Vibrio parahaemolyticus is a Gram-negative, halophilic pathogen that occurs naturally in coastal and estuarine environments. This human pathogen is frequently isolated from a variety of seafood, particular oysters, and is the leading cause of gastroenteritis associated with seafood consumption. Several outbreaks of V. parahaemolyticus infections linked to consumption of raw oysters have been documented. Contamination of oysters with V. parahaemolyticus is a concern for public health. This study investigated the efficacy of high pressure processing (HPP) in inactivating V. parahaemolyticus in raw Pacific oysters (Crassostrea gigas) and identified a process condition capable of achieving greater than 3.52-log reductions of V. parahaemolyticus in raw oysters for commercial application. Raw Pacific oysters were inoculated with a clinical strain of V. parahaemolyticus 10293 (O1:K56) to levels of 10⁴⁻⁵ cells per gram and processed at 293 MPa (43K PSI) for 90, 120, 150, 180 and 210 s. Populations of V. parahaemolyticus in oysters after processes were analyzed with the 5-tube most probable number (MPN) method. A minimum HPP of 293 MPa for 120 s at groundwater temperature (8±1 °C) was identified capable of achieving greater than 3.52-log reductions of V. parahaemolyticus in Pacific oysters. The HPP (293 MPa for 120 s at 8±1 °C) was validated at a commercial scale according to the FDA's National Shellfish Sanitation Program Post Harvest Processing (PHP) Validation/Verification Interim Guidance for Vibrio vulnificus and Vibrio parahaemolyticus. Negative results obtained by the MPN method were confirmed with a multiplex PCR detecting genes encoding thermolabile hemolysin (tl), thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). Oysters processed at 293 MPa for 120 sec had a shelf life of 6-8 days when stored at 5 °C or 16-18 days when stored in ice. This validated HPP was accepted by the FDA as a post harvest process to eliminate V. parahaemolyticus in raw oysters. / Graduation date: 2012

MENEZES, Francisca Gleire Rodrigues de. Caracterização fenotípica e genotípica de bactérias do gênero Vibrio isoladas em alguns estuários do Estado do Ceará. 2011. 94 f. : Tese (doutorado) - Universidade Federal do Ceará, Centro de Ciências Agrárias, Departamento de Engenharia de Pesca, Fortaleza-CE, 2011 / Submitted by Nádja Goes (nmoraissoares@gmail.com) on 2016-07-26T13:27:46Z No. of bitstreams: 1 2011_tese_fgrmenezes.pdf: 2453594 bytes, checksum: 0370cb8f6e2c333d91e3ccc575df6f2b (MD5) / Approved for entry into archive by Nádja Goes (nmoraissoares@gmail.com) on 2016-07-26T13:28:01Z (GMT) No. of bitstreams: 1 2011_tese_fgrmenezes.pdf: 2453594 bytes, checksum: 0370cb8f6e2c333d91e3ccc575df6f2b (MD5) / Made available in DSpace on 2016-07-26T13:28:01Z (GMT). No. of bitstreams: 1 2011_tese_fgrmenezes.pdf: 2453594 bytes, checksum: 0370cb8f6e2c333d91e3ccc575df6f2b (MD5) Previous issue date: 2011 / The frequent association of environmental aquatic contamination with vibriosis in humans suggests the need for systematic monitoring and study of environmental vibrio strains and their pathogenic potential and clinical significance. The objective of this study was to evaluate the diversity of vibrio species in four estuaries (Pacoti, Choró, Pirangi and Jaguaribe) in Ceará, Northeastern Brazil. Nineteen vibrio species were identified in 32 water samples and 32 sediment samples collected between January and April 2009. Overall, V. parahaemolyticus and V. alginolyticus were the most abundant (the former in Choró, the latter in Pacoti). The isolated strains were submitted to antibiogram testing with 15 antibiotics. All strains (n=197) were susceptible to sulfametoxazol-trimetoprim, ciprofloxacin, nalidixic acid and chloramphenicol. Resistance was observed to penicillin G (n=163; 82%), ampicillin (n=108; 54%), cephalothin (n=15; 7%), aztreonam (n=3; 1%), gentamicin, cefotaxime, ceftriaxone (1 each; 0.5%). Partial resistance was observed to cefalotin (n=52; 25%), ampicillin (n=28; 14%), aztreonam (n=10; 5%), tetracycline (n=8; 4%), oxytetracycline (n=2; 1%), and florfenicol, cefotaxime, ceftriaxone, streptomycin and gentamicin (1 each; 0.5%). Five species known to be pathogenic to humans were chosen for analysis of factors of pathogenicity. Strains belonging to the species V. parahaemolyticus (n=64) and V. cholerae (n=9) were submitted to molecular analysis using genes to confirm the species and indicate virulence. Sixty-three strains of V. parahaemolyticus were positive for species-specific tl, 57 were positive for tdh and 20 for trh. Five strains of V. cholerae were positive for species-specific ompW, but no strains presented the genes ctxA, zot, tcp or rfbO1. In conclusion, the estuaries surveyed presented a great diversity of vibrio species, the most abundant of which were V. parahaemolyticus and V. alginolyticus. Resistance to penicillin and ampicillin was elevated and positivity for virulence factors was considerable among strains of species pathogenic to humans. V. parahaemolyticus strains presented virulence genes indicating risk to public health. V. cholerae was identified in samples of both water and sediment / Muitas pesquisas têm associado contaminação aquática ambiental com infecções de Vibrio em humanos, sugerindo que a importância do monitoramento sistemático das cepas ambientais se faz necessário para definir seu possível potencial patogênico e sua significância clínica. O objetivo dessa pesquisa foi estudar a diversidade do gênero Vibrio isolado de quatro regiões estuarinas no Estado do Ceará, (Pacoti, Choró, Pirangi e Jaguaribe). As coletas realizadas resultaram num total de 32 amostras de água e 32 de sedimento, durante os meses de janeiro a abril de 2009. Foram catalogadas 19 espécies de bactérias pertencentes ao gênero Vibrio, das quais Vibrio parahaemolyticus e Vibrio alginolyticus foram as mais abundantes nos quatro estuários: V. parahaemolyticus no Rio Choró e V. alginolyticus no Rio Pacoti. As cepas identificadas foram submetidas a testes de susceptibilidade a quinze antimicrobianos. Todas as cepas analisadas (197) apresentaram susceptibilidade a sulfazotrim, ciprofloxacin, ácido nalidíxico e cloranfenicol, sendo que cento e sessenta e três (82%) apresentaram resistência a penicilina G, cento e oito (54%) a ampicilina, quinze (7%) a cefalotina, três (1%) a aztreonam, uma (0,5%) a gentamicina, a cefotaxima e a ceftriaxona. Cinquenta e uma cepas (25%) apresentaram comportamento intermediário frente à cefalotina, vinte e oito cepas (14%) a ampicilina, dez (5%) a aztreonam, oito (4%) a tetracilina, duas (1%) a oxitetraciclina e uma (0,5%) a florfenicol, a cefotaxima, a ceftriaxona, a estreptomicina e a gentamicina. Foram escolhidas cinco espécies patógenas ao homem para verificação de seus fatores de patogenicidade. As cepas identificadas como V. parahaemolyticus (64) e V. cholerae (9) foram analisadas através de técnicas de biologia molecular, usando genes que confirmam as espécies e genes que indicam virulência. Das 64 amostras de V. parahaemolyticus analisadas, 63 foram positivas para o gene tl, específico para espécie, 57 para o gene tdh e 20 para o trh, genes que indicam patogenicidade. Das nove cepas de V. cholerae, cinco foram positivas para o gene ompW, gene específico para espécie, porém, nenhuma amostra apresentou os genes de virulência ctxA, zot, tcp e rfbO1. Com isso conclui-se que os estuários dos rios analisados apresentam uma elevada abundância de espécies, tendo V. parahaemolyticus e V. alginolyticus como as mais abundantes. O antibiograma das cepas isoladas mostrou uma elevada resistência à penicilina e a ampicilina. Foram encontradas elevada positividade para a presença dos fatores de virulência nas cepas pertencentes às espécies de Vibrio patógenas a humanos. As cepas de V. parahaemolyticus apresentaram genes de virulência indicando que as cepas podem acarretar danos à saúde pública. A presença do V. cholerae foi confirmada nas águas e sedimento dos estuários

Marine vibrio MV-1 is a magnetotactic bacterium capable of aligning its cell in response to the Earth’s magnetic field. This ability is due to the presence of chainlike structures comprising magnetosomes, magnetite particles enclosed in a lipid membrane with associated proteins. Strain MV-1 differs from other, bettercharacterized strains of magnetotactic bacteria as the cells produce higher amounts of biomagnetite per litre of culture and its magnetosomes are unique in shape. This study investigates the presence and organisation of a gene cluster termed a “magnetosome island” within the genome of MV-1. In other magnetotactic bacteria this genomic region has been shown to contain many of the genes associated with magnetosome formation but has not been previously investigated for MV-1. One of the conserved fragments of this region was amplified using degenerate primers followed by extension of the known sequence using inverse PCR based technique and constructing plasmid libraries. Sequencing of the genome of strain MV-1 was accomplished as a part of this study. Significant work was done on comparison of the sequence quality obtained from SOLEXA, 454 and Sanger sequencing technologies. A number of obtained contigs were joined manually and the resulting sequence was automatically annotated using RAST. The obtained genome sequence of 3.6 Mb with a G+C content of 54.3 % was preliminarily analysed and used to search for magnetosome related genes. This study also analysed proteins associated with the magnetosomes of strain MV-1 using MALDI-TOF, LC-MS and Orbitrap mass spectrometry. These approaches allowed the identification of a number of proteins in the isolated magnetosome membrane fraction. Some of these proteins have very low similarity with other characterized proteins (either in magnetotactic bacteria or in other organisms). Another significant point is that genes that code for proteins such as MamR, MamK and MmsF were found to be present in several homologous copies within the “magnetosome island” of MV-1. Interestingly, this study shows that all homologous copies of these proteins were identified in the magnetosome membrane fraction. Generation of knock-out mutants of several specific genes from the “magnetosome island” of strain MV-1 was attempted; constructs were made based on suicide plasmids carrying the cre-lox or I-SceI systems. Despite altering numerous experimental conditions it was not possible to obtain conclusive evidence of the isolation of MV-1 transconjugants containing the integrated constructs. In order to investigate the cell localization of the magnetosome associated protein CAV30779.1, an enhanced green fluorescent protein (EGFP) fusion based construct was generated and transferred into MV-1 cells. The EGFP fluorescent protein fusions within the cells were detected by microscopy. This study reveals novel information about magnetosome formation in marine vibrio MV-1. The obtained results provide an important foundation for further investigation of this organism and contribute towards broadening the knowledge of the complex process of magnetosome formation in bacteria.

Evalúa la actividad antagonista de una colección de 25 actinomicetos de origen marino. La actividad inhibitoria se determina mediante pruebas in vitro sobre cepas de Vibrio aislados de Litopenaeus vannamei (Langostino Blanco). / Tesis

El objetivo del presente trabajo fue hacer un recuento de Vibrio parahaemolyticus Kanagawa positivo a partir de 50 muestras de pescados, moluscos y crustáceos crudos procedentes de terminales pesqueros, muelles y supermercados de consumo en Lima Metropolitana y Callao entre noviembre de 1999 y abril de 2000. El análisis microbiológico se realizó de acuerdo a la metodología recomendada por el Manual Bacteriológico Analítico (BAM, 7 ed.). Se halló Vibrio parahaemolyticus Kanagawa positivo en 4 muestras aislándose 5 cepas con las características bioquímicas correspondientes de un total de 568 cepas sospechosas. Los valores hallados de Número Más Probable (NMP) fueron en pescados y crustáceos de 3/g cada uno y en moluscos un valor mínimo de 3/g y un máximo de 7,4/g. Las muestras que presentaron Vibrio parahaemolyticus Kanagawa positivo (n=4) representaron un 8% del total de especies marinas (n=50), siendo los porcentajes hallados en pescados 2% (n=1), moluscos 4% (n=2) y crustáceos 2% (n=1). -- Palabras Clave: Vibrio parahaemolyticus, pescados, crustáceos, moluscos, Kanagawa positivo, NMP, Lima Metropolitana y Callao. / -- The aim of this research was performing a count of Vibrio parahaemolyticus Kanagawa positive out of 50 samples including raw fish, mollusks and crustaceans collected from fishermen’s wharf, fisheries, and supermarkets of edible character in Metropolitan Lima and Callao between november 1999 and april 2000. The microbiological analysis was performed according to Bacteriological Analytical Manual (BAM, 7 ed.). Vibrio parahaemolyticus Kanagawa positive was found in 4 samples from 5 strains with biochemical features that met those of Vibrio parahaemolyticus out of a total of 568 analized strains. The Most Probable Number (NMP) values found are as follows: Fish and crustaceans 3/g each, while molluscs had a minimum value of 3/g and a maximum of 7,4/g. Vibrio parahaemolyticus Kanagawa positive samples (n=4) represented 8% out of the total marine samples (n=50), being the percentages found in fish 2% (n=1), mollusks 4% (n=2) and crustaceans 2% (n=1) out of the total number of samples as well. -- Key Words: Vibrio parahaemolyticus, fish, mollusks, crustaceans, Kanagawa positive, MPN, Metropolitan Lima and Callao.

La investigación tuvo como objetivo incorporar una metodología de identificación rápida conocida como ARDRA (Amplified Ribosomal DNA Restriction Analysis) para identificar especies del género Vibrio. Se estandarizó la técnica con cepas referenciales. Luego, se aislaron cepas bacterianas asociadas con el cultivo de Litopenaeus vannamei. Posteriormente, se realizaron pruebas bioquímicas para encontrar cepas candidatas de pertenecer al género Vibrio. Al finalizar esta primera etapa, la técnica ARDRA estandarizada, fue aplicada en las cepas candidatas, confirmando de esta manera la factibilidad de la metodología bajo las condiciones estudiadas. En una segunda etapa, se secuenció la región 16S rDNA para confirmar e identificar las cepas candidatas por análisis filogenético. Se reportaron tres especies diferentes con alta similitud pertenecientes al Vibrio core group (Vibrio communis, Vibrio harveyi y Vibrio parahaemolyticus). Con estos resultados, fue posible diseñar una identificación rápida por ARDRA para identificar el Vibrio core group y la especie Vibrio communis. La metodología de diseño del ARDRA fue soportado por una valoración diagnóstica bioinformática, obteniendo de esta evaluación, una sensibilidad y una especificidad de 97,1 y 76,9%, respectivamente para la identificación del Vibrio core group, mientras que para identificar la especie Vibrio communis, se obtuvo una sensibilidad y una especificidad de 100 y 97,4%, respectivamente. Finalmente, se ha demostrado que es posible identificar ciertas especies del genero Vibrio asociados con la acuicultura de Litopenaeus vannamei, por ARDRA y esta metodología de identificación, tiene la ventaja de ser mucho más rápido y económico en comparación con la identificación por análisis filogenético, teniendo a su vez la desventaja de ser dependiente del uso del secuenciamiento en un primer momento para el diseño del ARDRA. Palabras clave: Litopenaeus vannamei, ARDRA, Vibrio communis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio core group, evaluación diagnóstica bioinformática.

Vibrio bacteria are emerging pathogens responsible for 80,000 illnesses and 100 deaths in the United States each year. Infections are directly linked to the marine environment and are acquired by consuming contaminated seafood or exposing wounds during aquatic activities. Florida has the highest national incidence of vibriosis, with 20% of its cases reported from the Indian River Lagoon region, a popular recreation destination. This study utilized a combination of cultivation and molecular techniques to investigate the local distribution of V. vulnificus, V. parahaemolyticus and V. cholerae in this local waterway. The targeted species were found in an array of samples which may facilitate their transmission to humans. Overall, these bacteria were abundant in estuarine sediments (Vp: 2,439 CFU/g, Vv: 303 CFU/g, Vc: 176 CFU/g), on the sharp edges of oyster shells (Vp: 82 CFU/cm, Vv: 102 CFU/cm, Vc: 41 CFU/cm), and in the water column (Vp: 3.78 CFU/ml, Vv: 5.51 CFU/ml, Vc: 2.46 CFU/ml). Vibrio also pose a hazard to recreational anglers as they were recovered from fish (Vp: 61%, Vv: 55%, Vc: 30%), live bait shrimp (Vp: 80%, Vv: 37%, Vc: 0%) and hooks (Vp: 32%, Vv: 18%, Vc: 0%). Additionally, a molecular analysis of the V. vulnificus virulence revealed that the local population was dominated by disease-causing (vcgC) strains, which may explain why wound-related infections are common in this region. Vibrio occurrence varied both spatially and temporally due to their relationship with salinity and temperature. These bacteria exhibited a strong negative correlation with salinity, being particularly abundant near freshwater discharge locations. Due to Florida’s year-round warm climate, these species were found to be permanent members of the local microbial community. Seasonal peaks in abundance occurred between August and October, a period which corresponds with the warmest water temperatures as well as frequent rainfall. Predictive models were constructed based on these parameters to provide a better understanding of how, when and where Vibrio spp. may be encountered by humans. This information is important for both water management and healthcare initiatives, with an overall goal of improving local recreational safety. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

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Bibliography: leaves 160-205. / 205, [128] leaves, [27] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to characterise the region of the chromosome associated with the gene encoding the MFRHA (Mannose-Fucose Resistant Haemagglutinin) / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1994

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 135-141).