Aim Because of the major problems that certain Vibrio specie, especially Vibrio harveyi, can cause the aquaculture industries a rapid method to identify Vibrio isolates is required. Early diagnosis of a V. harveyi infection could facilitate disease surveillance, treatment and prevention in cultured marine animals. Therefore, the use of PCR to aid in the identification of Vibrio is increasing and a way of extracting DNA in a cheap, fast and easy way is also of an important requirement to facilitate rapid diagnosis. Methods This report comprises biochemical profiling and PCR methods in the characterisation of four isolates of V. harveyi and single isolates of V. tubiashii, V. alginolyticus, V. anguillarum, V. splendidus, V. tapetis and V. parahaemolyticus. Strains were examined for adherence to a Hep-2 cell line. Four different DNA extraction methods were evaluated and compared. The detection limits and the analytical limits of two PCR methods for Vibrio were determined. Results The overall findings were that the use of a greater range of biochemical substrates than are in the API 20E is necessary to identify Vibrio strains, and that none of the strains tested adhered to Hep-2 cells. All extraction methods successfully produced DNA with the kit method giving the purest samples. RNA was a contaminant of the other techniques but this could be overcome by treating extracts with RNase. The rapid microwave extraction method gave appropriate PCR amplicons when tested. Conclusion PCR determination of the VH-sequence in combination with VHA and a distinguishable colonial morphology may be a good choice for the identifying of Vibrio harveyi.

Memoria para optar al Título Profesional de Médico Veterinario / La bacteria Vibrio parahaemolyticus corresponde a una de las principales ETA de la temporada de verano a lo largo de todo Chile, principalmente debido al consumo de productos del mar crudos o mal cocidos. Para su detección o recuento existen dos técnicas referenciales: Una cualitativa perteneciente a ISO (International Organization for Standardization), que expresa su resultado como presencia o ausencia de V. parahaemolyticus y otra cuantitativa perteneciente a FDA-BAM (Food & Drugs Administration's Bacteriological Analytical Manual) que expresa su resultado como NMP/g. Ambos métodos fueron implementados y verificados en dos matrices hidrobiológicas, cada matriz fue compuesta por 10 muestras de salmón y 10 muestras de chorito. El trabajo se realizó en el Laboratorio de Inocuidad de los Alimentos del Departamento de Medicina Preventiva Animal de la Facultad de Ciencias Veterinarias y Pecuarias de la Universidad de Chile. En el método cualitativo ISO 21782 se logró reconocer la presencia de la bacteria en el 100% de las muestras contaminadas, y a su vez todos los controles negativos arrojaron como resultado la ausencia de V. parahaemolyticus en ambas matrices. Para cumplir con la verificación del método cuantitativo FDA-BAM se debió realizar la contaminación de las muestras con tres niveles distintos de inóculo (101, 102, 103). Se logró apreciar directa relación entre el nivel de inóculo contaminado y el resultado obtenido posteriormente en la Tabla NMP del mismo método. Los resultados de ambos métodos cumplieron con la verificación ISO 16140. Los resultados pueden concluir que el Laboratorio de Inocuidad de los Alimentos del Departamento de Medicina Preventiva Animal de la Facultad de Ciencias Veterinarias y Pecuarias de la Universidad de Chile, se encuentra capacitado para la implementación de normas de referencia internacional, con el fin de cooperar y ser una herramienta válida para la detección y recuento de este patógeno en las matrices estudiadas. / The bacteria Vibrio parahaemolyticus corresponds to one of the main ETA of the summer season throughout Chile, mainly due to the consumption of raw or undercooked seafood. In order to detect and count that bacteria; there are two reference techniques: A qualitative, that belongs to ISO (International Organization for Standardization), which expresses its result in two terms; either as presence or absence of V. parahaemolyticus. Secondly, the quantitative technique, belonging to FDA-BAM (Food & Drug Administration's Bacteriological Analytical Manual), that expresses its result as NMP/g. Both methods were implemented and verified on two seafood matrixes, each of them was composed of 10 salmon samples and 10 chorito samples as well. This process was put into effect at the Laboratory of Food Safety Department of Animal Preventive Medicine of the Faculty of Veterinary and Animal Sciences at the University of Chile. In the qualitative method ISO 21782, the presence of the bacteria was recognized in 100% of contaminated samples, at the same time every negative control showed, as a result, the absence of V. parahaemolyticus in both matrixes. In order to fulfill the requirements of verification of FDA-BAM quantitative method, the contamination of samples with three different levels of inoculums (101, 102, 103) had to be done. Thanks to this; it was possible to appreciate the direct relationship between the level of contaminated inoculums and the result obtained in the Table NMP of the same method. The results of both methods meet the requirements of the ISO 16140 verification. The results obtained conclude that the Laboratory of Food Safety Department of Animal Preventive Medicine of the Faculty of Veterinary and Animal Sciences at the University of Chile is trained for the implementation of international reference standards methods in order to cooperate and be a valid tool for the detection and count of V. parahaemolyticus.

Cocos nucifera belong to the family Aracaceae (palm Family). The English name is coconut and it is used extensively as medicinal remedies against infections such as urinary tract infections, gastro intestinal infections, skin and wound infections. The in vitro antibacterial (including anti-listerial and anti-vibrio) properties as well as the evaluation of the combination potentials of the plant extract with six front-line antibiotics were evaluated in this study using standard procedures. The in vitro anti-listerial properties of the crude aqueous and n-Hexane extract of the husk of Cocos nucifera were carried out against 37 Listeria isolates. Twenty-nine of the test organisms were susceptible to the aqueous extract while thirty were susceptible to the n-Hexane extract both at the screening concentration of 25 mg/ml. Minimum Inhibitory Concentration (MIC) values for all the susceptible bacteria ranged between 0.6 - 5.0 mg/ml. For the aqueous extract, average log reduction in viable cell count ranged between 0.32 Log10 and 4.8 Log10 CFU/ml after 8 hours interaction in 1 × MIC and 2 × MIC. For the n-Hexane extract, the log reduction ranged between 2.4 Log10 and 6.2 Log10 CFU/ml after 8 hours interaction in 1 × MIC and 2 × MIC. The time-kill characteristics of the two extracts suggest that at higher concentration (2 × MIC) and longer duration of interaction (8 hr), more bacteria were killed. In vitro anti-vibrio and antibacterial properties experiment revealed that of all the 45 vibrio and 25 bacteria strains that was tested, 37 were susceptible to the aqueous extract and 38 to the n-Hexane extract, while 17 were susceptible to the aqueous extract and 21 to the n-Hexane extract. Minimum Inhibitory Concentration (MIC) values for all the susceptible bacteria ranged between 0.3 - 5.0 mg/ml. viii The time kill studies revealed that for the aqueous extract, average log reduction in viable cell count in time kill assay ranged between 0.12 Log10 and 4.2 Log10 CFU/ml after 8 hr interaction at 1 × MIC and 2 × MIC. For the n-Hexane extract, the log reduction ranged between 0.56 Log10 and 6.4 Log10 CFU/ml after 8 hr interaction in 1 × MIC and 2 × MIC. In the test for the combination interactions, the checkerboard method revealed synergy of 67% and indifferent of 33%, while the time kill assay detected synergy in 72% and indifferent in 28% of the combinations tested. The synergy detected was not specific to any of the antibiotics or the Gram reaction of the bacteria, and no antagonism was detected. We conclude that the aqueous and n-Hexane extract of the husk of C. nucifera contains potential broad spectrum antibiotics resistance modulating compounds that could be relevant in the treatment of infections caused by these pathogens. In addition, the husk which is being discarded as agro waste will opens up a vista of opportunities for utilization for therapeutic purposes

Vibrio parahaemolyticus (Vp) é uma bactéria naturalmente presente em regiões estuarinas, sendo a principal causa de gastrenterite de origem bacteriana associada a pescados, principalmente ostras cruas. Nesta pesquisa, foi desenvolvida uma avaliação quantitativa de risco para avaliar a probabilidade de Vp causar doença após o consumo de ostra crua, produzida e comercializada no Estado de São Paulo. O estudo incluiu a identificação e caracterização do perigo, a avaliação da exposição e a caracterização do risco. Um modelo matemático foi desenvolvido. Este modelo leva em consideração o comportamento de Vp em ostras na cadeia produtiva, em cada estação do ano, além da relação entre a dose de Vp ingerida e a probabilidade de desenvolver a doença. A avaliação da exposição foi desenvolvida em três etapas: cultivo, pós-coleta e consumo. Na etapa de cultivo foram considerados os fatores que influenciam a prevalência e o número de Vp em ostras no momento da coleta. Na etapa pós-coleta, foram descritas as práticas da indústria e foram considerados os fatores associados ao processamento, transporte e manipulação. Já na etapa de consumo foram considerados os fatores como a quantidade de ostras consumidas por porção, o peso médio por ostra consumida e a população de Vp patogênico no momento do consumo. O resultado do modelo quantitativo da avaliação da exposição foi, então, integrado ao modelo dose-resposta, Beta-Poisson, para se obter uma estimativa do risco. Esta estimativa expressa o impacto da exposição humana a Vp, sobre a saúde pública, associada ao consumo de ostras. A simulação de Monte Carlo foi utilizada para avaliar o efeito da variabilidade e incerteza das variáveis do modelo sobre a estimativa do risco. O modelo prediz uma probabilidade de ocorrência de doença de 4,6x10-4, por porção de ostra, consumida ao longo do ano. As variáveis que possuem maior influência sobre o risco de ocorrência de doença são a população de Vp em ostras no cultivo, a temperatura de transporte das ostras até o varejo e a porcentagem de Vp patogênico em ostra, no momento do seu consumo. O modelo evidencia que uma das maneiras de reduzir o risco de ocorrência de doença seria intervir nas condições de transporte de ostras até o varejo por meio da sua refrigeração. Com o modelo é possível identificar fatores e simular cenários para avaliar o comportamento de V. parahaemolytícus como um perigo microbiológico, ao longo da cadeia produtiva de ostra até o momento do seu consumo. Também é possível avaliar o impacto de medidas de intervenção na cadeia produtiva. As suposições adotadas limitam a aplicabilidade do modelo. Portanto, é necessário que o modelo seja validado, particularmente com relação ao número de casos de doença causados por Vp, cujos dados de vigilância epidemiológica inexistem no Brasil. / Vibrio parahaemolyticus (Vp) is naturally present in estuarine regions and is the main cause of gastroenteritis associated with the consumption of bivalve molluscan shellfish, specially raw oysters. In this research, a quantitative risk assessment was developed to evaluate the probability of Vp causing disease after consumption of raw oyster, produced and commercialized in the state of Sao Paulo. The study included the identification and characterization of the hazard, exposure assessment and risk characterization. A mathematical model was developed. This model takes into account the behavior of Vp in oysters in the productive chain, for each season of the year, besides the relationship between the number of cells of Vp ingested and the probability of developing the disease. The exposure assessment was done in three steps: farming, after harvesting and consumption. At the farming step, the factors that influence the prevalence and the population of Vp at the time of harvesting were considered. At the after harvesting step, the factors associated with transportation, handling and processing were considered. At the consumption step, factors related to the amount of oysters and the average weight per oyster consumed and the density of pathogenic Vp at the time of consumption were considered. Then, the quantitative model of exposure assessment was integrated to the dose-response model, BetaPoisson, in order to obtain a risk estimate. This calculation expresses the impact of the human exposure to Vp associated with the consumption of oysters on public health. The Monte Carlo simulation was used to evaluate the effect of variability and uncertainty of variables of the model in the risk estimation. The model predicts a probability of occurrence of the disease of 4,6x10-4 per serving of oyster consumed during one year. The variables showing the greatest influence on the risk of occurrence of disease are the density of Vp in oyster in the farming step, the temperature during transportation of oysters to the retail market and the percentage of pathogenic Vp strains in oysters,\' at the moment of consumption. The model indicates that the use of refrigeration during transportation of oysters to retail could reduce the risk of disease. The model allows the identification of factors and the simulation of scenarios in order to evaluate the behavior of V. parahaemolyticus, as a microbiologícal hazard, in the productive chain of oyster to the consumption. It is also possible to evaluate the impact of intervention measures in the productive chain. The assumptions Iimit the application of the model. Therefore, it is necessary to validate the model, particularly in relation to the number of cases of dísease caused by V. parahaemolyticus of which the data on epidemiologic surveillance do not exist in Brazil.

Vibrio cholerae y Shigella dysenteriae, entre otras bacterias enteropatógenas, causan enfermedades gastrointestinales, la segunda causa de muerte en niños menores de cinco años en el mundo. Los sucesos de gastroenteritis que desencadenan dichos microorganismos son mediados por la acción molecular de toxinas secretadas de tipo AB, CTX y STX, respectivamente para V, cholerae y S, dysenteriae. Dichas toxinas causan desbalances iónicos y lisis celular, generando deshidratación, pérdida de electrolitos y nutrientes. El correcto funcionamiento de ambas toxinas requiere una relación estricta de cinco subunidades de la proteína B por cada subunidad A, sin embargo, los mecanismos moleculares que regulan la expresión y mantienen la relación 5:1 entre las subunidades son poco entendidos. El objetivo de este estudio es analizar la regulación post transcripcional de las toxinas CTX y STX mediante aproximaciones experimentales in vitro y usando reacciones libres de células, en un entorno altamente puro y controlado. Nuestros resultados in vitro indican que CTX se expresa de manera más eficiente que STX. En el caso de CTX, la subunidad B tuvo mayor expresión que A, con una relación cercana a 5:1 como visto en estudios previos realizados in vivo. Por el contrario, STX fue sintetizada con baja eficiencia independientemente de la presencia o ausencia de la región codificantes para STXA, desviándose notablemente de una relación activa de 5 B por cada subunidad A. La adición de ppGpp, molécula implicada en la respuesta al estrés en bacterias, resultó en una ligera disminución de la expresión de ambas toxinas. En conclusión, nuestros resultados indican que la relación de 5 a 1 entre B y A de las toxinas CTX de V. cholerae es regulada durante la síntesis de proteínas. Por el contrario, la ausencia de regulación a nivel transcripcional o traduccional de STX del presente estudio in vitro, fue también observada en estudios in vivo, indicando que en la célula existen otros factores que podrían modular la estequiometría de síntesis. Si bien ambas toxinas, CTX y STX, permanecen a la misma familia de exotoxinas, los resultados del presente estudio indican que la regulación de su expresión difiere ampliamente entre ellas. Los resultados aquí reportados brindan nuevas perspectivas para el desarrollo de moléculas inhibidoras, con especial énfasis en perturbar el ribosoma de V, cholerae, para un desbalance en la síntesis de la toxina CTX y así reducir la patogenicidad de la bacteria. / Vibrio cholerae and Shigella dysenteriae, among other enteropathogenic bacteria, cause gastrointestinal diseases, the second cause of death in children under five years of age in the world. The events of gastroenteritis that trigger these microorganisms are mediated by the molecular action of secreted toxins of type AB, CTX and STX, respectively for V. cholerae and S. dysenteriae. These toxins cause ionic imbalances and cell lysis, generating dehydration, loss of electrolytes and nutrients. The correct functioning of both toxins requires a strict relationship of five subunits of protein B for each subunit A, however, the molecular mechanisms that regulate expression and maintain the 5: 1 ratio between the subunits are poorly understood. The objective of this study is to analyze the post transcriptional regulation of CTX and STX toxins by in vitro experimental approaches and using cell-free reactions, in a highly pure and controlled environment. Our in vitro results indicate that CTX is expressed more efficiently than STX. In the case of CTX, subunit B had greater expression than A, with a ratio close to 5: 1 as seen in previous studies conducted in vivo. In contrast, STX was synthesized with low efficiency regardless of the presence or absence of the region coding for STXA, deviating markedly from an active ratio of 5 B for each subunit A. The addition of ppGpp, a molecule involved in the stress response in bacteria, resulted in a slight decrease in the expression of both toxins. In conclusion, our results indicate that the ratio of 5 to 1 between B and A of CTX toxins of V. cholerae is regulated during protein synthesis. On the contrary, the absence of transcriptional or transcriptional STX regulation of the present in vitro study was also observed in in vivo studies, indicating that the cell may harbor other factors that could modulate the synthesis stoichiometry. Although both toxins, CTX and STX, belong to the same family of exotoxins, the results of the present study indicate that the regulation of their expression differs widely. The results reported here provide new perspectives for the development of inhibitory molecules, with special emphasis on disturbing the ribosome of V. cholerae, to provoke an imbalance in the synthesis of CTX toxin and thus to reduce the pathogenicity of the bacteria. / Tesis

Please read the abstract in the section, 00front, of this document / Dissertation (MSc)--University of Pretoria, 2001. / Microbiology and Plant Pathology / MSc / unrestricted

Quorum sensing is a mechanism used by many proteobacteria to regulate expression of target genes in a population-dependent manner. The quorum sensing system of Vibrio fischeri activates the luminescence (lux) operon when the autoinducer signaling molecule (N-3-oxohexanoyl homoserine lactone) is recognized and bound by the activator protein LuxR. LuxR subsequently binds to the lux box centered at à 42.5 bp upstream of the transcription initiation site and activates transcription from the lux operon promoter, resulting in the emission of light at high cell densities. LuxR consists of 250 amino acids arranged into an N-terminal (regulatory) domain and a C-terminal (activation) domain, and is thought to function as an ambidextrous activator capable of making multiple contacts with the alpha and sigma subunits of RNA polymerase (RNAP). Published work describing the results of alanine scanning mutagenesis performed on the C-terminal domain of LuxR (residues 190-250) has identified residues (K198, W201 and I206) that appear to play a role in positive control of transcription initiation. Additional mutagenesis of residues 180-189 has been undertaken via a three-primer or four-primer PCR-based method in this study. Variants of LuxR were screened for their ability to activate luciferase production and to repress transcription from an artificial promoter, and production of full-length LuxR was measured, in an attempt to identify additional positive control variants. No additional positive control variants were found in this study. Work has also been undertaken to identify intergenic suppressors between positive control variants of LuxR and the RNAP alpha subunit, RpoA. Starting with a recombinant Escherichia coli strain encoding the lux operon and LuxR variant I206E, a random chemical mutagenesis was performed on a vector encoding RpoA. Following transformation of the mutated plasmids encoding RpoA, high throughput luminescence assays were used to identify isolates with phenotypes brighter than the control. Isolation of an intergenic suppressor will confirm the existence of protein-protein interactions between LuxR and RpoA within the transcription initiation complex. The ability of other LuxR family members to establish productive protein-protein interactions with RNAP necessary for transcription initiation was also examined. LuxR homologues EsaR of Pantoea stewarti ssp. stewartii, a repressor of known function, and ExpR of Erwinia carotovora subsp. carotovora were also analyzed for their ability to activate the lux operon, as well as to repress transcription from an artificial promoter containing the lux box. / Master of Science

Final publication is available at: http://journal.frontiersin.org/article/10.3389/fmicb.2015.00270/full / 付記する学位プログラム名：グローバル生存学大学院連携プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19619号 / 医博第4126号 / 新制||医||1015(附属図書館) / 32655 / 京都大学大学院医学研究科医学専攻 / (主査)教授 中川 一路, 教授 木原 正博, 教授 松林 公蔵 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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