Vibrio vulnificus is a human pathogen commonly found in coastal and estuarine waters in temperate and subtropical regions across the world. The ecology of V. vulnificus has been studied in these regions primarily using cultured-based methods for enumeration of V. vulnificus from the environment. Optimal temperature and salinity ranges have been established, but relationships with other environmental parameters have not been studied as extensively. The primary objective of this study was to better understand the ecology of V. vulnificus and how environmental parameters found in south Texas bays and estuaries regulate its distribution. A recently developed molecular biological technique for the direct enumeration of V. vulnificus from estuarine water column samples was used to test three hypotheses: 1) V. vulnificus makes up a greater percentage of the total bacterial population in the water column under low oxygen conditions; 2) Powderhorn Lake serves as a pointsource for V. vulnificus in Matagorda Bay; 3) Higher V. vulnificus concentrations are found in the water column when oyster reefs are present. These hypotheses were formed to improve predictive models, identify potential hot-spots for V. vulnificus in the water column, and to better inform stakeholders as to when and where risk of infection might be greatest. Dissolved oxygen was rarely low enough in the environment to stress aerobic bacteria in the water column, so the first hypothesis could not be appropriately tested. Neither higher concentrations nor detection frequencies of V. vulnificus were found in Powderhorn Lake compared to the rest of the bay, so Powderhorn Lake was not identified as a point-source for V. vulnificus. Higher concentrations and detection frequencies of V. vulnificus were not found at sites with oyster beds, so oyster beds cannot be used as indicators of higher concentrations of V. vulnificus in the water column. Interestingly, patchiness of V. vulnificus was observed temporally and spatially throughout the sampling region of Matagorda Bay, on a scale that has not been frequently examined. Variation occurred between samples in close proximity to one another, as well as between sampling dates. This distribution exhibited a small scale patchiness not frequently reported in past studies.

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.

The nuclease gene of Vibrio vulnificus, vvn, is 696 bp long encoding a protein¡]Vvn¡^of 232 amino acids. Vvn is a periplasmic protein and is active in the oxidized form. DNA shuffling is a powerful method for in vitro mutational mechanism by homologous recombination with a low level of point mutation . DNA shuffling consists of four steps¡G¡]1¡^preparation of genes to be shuffled, ¡]2¡^random fragmentation with DNase I, ¡]3¡^fragment reassembly by primerless polymerase chain reaction¡]PCR¡^, and¡]4¡^amplification of reassembled products by a conventional PCR. The advantage of this process is that it can be used to rapidly evolve any protein, without any knowledge of its structure. The goal of this work was using DNA shuffling to generate a diversity of mutation in vvn within a short time. Followed by analyzing the DNase activity of periplasmic protein or in vivo, the mutants were divided into three groups for increase, decrease or no change in DNase activity. Randomly DNA sequencing vvn gene of fourteen transformed clones from the three groups showed only one clone has one base change with comparison to wild-type sequence. The mutation is at amino acid 22 of the N-terminus of Vvn, the change is from serine to isoleucine. The relative activity of mutant Vvn was 82 % in DNase and 59 % in RNase. The effect of a single amino acid change on the DNase and RNase activity of Vvn is different. It supports the postulation that there are two distinct but overlapping active sites exist in Vvn.

Dissertation (Ph.D.)--Auburn University, 2007. / Abstract. Vita. Includes bibliographic references.

Thesis (Ph. D.)--University of New South Wales, 2000. / Also available online.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 122-143).

Vibrio vulnificus is a marine pathogen of human health concern, capable of causing potentially fatal wound infections in a select group of the population. Previous studies have indicated this species’ strong negative correlation with salinity, not typically found above 30 ppt. This study assessed the ability of V. vulnificus to become Viable But Nonculturable in response to elevated salinity (35 ppt) as well as investigated novel methods for confirming their entrance into this state. Results showed a complete loss of culturability in both Environmental and Clinical strains of this bacterium by 9 days after inoculation. Using a High Content Imager, it was determined that these pathogens were not dying (< 10%) in response to the treatment and were partially becoming cocci (≈35%). This study indicates the importance of understanding the impact environmental parameters have on this human pathogen, and what it means for reliably detecting them. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

Vibrio vulnificus is an opportunistic human and animal pathogen with the highest death rate of any foodborne disease agent. The capsular polysaccharide (CPS) is essential for virulence. Over 100 CPS types (carbotypes) have been identified among natural isolates, yet little is known about the genetic mechanisms that drive such diversity. Chitin, the second most abundant polysaccharide in nature, induces competence in Vibrio species. We found that transformation frequency varies by strain and (GlcNAc)2 was the shortest chitin-derived polymer capable of inducing competence. We confirmed that V. vulnificus can undergo chitin-dependent carbotype conversion following the uptake and recombination of complete cps loci from exogenous genomic DNA. The acquisition of a partial locus was also demonstrated when internal regions of homology between the endogenous and exogenous loci existed. Thus, the same mechanism governing the transfer of complete cps loci also contributes to their evolution by generating novel combinations of CPS biosynthesis genes.

Vibrio vulnificus is an opportunistic human and animal pathogen with the highest death rate of any foodborne disease agent. The capsular polysaccharide (CPS) is essential for virulence. Over 100 CPS types (carbotypes) have been identified among natural isolates, yet little is known about the genetic mechanisms that drive such diversity. Chitin, the second most abundant polysaccharide in nature, induces competence in Vibrio species. We found that transformation frequency varies by strain and (GlcNAc)2 was the shortest chitin-derived polymer capable of inducing competence. We confirmed that V. vulnificus can undergo chitin-dependent carbotype conversion following the uptake and recombination of complete cps loci from exogenous genomic DNA. The acquisition of a partial locus was also demonstrated when internal regions of homology between the endogenous and exogenous loci existed. Thus, the same mechanism governing the transfer of complete cps loci also contributes to their evolution by generating novel combinations of CPS biosynthesis genes.

The periplasmic nuclease of Vibrio vulnificus, Vvn, has been purified to homogeneity by a one step purification procedure using chromatography on a SP Sepharose column. The purified enzyme showed different mobilities on reducing and non-reducing SDS-PAGE, suggesting that disulfide bonds are involved in the maintenance of a stable tertiary conformation of the protein. Vvn randomly cleaved single and double stranded DNA and RNA, and possessed endonucleolytic activity. The enzyme exhibited an optimal activity between pH 8.0 and pH 10.0, and the optimal temperatures for the DNase and RNase activity were 40 oC ¡V 60 oC and 40 oC ¡V 50 oC, respectively. The enzymatic activity was inhibited by EDTA and EGTA, indicating that Vvn was a metalloenzyme. The DNase and RNase activity of Vvn had different requirements for divalent cations. Chemical modification studies on Vvn revealed the involvement of lysine, arginine, tryptophan and carboxylate residues in the catalytic activity of the enzyme. The extents of inactivation of the DNase and RNase activity of Vvn by modification of the carboxylate group with EDC were different. Substrate DNA and RNA protected the DNase and RNase activity of Vvn from inactivation by PLP, PGO, NBS and EDC which modified lysine, arginine, tryptophan and the carboxylate group. Mg2+ could not protect the DNase and RNase activity of Vvn against the inactivation by PLP and PGO. Whereas Mg2+ protection was observed in NBS- and EDC-mediated inactivation of the DNase but not the RNase activity of Vvn . From these results, it is postulate that there may be two distinct but overlapping active sites, for the DNase and RNase activity, respectively.