Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Science , Discipline of Microbiology and Immunology, 2005. / "May, 2004" Includes corrigenda. includes bibliographical references (leaves 118-156).

A probability matrix for computer-assisted identification of vibrios has been constructed, based on the API20E system. Data were gathered from 173 strains representing 31 taxa of vibrios and related organisms, from a variety of sources. The matrix was tested internally by four statistical programs. Program OVERMAT and program MOSTTYP tested the separation and homogeneity of the taxa. Most of the taxa were satisfactory but a few were less so; reasons for this are discussed. Program CHARSEP and program DIACHAR tested the separation and diagnostic values, respectively, of the 50 characters used and from the results a shorter diagnostic test set was drawn up. The overall test error rate was 4.5%. The matrix was assessed externally by its performance in the identification of vibrio-like strains isolated from freshwater. The overall success rate for identification was 84%, using a Willcox score of ?0.99. The data generated for 172 of the reference strains and 243 wild strains were subjected to numerical taxonomic analysis. The main purpose of these analyses was to verify the quality of the identification scheme. Nevertheless the results produced were in good agreement with those of more detailed taxonomic studies. The taxonomic position of some named and some unnamed groups of Vibrio and Aeromonas was clarified, and taxa showing poor distinction were highlighted. An ecological survey was carried out to determine the distribution and seasonal occurrence of vibrios in various freshwater sites in the U.K. The results showed that species of vibrio both pathogenic and non-pathogenic for man, are widely distributed in rivers and canals. Some of the species considered to be indigenous to the waters. The role of these organisms in the freshwater environment remains to be determined.

The ability of bacteria to successfully adapt to changing environments allows for increased resistance to antibiotics, an increasing problem in drug development. Efflux via the cellular membrane is one of the major and most significant mechanisms bacteria employ for defense against antimicrobials. The outer membrane protein, TolC, is a nonspecific channel with broad substrate specificity and is a fundamental constituent of a number of multidrug resistance (MDR) efflux systems. TolC, which interacts with membrane bound antiporters to export various substrates, including antimicrobials, has been a focus for antibiotic development. The capacity to expel a wide range of compounds such as toxins, bile, dyes and detergents, further establishes the significance of these systems in aiding bacterial survival. Continued investigation and discovery of compounds secreted via TolC is required to fully understand the role of efflux systems. In addition to exporting compounds out of cells, TolC has been researched in connection with a number of metabolic processes including virulence, motility and quorum sensing. This work contributes to knowledge of interactions between these pathways by analysing phenotypic changes and alterations in gene expression in the tolC- mutant within Vibrio furnissii. Vibrio species are strongly associated with a number of reported phenotypic changes induced by mutations in tolC, particularly virulence and quorum sensing; the emerging pathogen V. furnissii is therefore a model organism for this study. Notably, this bacterium has become significant within the bio-fuel industry due to reports that it is able to synthesise large quantities of hydrocarbons. The potential for bacteria to produce hydrocarbons and fatty acids suitable for use in bio-fuels has considerable industrial applications. This work establishes the quantities of hydrocarbons and lipids present in V. furnissii and examines the role of TolC in the secretion of these compounds. Resistance nodulation division efflux pumps are a class of MDR systems that have been identified as targets for antimicrobial drug development. Understanding the consequence of disrupting the integral outer membrane component TolC, is therefore of significant interest. This work examines the connection between pathways disrupted within tolC- mutants, showing that quorum sensing gene regulation is altered within the mutant and subsequently, expression of the virulence factor AphB is increased. Reduced motility is also observed in the tolC- mutant, a phenotype that appears to result from disruption of the membrane and mis-assembled flagella. Investigation into cellular stress, recently reported to occur within tolC- mutants, was performed by comparing the capability of wild type and mutant cells to metabolise selenite, and cope with the subsequent increase in superoxides generated. Results show that tolC- mutants employ mechanisms which cope with the inevitable intracellular build up of substrates, particularly those that may be toxic to the cell.

Introdução - Vibrio fluvialis é um microorganismo que provoca a gastroenterite muito semelhante à cólera, mas também há relatos de casos extra-intestinais como sepse, ferida, peritonite e celulite hemorrágica e encefalite. Acredita-se que a infecção por esse microorganismo esteja vinculada ao consumo de peixes crus ou mal cozidos contaminados e / ou frutos do mar. A identificação dessa bactéria por métodos fenotípicos continua a ser um problema devido à sua grande semelhança com Aeromonas hydrophila e V.furnissii; por isso, a utilização de uma ferramenta de diferenciação entre essas espécies é importante. Nas últimas décadas, o aumento da resistência aos antimicrobianos tem sido um fator preocupante, porque ela interfere na escolha dos medicamentos para o tratamento eficaz e há uma necessidade de rápida produção de novos antibióticos. Ambientes costeiros e estuários estão em perigo de serem contaminados por esgoto, que pode conter drogas que agem de forma seletiva, permitindo o desenvolvimento de resistência aos antimicrobianos. Vários estudos demonstraram que estirpes clínicas de V. fluvialis são resistentes a múltiplas drogas. Objetivos - Desenvolver um marcador molecular baseado no 16S rDNA capaz de detectar o grupo V. fluvialis-V. furnissii, e avaliar a susceptibilidade a antibióticos destas espécies, principalmente a partir de amostras ambientais. Métodos - Após a elaboração dos iniciadores a partir do alinhamento das espécies do gênero Vibrio, foram utilizadas cepas identificadas fenotipicamente como V. fluvialis e de V. furnissii para a sua detecção molecular. O perfil de susceptibilidade a antibióticos pelo método da disco-difusão foi realizada, assim como a investigação molecular da presença do elemento SXT e de seus genes de resistência a antimicrobianos. Resultados: Com a utilização dos iniciadores desenvolvidos foi possível confirmar corretamente as espécies. Observou-se alta porcentagem de resistência a ampicilina e cefalotina, sendo que 65,9por centode V. fluviais e 43,24por centode V. furnissi apresentaram resistência a pelo menos dois dos antibióticos utilizados. Somente em uma cepa de V. fluvialis detectou-se a presença de SXT e houve uma banda desconhecida de alto peso molecular quando da pesquisa do gene sulII. Conclusões: O método molecular mostrou ser um importante instrumento para se detectar espécies altamente relacionadas. Foram detectadas cepas resistentes a múltiplos antibióticos, indicando que o meio ambiente é um provável reservatório de genes de resistência; porém necessita-se de futuras investigações moleculares para se determinar o papel destes e sua possível associação com elementos genéticos. A detecção do elemento SXT sem a presença dos seus genes de resistência conhecidos atualmente reforça a idéia da extensão de seu papel adaptativo, além de ser o primeiro relato de sua existência na América do Sul / Introduction - Vibrio fluvialis is a microorganism that causes gastroenteritis very similar to cholera, however there are also reports of extraintestinal cases as sepse, skin wounds, peritonitis and hemorrhagic cellulitis and cerebritis. It is believed that infection by this organism is linked to the consumption of raw or undercooked contaminated fish and / or seafood. Identification of this bacteria by phenotypic methods remains a problem due to its great similarity with Aeromonas hydrophila and V.furnissii, therefore the use of a tool to differentiate these species is important. In recent decades, increasing antimicrobial resistance has been a concerning factor because it interferes in the choice of drugs for effective treatment and there is a need for rapid production of new antibiotics. Coastal and estuarine environments are in danger of being contaminated by sewage, which may contain drugs that will act selectively, allowing the development of antimicrobial resistance. Several studies have demonstrated that clinical strains of V. fluvialis are resistant to multiple drugs. Objectives - To develop a 16S rDNA - molecular marker able to detect the group V. fluvialis-V.furnissii, and to evaluate the antibiotic susceptibility of this species mainly from environmental samples. Methods - After the development of primers from alignment of the genus Vibrio strains phenotypically identified as V. fluvialis and V. furnissii were used for their molecular identification. The profile of antibiotic susceptibility was performed by the disk diffusion method, and the molecular investigation of the presence of the SXT element and their antimicrobial resistance genes. Results - The primers developed were able to confirm correctly the species. A high percentage of resistance to ampicillin and cephalothin was observed, V. fluvialis and V. furnissii showed resistance to at least two of the antibiotics used, 65.9 per cent and 43.24 per cent respectively. Only in one strain of V. fluvialis we detected presence of SXT and there was an unknown band of high molecular weight when we investigated gene sulII. Conclusions - Molecular identification has proved to be an important tool for differentiating highly related species. Strains resistant to multiple antibiotics were detected, indicating that the environment is likely a reservoir for resistance genes, but it is needed further molecular investigations to determine their role and their possible association with genetic elements. Detection of the SXT element without the presence of its known resistance genes reinforces the idea of the extent of its adaptive role, and this is the first report of its existence in South America

Li Jun. / "March 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 189-216). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Introdução - Vibrio fluvialis é um microorganismo que provoca a gastroenterite muito semelhante à cólera, mas também há relatos de casos extra-intestinais como sepse, ferida, peritonite e celulite hemorrágica e encefalite. Acredita-se que a infecção por esse microorganismo esteja vinculada ao consumo de peixes crus ou mal cozidos contaminados e / ou frutos do mar. A identificação dessa bactéria por métodos fenotípicos continua a ser um problema devido à sua grande semelhança com Aeromonas hydrophila e V.furnissii; por isso, a utilização de uma ferramenta de diferenciação entre essas espécies é importante. Nas últimas décadas, o aumento da resistência aos antimicrobianos tem sido um fator preocupante, porque ela interfere na escolha dos medicamentos para o tratamento eficaz e há uma necessidade de rápida produção de novos antibióticos. Ambientes costeiros e estuários estão em perigo de serem contaminados por esgoto, que pode conter drogas que agem de forma seletiva, permitindo o desenvolvimento de resistência aos antimicrobianos. Vários estudos demonstraram que estirpes clínicas de V. fluvialis são resistentes a múltiplas drogas. Objetivos - Desenvolver um marcador molecular baseado no 16S rDNA capaz de detectar o grupo V. fluvialis-V. furnissii, e avaliar a susceptibilidade a antibióticos destas espécies, principalmente a partir de amostras ambientais. Métodos - Após a elaboração dos iniciadores a partir do alinhamento das espécies do gênero Vibrio, foram utilizadas cepas identificadas fenotipicamente como V. fluvialis e de V. furnissii para a sua detecção molecular. O perfil de susceptibilidade a antibióticos pelo método da disco-difusão foi realizada, assim como a investigação molecular da presença do elemento SXT e de seus genes de resistência a antimicrobianos. Resultados: Com a utilização dos iniciadores desenvolvidos foi possível confirmar corretamente as espécies. Observou-se alta porcentagem de resistência a ampicilina e cefalotina, sendo que 65,9por centode V. fluviais e 43,24por centode V. furnissi apresentaram resistência a pelo menos dois dos antibióticos utilizados. Somente em uma cepa de V. fluvialis detectou-se a presença de SXT e houve uma banda desconhecida de alto peso molecular quando da pesquisa do gene sulII. Conclusões: O método molecular mostrou ser um importante instrumento para se detectar espécies altamente relacionadas. Foram detectadas cepas resistentes a múltiplos antibióticos, indicando que o meio ambiente é um provável reservatório de genes de resistência; porém necessita-se de futuras investigações moleculares para se determinar o papel destes e sua possível associação com elementos genéticos. A detecção do elemento SXT sem a presença dos seus genes de resistência conhecidos atualmente reforça a idéia da extensão de seu papel adaptativo, além de ser o primeiro relato de sua existência na América do Sul / Introduction - Vibrio fluvialis is a microorganism that causes gastroenteritis very similar to cholera, however there are also reports of extraintestinal cases as sepse, skin wounds, peritonitis and hemorrhagic cellulitis and cerebritis. It is believed that infection by this organism is linked to the consumption of raw or undercooked contaminated fish and / or seafood. Identification of this bacteria by phenotypic methods remains a problem due to its great similarity with Aeromonas hydrophila and V.furnissii, therefore the use of a tool to differentiate these species is important. In recent decades, increasing antimicrobial resistance has been a concerning factor because it interferes in the choice of drugs for effective treatment and there is a need for rapid production of new antibiotics. Coastal and estuarine environments are in danger of being contaminated by sewage, which may contain drugs that will act selectively, allowing the development of antimicrobial resistance. Several studies have demonstrated that clinical strains of V. fluvialis are resistant to multiple drugs. Objectives - To develop a 16S rDNA - molecular marker able to detect the group V. fluvialis-V.furnissii, and to evaluate the antibiotic susceptibility of this species mainly from environmental samples. Methods - After the development of primers from alignment of the genus Vibrio strains phenotypically identified as V. fluvialis and V. furnissii were used for their molecular identification. The profile of antibiotic susceptibility was performed by the disk diffusion method, and the molecular investigation of the presence of the SXT element and their antimicrobial resistance genes. Results - The primers developed were able to confirm correctly the species. A high percentage of resistance to ampicillin and cephalothin was observed, V. fluvialis and V. furnissii showed resistance to at least two of the antibiotics used, 65.9 per cent and 43.24 per cent respectively. Only in one strain of V. fluvialis we detected presence of SXT and there was an unknown band of high molecular weight when we investigated gene sulII. Conclusions - Molecular identification has proved to be an important tool for differentiating highly related species. Strains resistant to multiple antibiotics were detected, indicating that the environment is likely a reservoir for resistance genes, but it is needed further molecular investigations to determine their role and their possible association with genetic elements. Detection of the SXT element without the presence of its known resistance genes reinforces the idea of the extent of its adaptive role, and this is the first report of its existence in South America

Includes bibliography.

Nine isolates of bacteria recovered from fish dying at marine facilities were collected from different geographic areas. The strains included: an isolate from chinook salmon (Oncorhynchus tshawytscha) reared in net pens in New Zealand, an isolate from chum salmon (Oncorhynchus keta) held at a laboratory in Oregon, USA., and seven strains recovered from tilapia (Oreochromis spilurus), silvery black porgy (Acanthopagrus cuvieri), and greasy grouper (Epinenhelus tauvina) cultured in Kuwait. All isolates were characterized by examination of morphological and biochemical properties and were confirmed to be members of the genus Vibrio. All isolates differed phenotypically from each other, from vibrios known to be pathogenic for fish, and from other named Vibrio species. Analysis of key phenotypic characteristics used to establish existing species suggested that the isolates tested were new Vibrio species. Four of the isolates (two from coldwater fish and two from warmwater fish) were selected for further study. This included determination of percent guanine plus cytosine (%G+C), comparison of growth characteristics, analysis of major 0 antigens and testing of pathogenicity. The four isolates examined had an absolute requirement for NaCl. Optimum growth temperatures varied among the isolates and were consistent with the temperature optima of the hosts from which the isolates were obtained. Serological analysis using slide agglutination, microtiter agglutination, and Ouchterlony double diffusion tests detected specific thermostable (0) antigens unique for each of the four isolates. A common minor antigen was observed between two of the other isolates from Kuwait. Experimental infections were produced in fingerling rainbow trout (Salmo gairdneri) using intraperitoneal injection of the four isolates. The pathogenicity of the two isolates from Kuwait was higher than that of the two salmonid isolates. The strains from Kuwait were used to challenge juvenile chinook salmon by waterborne exposure. The pathology produced by infection was characteristic Gram-negative hemorrhagic septicemia. / Graduation date: 1988

Thesis (M.Sc.)(Microbiology)--University of Pretoria, 2005. / Includes summary. Includes bibliographical references. Available on the Internet via the World Wide Web.

To overcome the membrane permeability barrier and low nutrient availability in the environment, Gram-negative organisms have evolved many mechanisms dedicated to the intake of nutrients. One such mechanism is the long-chain fatty acid uptake pathway. This pathway involves machinery that transports fatty acids across the outer membrane and into the cell, where the lipid can be used for either nutrition or for remodeling the structure of the membrane. Interestingly, the fatty acid species that can be recognized by this machinery differ between organisms; the aquatic pathogen Vibrio cholerae demonstrates a much wider substrate recognition profile than other Gram-negative species. In this work we elaborate on the lipid nutrients accessible to V. cholerae, demonstrating that it can use lysophosphatidylcholine as both a carbon source as well as a source of fatty acids for remodeling its outer membrane. We identify the enzyme responsible for the breakdown of lysophosphatidylcholine, VolA (Vibrio outer membrane lysophospholipase A). VolA is conserved in many Vibrio species as well as other aquatic Gram-negatives, annotated as a putative lipase. We show VolA is co-expressed with the outer membrane fatty acid transporter FadL; FadL serves to transport across the outer membrane those long-chain fatty acids freed from lysophosphatidylcholine by VolA. VolA is expressed on the surface of the cell as a membrane anchored lipoprotein; this is novel as VolA is the first identified example of a surface-localized lipoprotein phospholipase. Biochemical characterization of VolA shows that it acts as a canonical lysophospholipase in vitro, suggesting that it works in tandem with the FadL transporter, freeing fatty acids from lysophosphatidylcholine at the surface of the cell to be brought in via the fatty acid uptake pathway. This work expands on the currently understood lipid uptake abilities of Vibrio cholerae, demonstrating a novel mechanism for utilizing a nutrient not previously thought to metabolized. VolA is an important to our understanding of the larger picture of lipid uptake and how it contributes to the survival of Gram-negative organisms. / text