 from an out-of-hospital setting) were analyzed using the multilocus typification scheme and BLASTn in the search for virulence genes. Fifteen different sequence types were identified. It was found that the ST3 genotype, which is found in the pandemic clone, was the most abundant, with 52% (n=53); followed by ST120, with 23.5% (n=24); and the CC345 clonal complex, with 11.8% (n=12). A total of 89 analyzed strains presented genes encoding the pathogenicity island VpaI-7 (87.3%), while 96 presented the tdh gene (94.1%), and 6 the trh gene (5.9%). The ST3 genotype was the predominant one during the evaluated period, this genotype was the cause of a major outbreak in Peru's past history. Other pathogenic genotypes found represent a latent public health risk associated with seafood consumption.

Pathogenic potential of Vibrio parahaemolyticus isolated from tropical estuarine environments in Ceará, Brazil

Vibrio parahaemolyticus is a potentially pathogenic bacterium that occurs naturally in estuarine environments worldwide. This research aimed to investigate the occurrence of V. parahaemolyticus in estuarine environments and determine the virulence profile in an aquaculture environment by molecular techniques and conventional microbiological methods. Sampling was conducted in four estuaries in the State of Ceará (Pacoti, Choró, Pirangi and Jaguaribe), Brazil, between January and April 2009. The analysis included 64 samples of water (n=32) and sediment (n=32) collected from the estuaries. The samples yielded 64 isolates suspected to be V. parahaemolyticus. The isolates were submitted to biochemical identification using a dichotomous key and PCR for the detection of the species-specific tlh gene. Virulence was assessed by testing for urea hydrolysis and ß-hemolysis in erythrocytes (Kanagawa phenomenon) and simultaneous detection of the tdh and trh genes. All but one of the isolates (63/64) were confirmed to be V. parahaemolyticus by genotypic detection of tlh gene. The tdhand trh genes were detected in 57 and 19 isolates, respectively. The Kanagawa test was positive for 51 isolates. Only one isolate was positive for urease. The incidence of tdh/trh-positivity was very high in isolates recovered from the environment. The present study demonstrates the need to increase knowledge of the ecology and pathogeny of V. parahaemolyticus

Genomic basis of antibiotic resistance in Vibrio parahaemolyticus strain JPA1

A multi-resistant strain of Vibrio parahaemolyticus was isolated from a tropical estuary in Rio de Janeiro, Brazil. Genome sequencing was conducted to establish the molecular basis of antibiotic resistance in this organism. The genetic content of this strain revealed it to be a non-virulent lineage that nevertheless possesses several antibiotic resistance determinants.

Distribution of Pathogenic Vibrio Species in the Coastal Seawater of South Korea (2017–2018)

OBJECTIVES: Pathogenic Vibrio species are widely distributed in warm estuarine and coastal environments, and can infect humans through the consumption of raw or mishandled contaminated seafood and seawater. For this reason, the distribution of these bacteria in South Korea was investigated.METHODS: Seawater samples were collected from 145 coastal area points in the aquatic environment in which Vibrio species live. Environmental data (i.e., water temperature, salinity, turbidity, and atmospheric temperature) was collected which may help predict the distribution of the species (data not shown). Seawater samples were filtered, and incubated overnight in alkaline peptone water, at 37°C. Using species-specific polymerase chain reaction methods, screening tests were performed for the hlyA, ctxA, vvhA, and tlh genes. Clones of pathogenic Vibrio species were isolated using 3 selective plating media.RESULTS: In 2017, total seawater isolation rates for Vibrio vulnificus, Vibrio cholerae (non-pathogenic, non-O1, non-O139 serogroups), and Vibrio parahaemolyticus were 15.82%, 13.18%, 65.80%, respectively. However, in 2018 isolation rates for each were 21.81%, 19.40%, and 70.05%, respectively.CONCLUSION: The isolation rates of pathogenic Vibrio species positively correlated with the temperature of seawater and atmosphere, but negatively correlated with salinity and turbidity. From 2017 to 2018, the most frequent seawater-isolated Vibrio species were V. parahaemolyticus (68.10 %), V. vulnificus (16.54%), and non-toxigenic V. cholerae (19.58%). Comprehensive monitoring, prevention, and control efforts are needed to protect the public from pathogenic Vibrio species.

Assessment of homeopathic medicines on survival and antioxidant response in white shrimp Litopenaeus vannamei / Evaluación de medicamentos homeopáticos en la supervivencia y respuesta antioxidante del camarón blanco Litopenaeus vannamei infectado con Vibrio parahaemolyticus

ABSTRACT Objective. Evaluating the effect of homeopathic medicines on survival and activity of the superoxide dismutase (SOD) enzyme in white shrimp Litopenaeus vannamei subjected to infection with the pathogenic Vibrio parahaemolyticus. Materials and methods . The average lethal dosage (LD50) was determined for the pathogen strain in L. vannamei juveniles under immersion (Imm), injection (Inj) and incision + immersion (Inc+Imm) methods. Four treatments were applied: (1) Mix CIB®-HOM Heel-Mix (TH1), constituted by equal v/v ratio, of Cyme-Heel®, Gal-Heel®, Hepa-Heel®, Mucs-Heel® and Chol-Heel®; (2) Mix CIB®-HOM Pav-Mix (TH2), constituted by the same v/v ratio of Passiflora incarnata, Valeriana officinalis, Zincum valerianicum and Ignatia amara (Similia®); (3) Heel-Mix/Pav-Mix (TH3) consisting of a 1:1 v/v combination of the TH1 and TH2 treatments, and (4) ViT-Mix (TH4), constituted by Vidatox®, and a control (not treated/infected). Results. While applying the methods Imm, Inj and Inc+Imm, LD50 was 0.9 x 106; 0.6 x 106 and 0.5 x 106 UFC.mL-1, respectively. At the end of the challenge, the groups treated with TH2, TH3 and TH4 had a greater survival rate to that of the control group (p<0.05). Moreover, these two last treatments showed a greater SOD activity with respect to the control group (p<0.05). Conclusions. The homeopathic treatments (TH3 and TH4) increased survival and SOD activity in L. vannamei juveniles challenged with V. parahaemolyticus, which suggests that the homeopathic treatments employed had the potential as an alternative for the control of V. parahaemolyticus and its associated diseases, including the early mortality syndrome in shrimp farming.

RESUMEN Objetivo. Evaluar el efecto de medicamentos homeopáticos sobre la supervivencia y actividad de la enzima superóxido dismutasa (SOD) del camarón Litopenaeus vannamei sometido a infección con Vibrio parahaemolyticus. Materiales y métodos. Se determinó la dosis letal media (DL50) para la cepa patógena en juveniles de L. vannamei, bajo los método de inmersión (Inm), inyección (Iny) e incisión + inmersión (Inc+Inm). Luego el efecto de cuatro medicamentos homeopáticos sobre juveniles de L. vannamei retados con Vibrio parahaemolyticus fue evaluado usando el índice la supervivencia y la actividad SOD. Se aplicaron cuatro tratamientos: (1) Mezcla CIB®-HOM Heel-Mix (TH1), constituido por igual proporción v/v, de Cyme-Heel®, Gal-Heel®, Hepa-Heel®, Mucs-Heel® y Chol-Heel®; (2) Mezcla CIB®-HOM Pav-Mix (TH2), constituido por igual proporción v/v de Passiflora incarnata, Valeriana officinalis, Zincum valerianicum e Ignatia amara (Similia®); (3) Heel-Mix/Pav-Mix (TH3) constituido por una combinación 1:1 v/v de los tratamientos TH1 y TH2, y (4) ViT-Mix (TH4), constituido por Vidatox®, y un control (no tratado/infectado). Resultados. Al aplicar los método Inm, Iny e Inc+Inm la DL50 fue de 0.9 x 106; 0.6 x 106 y 0.5 x 106 UFC.mL-1, respectivamente. Los camarones tratados con TH3 y TH4 presentaron una mayor actividad de SOD con respecto al grupo control (p<0.05). Al final del reto, los grupos TH2, TH3 y TH4 tuvieron una supervivencia mayor a la del grupo control (p<0.05). Conclusiones. Los tratamientos homeopáticos (TH3 y TH4), aumentaron la actividad de la enzima SOD y la supervivencia en juveniles de L. vannamei, retados con V. parahaemolyticus. Esto sugiere que los tratamientos homeopáticos empleados tienen potencial como alternativa para el control de V. parahaemolyticus y sus enfermedades asociadas, incluido el síndrome de mortalidad temprana en el cultivo del camarón.

Pesquisa de Listeria monocytogenes e Vibrio parahaemolyticus em amostras de pescada amarela (Cynoscion acoupa) comercializadas na cidade de São Luís, MA / Search of Listeria monocytogenes and Vibrio parahaemolyticus in samples of fish (Cynoscion acoupa) marketed in the city of Sao Luis, MA

[{"text": "O aumento no consumo de pescados\r\ndevido a mudanças nas dietas alimentares\r\nfez com que a pescada amarela\r\n(Cynoscion acoupa) se tornasse\r\numa das espécies mais consumidas\r\nno Maranhão. Apesar dos benefícios\r\nnutricionais, esse consumo traz\r\nconsigo riscos à saúde pública quando\r\nocorre contaminação. O objetivo\r\ndesta pesquisa foi verificar a presença\r\nde Listeria monocytogenes e Vibrio\r\nparahaemolyticus em amostras\r\nde pescada amarela (C. acoupa) vendidas\r\nnas feiras e supermercados de\r\nSão Luís/ MA. Foram coletadas 30\r\namostras de filés de pescada amarela\r\nem feiras e supermercados e o\r\nprocessamento dessas amostras foi\r\nfeito no Laboratório de Microbiologia\r\nde Alimentos e Água da Universidade\r\nEstadual do Maranhão.\r\nAs análises microbiológicas foram\r\nrealizadas segundo o Manual de Métodos\r\nde Análise Microbiológica de\r\nAlimentos. Constatou-se a ausência\r\nde V. parahaemolyticus e ausência\r\nde L. monocytogenes em 100% das\r\namostras. Pode-se concluir, assim,\r\nque os resultados estão de acordo\r\ncom a RDC nº 12 de 2001, da ANVISA,\r\ne que, apesar das amostras não\r\napresentarem os patógenos investigados,\r\nfaz-se necessária a criação\r\nde parâmetros para essas bactérias,\r\ncomo forma de prevenção dos riscos\r\nà saúde pública.(AU)", "_i": "pt"}]

A foodborne outbreak of gastroenteritis caused by Vibrio parahaemolyticus associated with cross-contamination from squid in Korea / 한국역학회지

OBJECTIVES: Water-borne diseases caused by Vibrio parahemolyticus are often known to cause gastritis when raw or undercooked seafood is eaten. It is very rare that Vibrio gastritis caused by ingesting non-seafood products occurs on a large scale. On September 19, 2017, a large-scale Vibrio gastritis occurred after the city residents consumed food at a bazaar held in a welfare center in Jungnang-gu, Seoul. METHODS: The total number of visitors was approximately 299, and 237 (79.3%) of them showed symptoms. Among those who showed symptoms, 116 (48.9%) consulted the hospital, and 53 (45.6%) were hospitalized. Among the 299 exposed individuals, 174 (58.1%) responded to this survey: 163 (93.6%) with and 11 (6.4%) without symptoms. This study was retrospectively conducted by investigating the exposed individuals. To investigate the spread of infection, medical staff of hospitals in the epidemic area were interviewed, exposed individuals surveyed, microbiological testing conducted, and ingredient handling and cooking processes investigated. RESULTS: A total of 237 individuals, including 6 food handlers, were affected (prevalence, 79.2%). During the microbiological testing, V. parahemolyticus was found in 34 patients and 4 food handlers. In the consumption analysis, the relative risk of kimbap was 6.79 (confidence interval 1.10 to 41.69). In-depth investigation found that squid, an ingredient of Korean pancake, and egg sheets, an ingredient of kimbap, were prepared using the same cutting board and knife, which were thought to be the cause of cross-contamination that led to a large-scale outbreak of Vibrio gastritis. CONCLUSION: A recent large-scale outbreak of Vibrio gastritis occurred due to the cross-contamination with kimbap during the preparation process of squid rather than the actual consumption of seafood. Thus, a more stringent hygiene management is necessary during the processing and management of food to prevent infections associated with V. parahemolyticus.

Anti-Bacterial Effect of Lactobacillus rhamnosus Cell-Free Supernatant Possessing Lysozyme Activity Against Pathogenic Bacteria

Recently, there has been a growing demand for natural preservatives because of increased consumer interest in health. In this study, we produced Lactobacillus rhamnosus cell-free supernatant (LCFS) and evaluated and compared its antimicrobial activity with existing natural preservatives against pathogenic microorganisms and in chicken breast meat contaminated with Escherichia coli and Staphylococcus aureus. Lactobacillus rhamnosus cell-free supernatant possessed 30 units of lysozyme activity and contained 18,835 mg/L of lactic acid, 2,051 mg/L of citric acid and 5,060 mg/L of acetic acid. Additionally, LCFS inhibited the growth of fourteen pathogenic bacteria, S. aureus, Bacillus cereus, Listeria monocytogenes, Vibrio parahaemolyticus, Listeria innocua, S. epidermidis, L. ivanovii, E. coli, Pseudomonas aeruginosa, Shigella sonnei, Shi. flexneri, Proteus vulgaris, Pseudomonas fluorescens, and Klebsiella pneumoniae. The antibacterial activity of LCFS was stronger than that of egg white lysozyme (EWL), Durafresh (DF) and grapefruit seed extract (GSE). Additionally, LCFS maintained its antimicrobial activity after heat treatment at 50℃~95℃ and at pH values of 3~9. Moreover, LCFS inhibited the growth of E. coli and S. aureus in chicken breast meat. In conclusion, it is expected that LCFS, which contains both lysozyme and three organic acids, will be useful as a good natural preservative in the food industry.

A foodborne outbreak of gastroenteritis caused by Vibrio parahaemolyticus associated with cross-contamination from squid in Korea / 한국역학회지

OBJECTIVES: Water-borne diseases caused by Vibrio parahemolyticus are often known to cause gastritis when raw or undercooked seafood is eaten. It is very rare that Vibrio gastritis caused by ingesting non-seafood products occurs on a large scale. On September 19, 2017, a large-scale Vibrio gastritis occurred after the city residents consumed food at a bazaar held in a welfare center in Jungnang-gu, Seoul.METHODS: The total number of visitors was approximately 299, and 237 (79.3%) of them showed symptoms. Among those who showed symptoms, 116 (48.9%) consulted the hospital, and 53 (45.6%) were hospitalized. Among the 299 exposed individuals, 174 (58.1%) responded to this survey: 163 (93.6%) with and 11 (6.4%) without symptoms. This study was retrospectively conducted by investigating the exposed individuals. To investigate the spread of infection, medical staff of hospitals in the epidemic area were interviewed, exposed individuals surveyed, microbiological testing conducted, and ingredient handling and cooking processes investigated.RESULTS: A total of 237 individuals, including 6 food handlers, were affected (prevalence, 79.2%). During the microbiological testing, V. parahemolyticus was found in 34 patients and 4 food handlers. In the consumption analysis, the relative risk of kimbap was 6.79 (confidence interval 1.10 to 41.69). In-depth investigation found that squid, an ingredient of Korean pancake, and egg sheets, an ingredient of kimbap, were prepared using the same cutting board and knife, which were thought to be the cause of cross-contamination that led to a large-scale outbreak of Vibrio gastritis.CONCLUSION: A recent large-scale outbreak of Vibrio gastritis occurred due to the cross-contamination with kimbap during the preparation process of squid rather than the actual consumption of seafood. Thus, a more stringent hygiene management is necessary during the processing and management of food to prevent infections associated with V. parahemolyticus.

Formação de biofilme por Vibrio parahaemolyticus isolados de pescados / Biofilm formation by Vibrio parahaemolyticus isolated from fish

O pescado é um alimento altamente perecível, possui pH próximo a neutralidade, elevada atividade de água e alto teor de nutrientes facilmente utilizáveis por micro-organismos. Vibrio parahaemolyticus pode ser encontrado em ambientes com salinidade entre 3% e 8% e tem pH ideal para multiplicação entre 7,8 e 8,6. É um patógeno que pode causar gastrenterite aguda pelo consumo de frutos do mar contaminados, crus ou mal cozidos. Mesmo os processos de tratamento de água como cloração, adição de antibióticos e filtros apresentam dificuldade em reduzir a contaminação por Vibrio, sendo suposto que este gênero bacteriano pode formar biofilmes em diferentes superfícies. O objetivo do trabalho foi verificar a capacidade de V. parahaemolyticus isolados de pescados formarem biofilme após estresse subletal. No decorrer de um ano, foram realizadas 12 coletas mensais de amostras de peixes capturados no estuário da Lagoa dos Patos, as quais foram analisadas quanto à presença de V. parahaemolyticus. Concomitantemente, foram coletadas assepticamente amostras de água do estuário para análise de sanilidade e pH. Os isolados de Vibrio foram analisados pela reação em cadeia da polimerase (PCR) para identificação da espécie pela presença dos genes toxR. Além dos isolados obtidos no presente trabalho, também foram estudadas 15 outras cepas de V. parahaemolyticus previamente isoladas em outros trabalhos. As cepas foram avaliadas quanto à capacidade de produção de biofilme em placas de microtitulação. A capacidade de produção de biofilme após as cepas serem submetidas a diferentes tipos de estresse subletal (42ºC, 20ºC, 4ºC e pH ácido) também foi testada. Dentre os 120 peixes analisados, foram isolados V. parahaemolyticus de quatro (3,33%) pescados, sendo Mugil platanus a única espécie de peixe na qual o micro-organismo foi encontrado. Das 19 cepas analisadas, 89,5% foram capazes de formar biofilme, o que parece indicar que essa capacidade tem um papel importante na sobrevivência do micro-organismo nos pescados. Dessas, 25% das cepas aumentaram a capacidade de formar biofilme. Com base nos resultados, conclui-se que peixes da espécie M. platanus do estuário da Lagoa dos Patos são hospedeiros de V. parahaemolyticus e que a quase totalidade das cepas são formadoras de biofilme. A exposição a condições subletais de estresse tem efeito distinto sobre as diferentes cepas, induzindo aumento na capacidade de formar biofilme em algumas. Este foi o primeiro estudo realizado com V. parahaemolyticus, para avaliar o efeito de fatores de estresse sobre a formação de biofilme.(AU)

Fish is a highly perishable food, has a neutral pH, high water activity and high content nutrient, which makes it favorable to the microorganisms multiplication. Vibrio parahaemolyticus may be found in environments with a salinity of 3% and 8% and has optimal pH for multiplication between 7.8 and 8.6. This pathogen can cause acute gastroenteritis by consumption of contaminated raw or undercooked seafood. There is difficulty in reducing Vibrio contamination during fish processing, being supposed that this bacterial genus can form biofilm on different surfaces. The aim of this study was to verify the ability of V. parahaemlyticus isolated from fish from biofilm after sublethal stress. In the course of one year, 12 monthly samples of fish caught in the Lagoa dos Patos Estuary were analyzed for the presence of V. parahaemolyticus. Concurrently, water samples from estuary were collected aseptically for salinity analysis and pH. Vibrio isolates were analyzed by polymerase chain reaction (PCR) to identification of the species by presence of the toxR gene. In addition to the isolates obtained in this study were also studied 15 other strains of V. parahaemolyticus previously isolated in other works. The strains were evaluated for biofilm production capacity in microtiter plates. The biofilm production capacity after the strains had being subjected to different types of sublethal stress (42oC, 20°C, 4°C and acid pH) was also tested. Among the 120 analyzed fish, V. parahaemolyticus were isolated from four (3.33%) fishes, and Mugil platanus was the only species in which the microorganism was found. Among the 19 strains analyzed, 89.5% were able to form biofilm, which seems to indicate that this ability has an important role in the microorganism survival in the fish. Among these strains, 25% increased the ability to form biofilm after sublethal exposure. Based on the results, we concluded that fish of the species M. platanus of the Lagoa dos Patos Estuary are hosts of V. parahaemolyticus and that almost all of these strains are forming biofilm. Exposure to sublethal stress conditions has distinct effect on different strains, inducing an increase in the ability to form biofilm in some. This was the first study about the effects of stress on the V. parahaemolyticus biofilms formation.(AU)

Fatores de patogenicidade de Vibrio spp. de importância em doenças transmitidas por alimentos / Vibrio spp. pathogenicity factors of importance in foodborne diseases

As bactérias do gênero Vibrio habitam ambiente tipicamente marinho e estuarino, sendo comumente isoladas de pescados. As principais espécies de Vibrio reportadas como agentes de infecções em humanos são V. vulnificus , V. parahaemolyticus , V. cholerae e V. mimicus . V. vulnificus é considerado o mais perigoso, podendo causar septicemia e levar à morte. V. parahaemolyticus é um patógeno importante nas regiões costeiras de clima temperado e tropical em todo o mundo e tem sido responsável por casos de gastroenterites associadas ao consumo de peixes, moluscos e crustáceos marinhos. V. cholerae causa surtos, epidemias e pandemias relacionados com ambientes estuarinos. V. mimicus pode causar episódios esporádicos de gastroenterite aguda e infecções de ouvido. A patogenicidade das bactérias está ligada à habilidade do micro-organismo em iniciar uma doença (incluindo entrada, colonização e multiplicação no corpo humano). Para que isso ocorra, os micro-organismos fazem uso de diversos fatores. O objetivo desta revisão foi sintetizar o conhecimento disponível na literatura sobre os fatores de patogenicidade de V. vulnificus , V. parahaemolyticus , V. cholerae e V. mimicus.(AU)

Bacteria of the genus Vibrio typically habitat marine and estuarine environment and are commonly isolated from fish. The main Vibrio species reported as agents of infections in humans are V. vulnificus , V. parahaemolyticus , V. cholerae and V. mimicus . V. vulnificus is considered the most dangerous, may cause sepsis and lead to death. V. parahaemolyticus is an important pathogen in coastal regions of temperate and tropical climates around the world and has been responsible for cases of gastroenteritis associated with consumption of fish, shellfish and marine crustaceans. V. cholerae causes outbreaks, epidemics and pandemics related to estuarine environments. V. mimicus can cause sporadic episodes of acute gastroenteritis and infections. The pathogenicity of the bacteria is linked to the ability of the micro-organism to initiate a disease (including entry, colonization and multiplication in the human body). For this to occur, the micro-organisms make use of several factors. The objective of this review is to summarize the knowledge available in the literature on the factors of pathogenicity of V. vulnificus , V. parahaemolyticus , V. cholerae and V. mimicus.(AU)

The expression of heterologous MAM-7 in Lactobacillus rhamnosus reduces its intrinsic capacity to inhibit colonization of pathogen Vibrio parahaemolyticus in vitro

BACKGROUND: Vibrio parahaemolyticus (V. parahaemolyticus) is a Gram-negative, halophilic bacterium recognized as one of the most important foodborne pathogen. When ingested, V. parahaemolyticus causes a self-limiting illness (Vibriosis), characterized mainly by watery diarrhoea. Treatment is usually oral rehydration and/or antibiotics in complicated cases. Since 1996, the pathogenic and pandemic V. parahaemolyticus O3:K6 serotype has spread worldwide, increasing the reported number of vibriosis cases. Thus, the design of new strategies for pathogen control and illness prevention is necessary. Lactobacillus sp. grouped Gram positive innocuous bacteria, part of normal intestinal microbiota and usually used as oral vaccines for several diarrheic diseases. Recombinants strains of Lactobacillus (RL) expressing pathogen antigens can be used as part of an anti-adhesion strategy where RL block the pathogen union sites in host cells. Thus, we aimed to express MAM-7 V. parahaemolyticus adhesion protein in Lactobacillus sp. to generate an RL that prevents pathogen colonization RESULTS: We cloned the MAM-7 gene from V. parahaemolyticus RIMD 2210633 in Lactobacillus expression vectors. Recombinant strains (Lactobacillus rhamnosus pSEC-MAM7 and L. rhamnosus pCWA-MAM7) adhered to CaCo-2 cells and competed with the pathogen. However, the L. rhamnosus wild type strain showed the best capacity to inhibit pathogen colonization in vitro. In addition, LDH-assay showed that recombinant strains were cytotoxic compared with the wild type isogenic strain CONCLUSIONS: MAM-7 expression in lactobacilli reduces the intrinsic inhibitory capacity of L. rhamnosus against V. parahaemolyticus.

Amplification of tlh gene in other Vibrionaceae specie by specie-specific multiplex PCR of Vibrio parahaemolyticus

Background The surveillance of Vibrio parahaemolyticus in the Chilean coast has been mainly performed by multiplex PCR amplification of three different hemolysin genes, which are specie-specific virulence factors. These genes are also employed in the determination of V. parahaemolyticus pathogenic load in seafood and for characterization of pathogenic strains associated to diarrhea cases in human. During environmental surveillance that we performed every summer, we occasionally observed a thermolabile hemolysin (tlh) PCR product of a slightly smaller size than expected, which was coincident with low loads of V. parahaemolyticus in the environment. In order to understand this observation, we probed the specificity of tlh primers for the detection of V. parahaemolyticus at different bacterial loads and DNA concentrations. Results Primers used for the detection of V. parahaemolyticus specific tlh amplified a slightly smaller tlh gene, which is found in Vibrio alginolyticus and other related strains. These amplicons were observed when V. parahaemolyticus was absent or in undetectable loads in the environment. Conclusions Surveillance of V. parahaemolyticus using tlh primers can be imprecise because amplification of a V. parahaemolyticus specific marker in V. alginolyticus and other related strains occurs. This situation complicates potentially the estimation of bacterial load in seafood, because do not ensure the correct identification of V. parahaemolyticus when his load is low. Additionally, it could complicate the tracking of outbreaks of V. parahaemolyticus infections, considering the genetic markers used would not be specie-specific.

Vibrio parahaemolyticus isolados de pescados do estuário da Lagoa dos Patos / Vibrio parahaemolyticus isolated from fishes captured from the Lagoa dos Patos estuary

O estuário da Lagoa dos Patos é um importante polo pesqueiro artesanal por constituir área de criação de diversas espécies de peixes e crustáceos. Suas águas são influenciadas diretamente por fatores meteorológicos, os quais interferem na entrada de água oceânica no estuário e na vazão, refletindo na sua salinidade. Por esta razão, há a possibilidade de V. parahaemolyticus ser encontrado no estuário da lagoa e, consequentemente, nos pescados nele capturados, o que ofereceria risco aos consumidores. O objetivo do estudo foi de avaliar a presença de V. parahaemolyticus em pescados do estuário da Lagoa dos Patos, capturados e processados artesanalmente. Cinquenta e seis amostras de pescados oriundos do estuário da Lagoa dos Patos foram analisadas quanto à presença de V. parahaemolyticus. Três das cinco espécies analisadas, camarão-rosa (Farfantepenaeus paulensis), tainha (Mugil platanus) e linguado (Paralichthys orbignyanus) albergavam o micro-organismo pesquisado. Estes resultados servem de alerta para a necessidade de maior fiscalização na produção de pescados, bem como da adoção de medidas higiênico-sanitárias que previnam o risco de transmissão deste patógeno para os consumidores. Este é o primeiro registro de isolamento deV. parahaemolyticus de F. paulensis, M. platanus e P. orbignyanus.

Lagoa dos Patos estuary is an important artisanal fishing pole because it represents an area for breeding several species of fish and shellfish. Water is directly influenced by meteorological factors, which interfere in the ocean water into the estuary and flow, reflecting on the salinity. For this reason, there is the possibility of V. parahaemolyticus be found in the lagoon estuary; consequently the fish caught from these place could offer risks to the health of consumers. The objective of this study was to evaluate the presence of V. parahaemolyticus in the Lagoa dos Patos estuary, and in the fishes captured from small-scale fishing and processing. Fifty-six samples of fish caught from the Lagoa dos Patos estuary were analyzed for the presence of V. parahaemolyticus. Three of five species analyzed, shrimp (Farfantepenaeus paulensis), mullet (Mugil platanus) and sole (Paralichthys orbignyanus) harbored this microorganism. These results serve as a warning for providing the best technical oversight to the production of fish, and also the adoption of hygienic and sanitary measures to prevent the risk of transmission of this pathogen to consumers. This is the first report on the isolation of V. parahaemolyticus from F. paulensis, M. platanus and P.orbignyanus.

Crecimiento y sobrevivencia de Vibrio parahaemolyticus en ostión americano (Crassostrea virginica) almacenado en refrigeración / Growth and survival of total and pathogenic Vibrio parahaemolyticus in American oyster (Crassostrea virginica) under cold storage

Objetivo. Cuantificar las densidades de Vibrio parahaemolyticus en ostión americano (Crassostrea virginica) almacenado en refrigeración. Material y métodos. Se almacenaron 320 ostiones a 7 °C durante nueve días y se determinaron las densidades totales y patogénicas mediante la técnica NMP-PCR. Resultados. Se observaron densidades de V. parahaemolyticus tlh+ en los días 0,3 y 6 de almacenamiento con 1. 134,2.764 y 0.785 log10NMP/g, respectivamente, y en los días 0 y 3 la densidad patogénica trh+ con 0.477 y 0.519 log10NMP/g, respectivamente; las densidades patogénicas tdh+ (0.519 log10NMP/g), tdh+/trh+ (0.519 log10 NMP/g) y tdh+/orf8+ (-0.444 log10NMP/g) se detectaron al tercer día de almacenamiento. Conclusión. Los resultados sugieren que el crecimiento de V. parahaemolyticus y la ocurrencia de genes patogénicos a 7 °C involucran cambios en la expresión génica como una respuesta al estrés por frío. Esto contribuye a la sobrevivencia y virulencia de V. parahaemolyticus, lo cual representa un riesgo a la salud pública.

Objective. To quantify Vibrio parahaemolyticus densities in American oyster (Crassostrea virginica) under cold storage. Materials and methods. 320 oysters were stored at 7°C for nine days and total and pathogenic densities were determined by the NMP-PCR methodology. Results. V. parahaemolyticus tlh+ densities were observed on 0,3, and 6 days of storage at 1.134, 2.764 and 0.785 log10NMP/g, respectively, and pathogenic density trh+ on 0 and 3 days at 0.477 and 0.519 log10NMP/g, respectively; the pathogenic densities tdh+ (0.519 log10NMP/g), tdh+/trh+ (0.519 log10NMP/g), and tdh+orf8+ (-0.444 log10NMP/g) were detected on day 3 of storage. Conclusion.The results suggest that V. parahaemolyticus growth and pathogenic genes occurrence at 7°C involve changes in the genetic expression as a cold shock response, favoring V. parahaemolyticus survival and virulence, representing a health risk.

RAW TROPICAL OYSTERS AS VEHICLES FOR MULTIDRUG-RESISTANT Vibrio parahaemolyticus / Ostras tropicais cruas como fonte de Vibrio parahaemolyticus multirresistentes

The following study aimed to determine the antimicrobial susceptibility profile of Vibrio parahaemolyticus strains from fresh and frozen oysters Crassostrea rhizophorae sold in Fortaleza-Brazil. An antibiogram was performed on 87 isolates using nine antibiotics: gentamicin (Gen 10 µg), ampicillin (Amp 10 µg), penicillin G (Pen 10U), ciprofloxacin (Cip 5 µg), chloramphenicol (Chl 30 µg), nalidixic acid (Nal 30 µg), tetracycline (Tet 30 µg), vancomycin (Van 30 µg) and erythromycin (Ery 15 µg). All strains were resistant to at least one antibiotic, and 85 (97.7%) were multi-resistant, with predominance of the Van+ Pen+Amp resistance profile (n = 46). Plasmid resistance to Pen, Amp and Ery was detected. Thus, the risk that raw oyster consumption poses to the health of consumers is highlighted, due to the fact that these bivalves may host antibacterial-resistant microorganisms.

O presente estudo objetivou determinar o perfil de suscetibilidade a antimicrobianos de cepas de Vibrio parahaemolyticus oriundas de ostras “in natura” e congeladas comercializadas em Fortaleza-Brasil. Oitenta e sete (87) cepas foram submetidas ao antibiograma com emprego de nove antibióticos: gentamicina (Gen 10 µg), ampicilina (Amp 10 µg), penicilina G (Pen 10U), ciprofloxacin (Cip 5 µg), cloranfenicol (Clo 30 µg), ácido nalidíxico (Nal 30 µg), tetraciclina (Tet 30 µg), vancomicina (Van 30 µg) e eritromicina (Eri 15 µg). Todas as cepas mostram-se resistentes a pelo menos um antibiótico, e 85 (97,7%) apresentaram multirresistência, com predomínio do perfil Van+ Pen+Amp (n = 46). Foi detectada resistência plasmidial a Pen, Amp e Eri. Dessa forma, o risco que o consumo de ostras cruas representa para a saúde dos consumidores merece ser destacado, uma vez que esses bivalves podem ser veículos de transmissão de micro organismos multirresistentes a fármacos antibacterianos.