Graduation date: 1972

Vibrio vulnificus (Vv) and V. parahaemolyticus (Vp) are gram negative, halophilic bacteria that occur naturally in estuarine waters of Galveston Bay. Both bacteria have the potential to cause infections in humans either via consumption or direct contact. Finfish are a potential vector for these bacteria. Previous work by Brinkmeyer determined that these bacteria are present on the benthic dwelling catfish, Ariopsis felis and Bagre marinus, using a conventional microbial method. The present work focused on using Quantitative Polymerase Chain Reaction (QPCR) and Terminal Restriction Fragment Length Polymorphism (T-RFLP) to not only determine presence of these bacteria, but also to quantify them and look at community structure. QPCR was able to detect bacteria presence in 34 percent, 31.6 percent, and 0 percent for V.vulnificus, V.parahaemolyticus. thermolabile hemolysin (tlh) and thermostable direct hemolysin (tdh) genes, respectively. Statistical analysis of the QPCR results found that there was no significant difference between the length of fish, location of catch or species of fish in relation to the abundance of bacteria. T-RFLP was able to detect the presence of bacteria in approximately 70 percent of the samples surveyed. Bands produced from T-RFLP were able to be grouped into five different ranges. The most frequently occurring band fell in the range of 213-219 base pairs, and the most common number of bands per sample was 1 band. This study found that both QPCR and T-RFLP were better assays than conventional microbial methods for detecting the presence of V. vulnificus and V. parahaemolyticus on catfish fins. QPCR proved to be the most rapid detection method. Based on this study, it was determined that these Vibrio spp. bacteria have some type of relationship with A. felis and B. marinus. This information may be useful to the medical community for determining when there is a greater risk of infection via catfish puncture wounds.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references. Also available online.

The halophilic bacterium Vibrio parahaemolyticus is widely distributed as a natural inhabitant of marine and estuarine environments. Some strains cause gastroenteritis in humans. Clinical isolates are thought to possess virulence factors that are absent from the majority of environmental isolates. Studies were undertaken to differentiate clinical (virulent) and environmental (mainly avirulent) forms of Vibrio parahaemolyticus. Initially, identification and confirmation of a total of 55 V. parahaemolyticus isolates (23 clinical and 32 environmental) was carried out by using selective media, biochemical and nutritional tests. Identity was confirmed by specific polymerase chain reaction (PCR) targeting the toxR gene. In an attempt to differentiate between virulent and avirulent forms V. parahaemolyticus, potential virulence factors (enzyme activities), presence of plasmids and analyses of whole cell protein profiles and extracellular products (ECPs) by SDS-PAGE were performed. The results suggested that the presence of plasmids in isolates was not linked to virulence and SDS-PAGE profiles did not differentiate between virulent and avirulent forms, but a combination of enzyme activities may contribute to virulence. The ECPs of all 55 V. parahaemolyticus isolates were tested for their cytotoxicity towards two types of cell lines, the clinical isolates showed that 21 out of 23 (91%) and 2 out of 23 (8.69%) showed high and medium cytotoxicity, respectively. Amongst the environmental isolates 2 out of 32 (6.25%), 2 out of 32 (6.25%) and 28 out of 32 (87.5%) showed high, medium and low cytotoxicity, respectively. Randomly amplified polymorphic DNA (RAPD)-PCR was used to analyse the two groups of isolates. Firstly a 600 bp band was recognised in mainly clinical isolates. This DNA fragment was cloned and sequenced and found to code for an outer membrane protein (OMP). Two PCR primers were designed to specifically amplify a 200bp unique sequence from presumptive virulent strains (PCR-OMP); however, not all clinical isolates were positive (21 out of 23, 91%). A second RAPD-PCR identified a further unique band of approximately 310 bp in mostly clinical isolates. After cloning this band’s DNA, the DNA sequence revealed a hypothetical gene, htp, whose function is not known. Specific primers, VPHTP1 and VPHTP2 were developed for the detection of the htp sequence, but again not all clinical isolates were positive (19 out of 23, 82.6%). This led to the development of a multiplex M-PCR which detects all isolates of V. parahaemolyticus and differentiates them into potentially virulent and avirulent forms. The M-PCR works by targeting the toxR gene, and sequences for omp and htp. The M-PCR was performed on all V. parahaemolyticus isolates used in this study, as well as other Vibrio species and a selection of non-Vibrio species. The amplification of toxR gene 367 bp fragment was found in all V. parahaemolyticus tested; all clinical isolates (100%) showed amplification of omp and/or htp. This multiplex PCR detected 3 (9.37%) environment isolates, which may potentially be able to cause disease. No amplification was seen for the other species tested. Thus, the M-PCR could be used for identifying V. parahaemolyticus and detecting / differentiating potentially virulent and avirulent forms. This method should be

Seventeen strains of multi drug-resistant strains were isolated from aquaculture farms in previous study, and 82% (14/17) of these strains are belong to Vibrio species, which is one of the major pathogens in the aquaculture farms. Vibrio disease (vibriosis) which caused by the Vibrio will cause the fish skin ulcer with bleeding and gastrointestinal symptoms such as swelling. The highest mortality rate of Vibrio infected marine fish is up to 100%. Two vibriophages, Vibrio parahaemolyticus vibriophage £pVP2 and Vibrio alginolyticus vibriophage £pVA2, were isolated from aquaculture environments in this study. The morphology of vibriophage was examined by electron microscopy. The £pVP2 phage has an oval head with a tail and six tail fibers. As £pVA2, an icosarhedral head with an short tail is observed. In the DNA electrophoresis analysis, a fragment at 17.8k bp was observed in both £pVA2 and £pVP2 sample. Restriction endonuclease digestion pattern revealed that the £pVA2 vibriophage DNA can be cut into various small size of DNA fragments by EcoR I and Xba I endonuclease. In the host-range analysis, £pVP2 can infect Vibrio parahaemolyticus and Vibrio alginolyticus, while £pVA2 and £pVP2 cannot infect other Vibrio spp., Pasteurella, E. Coli, and antibiotic-resistant bacteria isolated from aquaculture farms. £pVA2 and £pVP2 can survive at the temperature below 65¢XC and 63¢XC, respectively. To obtain the maximum proliferation of vibriophage, different conditions were tested, including infection time point, host bacteria age, MOI (multiplicity of infection) and various mediums. Vibriophage £pVA2 was proliferated under following conditions: (1) phage was add to the medium with host seed together; (2) the overnight cultured host bacteria was used as seed; (3) the host cells were infected at MOI= 10-6; (4) MSWYE media was used as culture media. Vibriophage £pVP2 was proliferated following £pVA2 conditions but MOI values was 10-4.

Objetivos. Determinar las características epidemiológicas y clínicas de la gastroenteritis causada por Vibrio parahaemolyticus del grupo pandémico en el Perú. Materiales y métodos. Se examinó las historias clínicas y registros de laboratorio de cien casos de gastroenteritis en los cuales se aisló V. parahaemolyticus del grupo pandémico y no pandémico. Se recolectó información epidemiológica y clínica y se realizó el análisis estadístico de los datos para evaluar si la gravedad de la enfermedad se asoció con la presencia de las cepas del grupo pandémico. Resultados. Se logró colectar información epidemiológica en 85% de los casos e información clínica sólo en 37% de los casos, principalmente de los hospitalizados. Los casos del grupo pandémico tuvieron una mayor probabilidad de tener deposiciones líquidas (96,3% frente a 62,5%, p<0,05), presentar deshidratación moderada o grave (100% frente a 60%, p<0,05) y requerir atención hospitalaria (98% frente a 42,9%, p<0,0001). Fue más probable aislar una cepa pandémica en personas de 30 o más años de edad (63% frente a 39,5%, p<0,05). Conclusiones. El Vibrio parahaemolyticus del grupo pandémico causa enfermedad gastrointestinal de mayor gravedad que las cepas no pandémicas, con mayor probabilidad de requerir atención hospitalaria. Basados en este reporte, se recomienda incluir la identificación de V. parahaemolyticus en el diagnóstico etiológico de agentes causantes de gastroenteritis grave en el sistema de salud del Perú. / Objective. To determine the epidemiological and clinic characteristics of gastroenteritis caused by Vibrio parahaemolyticus strains of the pandemic group in Peru. Material and methods. Clinical and laboratory records were searched in 100 cases of gastroenteritis caused by V parahaemolyticus, either of the pandemic or non pandemic group. Clinical and epidemiological data were collected and statistical analysis was done to evaluate if the severity of illness was associated with the pandemic group. Results. Epidemiological data were collected in 85% of cases, and clinical data were only available in 37% of cases, mainly on those hospitalized. Cases associated with the pandemic strains had a higher probability of liquid stools (96.3% vs. 62.5%, p<0.05), moderate or severe dehydration (100% vs. 60%, p<0.05), and hospital care (98% vs. 42.9%, p<0.0001). Cases aged thirty or older were associated with the pandemic strains (63% vs. 39.5%, p<0.05). Conclusions. Vibrio parahaemolyticus of the pandemic group causes more severe gastrointestinal disease than none pandemic strains, with higher probability of requiring hospital care. Based on this report, it is advisable to include the identification of V. parahaemolyticus in the etiological diagnosis of agents causing severe gastroenteritis in the Peruvian health system.

Vibrio parahaemolyticus is considered to be an emerging, yet understudied, human pathogen. The V. parahaemolyticus BB22OP genome was sequenced to allow for a comparative analysis between the genome of BB22OP and another previously sequenced, pathogenic strain of V. parahaemolyticus, RIMD2210633. V. parahaemolyticus BB22OP is interesting because it exhibits a spontaneous phenotypic switch in colony morphology due to the loss of a functional OpaR; this also influences virulence. OpaR is the major quorum-sensing regulator in V. parahaemolyticus homologous to LuxR from V. harveyi. When opaR is removed from the RIMD2210633 genome, the same phenotypic switch is not seen indicating a difference between the quorum-sensing systems in these two strains. Understanding the regulatory variation in these two strains has the potential to provide key insights into the control of pathogenesis in this organism. Initially, the BB22OP genome sequencing results aligned into 125 contigs. The genome has now been assembled into two distinct chromosomes with only two gaps remaining to be filled. These gaps are located in the integron region, which is difficult to assemble due to its structure. The integron is a series of gene cassettes separated by inverted repeats that facilitate recombination events that build the integron. The integron region is further evidence of genetic differences between the two strains. The integron in the RIMD2210633 strain is comprised of 69 gene cassettes, while the BB22OP integron contains at least 86 gene cassettes. There are 313 genes novel to the BB22OP genome, which could result in the phenotypic differences seen in these two strains. Additionally five of the 313 genes are predicted to be transcriptional regulators indicating the potential for differential gene regulation. Further comparative analysis will likely reveal more phenotypic divergence between the physiology of RIMD2210633 and BB22OP. Additionally, the CsrA regulatory network was explored in RIMD2210633. CsrA was first characterized in E. coli as a global regulator of carbon storage and metabolism. RIMD2210633 contains a CsrA homolog and was predicted to contain four CsrA-regulating sRNAs (CsrB1-3 and CsrC), and this work confirmed that these sRNAs regulate CsrA in the same manner as in E. coli. CsrA and the same CsrA-regulating sRNAs were found in the BB22OP genome as well. Since CsrA is known to regulate glycogen production, a qualitative iodine-staining plate assay and a quantitative glycogen assay were used to indirectly measure CsrA activity in the presence and absence of individual regulatory sRNAs. The RIMD2210633 CsrA, CsrB1, CsrB2, CsrB3 and CsrC were shown to have the predicted physiological role in recombinant E. coli, with higher glycogen levels observed when CsrA was active and lower levels when each of the sRNAs was overexpressed. CsrA is also known to regulate biofilm production and virulence factors. In an attempt to develop a screening method for potential CsrA targets, a transcriptional/translational fusion system was developed. Transcriptional and translational fusions to β-galactosidase were created to PdksA, PglgC1 and PtoxR from RIMD2210633. CsrA or CsrB2 was overexpressed in recombinant E. coli containing each of the fusion constructs in order to see what happens to the gene expression from these promoters at low and high CsrA activity levels. Surprisingly, changing the activity levels of CsrA impacted both transcriptional and translational levels making the results of the assay difficult to interpret. Collectively these efforts have enhanced our understanding of V. parahaemolyticus. In particular, the sequencing of BB22OP has allowed for a comparative analysis between the BB22OP and RIMD2210633 strains. These strains have remarkably conserved genomes despite the phenotypic differences they exhibit. It appears there is variation in the quorum-sensing systems of these two strains. Further analysis will reveal how the quorum-sensing regulons differ and how this impacts the virulence of these two pathogenic V. parahaemolyticus strains. / Master of Science

Vibrio parahaemolyticus is a Gram-negative, halophilic, human pathogenic bacterium ubiquitous in the marine environment. Like many Vibrio species, V. parahaemolyticus commonly associates with shellfish, particularly oysters. Ingestion of a raw or under cooked oysters contaminated with V. parahaemolyticus can cause gastroenteritis, which is typically self-limiting and rarely causes death. Globally, oyster production is highly lucrative, especially on the West Coast of the United States where approximately 60% of oyster production occurs each year. Outbreaks of V. parahaemolyticus can result in a significant public health problem as well as an economic burden for the oyster farms implicated in the outbreak. With the increase in overall V. parahaemolyticus outbreaks, improved post-harvest processing strategies have been developed to reduce this natural contaminant. Depuration was developed to allow shellfish to purge contaminants from their tissues into the clean, flowing seawater where they are held. This post-harvest processing technique can typically reduce fecal contaminants from the oyster tissues but is relatively ineffective at eliminating V. parahaemolyticus and other Vibrio species.. Thus, improved methods for reducing this and other human pathogenic Vibrio are needed to effectively produce safer oysters for the consumer. To develop more effective and novel V. parahaemolyticus intervention strategies, first we must identify the factors that are involved in V. parahaemolyticus colonization of the oyster, allowing them toresist depuration. This study sought to investigate specific factors utilized by V. parahaemolyticus and, in the process, determined that various strains of V. parahaemolyticus have different alleles of the Type IV pili, mannose-sensitive hemagglutinin (MSHA)and chitin-regulated pilus (PilA). In addition, we expanded our investigations into the allelic diversity of MSHA and PilA from Vibrio cholerae and Vibrio vulnificus and found that V. cholerae strains that possess the Type IV toxin co-regulated pilus (TCP) maintained highly conserved MSHA and PilA sequences while strains of V. cholerae without TCP, and all of the V. vulnificus and V. parahaemolyticus strains examined, had highly divergent sequences with no discernable connection to isolation source or observed phenotype. Following that discovery, we determined that Type I, and Type IV pili, as well as polar and lateral flagellar systems contribute to V. parahaemolyticus persistence in the Pacific oyster during depuration, while Type III secretion systems and phase variation do not. Overall, we have identified factors involved in colonization of the Pacific oyster by V. parahaemolyticus. Future studies investigating conditions that affect pili and flagella production in V. parahaemolyticus may provide novel depuration conditions that could easily and effectively increase the efficiency of oyster depuration, ultimately reducing the risk of seafood-borne illness by V. parahaemolyticus associated with oysters. / Graduation date: 2013

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.

Com o objetivo de avaliar a presença de Vibrio parahaemolyticus em atum, foram coletadas 112 amostras, sendo 56 durante o inverno de 2003 (junho a julho) e 56, durante o verão de 2003-2004 (dezembro a janeiro), vendidos em diversos pontos comerciais da zona sul da cidade de São Paulo. Foi determinado o Número Mais Provável (NMP) de Vibrio parahaemolyticus, comparando a contaminação observada durante os dois períodos. As cepas foram estudadas quanto à produção de urease, ao fenômeno de Kanagawa e à sensibilidade a antibióticos. Apenas 2,68% das amostras foram positivas (3/112). Dessas, duas amostras foram coletadas no verão e uma, no inverno. A amostra positiva obtida no inverno apresentou 3 NMP/g, as outras duas, coletadas durante o verão, apresentaram respectivamente, 3 e 4 NMP/g. Todas as cepas isoladas de Vibrio parahaemolyticus foram negativas ao teste de Kanagawa e não produtoras de Urease, não apresentando nenhuma característica patogênica. Todas as cepas foram resistentes a ampicilina, eritromicina, estreptomicina penicilina G, polimixina B e vancomicina. Apresentaram susceptibilidade intermediária a ciprofloxacina, kanamicina e gentamicina, e sensibilidade a ácido nalixídico, cloranfenicol e tetraciclina. Conclui-se que, nas condições desse estudo, o sashimi de atum revelou-se um alimento de baixo risco ao consumidor, no que se refere a infecção (toxigênica) por Vibrio parahaemolyticus / In order to evaluate the presence of Vibrio parahaemolyticus in tuna traded in retail stores in the south region of the city of São Paulo, 112 samples were collected, 56 during the winter of 2003 (June and July) and 56, during the summer of 2003-2004 (December to January). Most probable number (MPN) of Vibrio parahaemolyticus was determined, comparing the contamination level observed in the two periods. Strains were analyzed in relation to urease production, Kanagawa phenomenon and sensitivity to antibiotics. Only 2.68% of the samples were positive (3/112), two of them collected in the summer, and one of them, in the winter. The positive sample obtained in the winter presented 3 NMP/g, and the other two samples, collected in the summer, presented 3 and 4 NMP/g, respectively. All Vibrio parahaemolyticus samples isolated were Kanagawa, urease negative. Therefore, they did not present any pathogenic characteristic. All strains were resistant to ampicillin, erythromycin, streptomycin, penicillin G, polymyxin B and vancomycin. They presented intermediate susceptibility to ciprofloxacin, kanamycin and gentamicin, and sensitivity to nalidixic acid, chloramphenicol and tetracycline. It may be concluded that, in the conditions of this study, tuna sashimi is considered low risk food in relation to toxigenic infection by Vibrio parahaemolyticus