Thesis (Ph. D.)--Ohio State University, 2009. / Title from first page of PDF file. Includes vita. Includes bibliographical references (p. 192-231).

No description available.

Techn. Univ., Diss., 2010--München.

Universiẗat, Diss., 2003--München.

Universiẗat, Diss., 2004--München.

Tierärztliche Hochsch., Diss., 2005--Hannover.

Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-06-19T11:54:47Z No. of bitstreams: 1 resumo.pdf: 19644 bytes, checksum: 54f0c36ee9445a27674f067adaa45848 (MD5) / Made available in DSpace on 2017-06-19T11:54:47Z (GMT). No. of bitstreams: 1 resumo.pdf: 19644 bytes, checksum: 54f0c36ee9445a27674f067adaa45848 (MD5) Previous issue date: 2005-02-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O crescimento de 14 isolados de Escherichia coli, Yersinia enterocolitica e Salmonella spp., obtidos de carnes, carcaças e fezes suínas, foi avaliado em diferentes condições de cultivo. Treze isolados (92,8%) não cresceram na presença de 0,5% de ácido lático e de 10% de cloreto de sódio em meio Infusão Cérebro e Coração (BHI). As concentrações de 3,5% e, mais acentuadamente, 5% de cloreto de sódio reduziram o crescimento de todos os isolados nesse mesmo meio. A adição de até 0,02% de nitrito de sódio não afetou o crescimento dos isolados em BHI. O crescimento das estirpes E. coli CS5 e Salmonella sp. CCS1 e CCS2 em homogenato de carne suína foi menor na presença de 3,5% de cloreto de sódio. Os isolados E. coli CS3, Y. enterocolitica CS6 e Salmonella sp. CCS1 mostraram redução no crescimento com a adição de 0,015% de nitrito de sódio ao homogenato. Quando 3,5% de cloreto de sódio e 0,015% de nitrito de sódio foram adicionados ao homogenato, os isolados E. coli CS1 e Salmonella sp. FS3, FS7 e CCS3 foram inibidos. O crescimento dos demais isolados não foi afetado pela adição desses sais. A sobrevivência de seis isolados de Salmonella spp. inoculados em carne suína mantida a -15 °C por 20 semanas também foi avaliada. Após o período de estocagem, constatou-se redução média de 1,4 ciclo logarítmico na população. Salmonella sp. CCS4 apresentou a menor redução, aproximadamente 1,16 ciclo logarítmico. Salmonella sp. FS10 perdeu mais que 99% de culturabilidade, após incubação em solução salina com 0,85% e 3,5% de cloreto de sódio a 4 °C por 30 dias. A susceptibilidade dos isolados a dez antimicrobianos foi investigada. Os quatorze isolados apresentaram resistência à tetraciclina. Sete isolados (50%) foram resistentes a pelo menos dois antimicrobianos. Seis isolados (42,8%) exibiram resistência a três antimicrobianos: um isolado de E. coli, proveniente de carne suína (E. coli CS4) e cinco de Salmonella spp., sendo um obtido de fezes suínas (Salmonella sp. FS7) e os demais de carcaças suínas (Salmonella sp. CCS1, CCS2, CCS3 e CCS4). A multirresistência foi observada para os antimicrobianos tetraciclina, sulfametoxazol/trimetoprim, cloranfenicol e ácido nalidíxico. As concentrações mínimas inibitórias (CMI) do promotor de crescimento olaquindox foram determinadas para cada isolado e variaram de 15 a 60 μg/mL. Os maiores valores de CMI foram de 60 μg/mL para Y. enterocolitica CS6 e 50 μg/mL para Salmonella sp. FS7 e CCS3. / The growth of fourteen isolates of Escherichia coli, Yersinia enterocolitica and Salmonella spp. isolated from pork, swine carcasses and feces was evaluated at different conditions. In the presence of lactic acid 0,5 % and sodium chloride 10 % in brain heart infusion medium (BHI) 13 isolates (92,8 %) did not grow. Sodium chloride 3,5 % and 5 % concentration, more strongly, inhibited the growth of all isolates in the same medium. Addition of up to 0,02% of sodium nitrite did not affect the growth of isolates in BHI. The growth of E. coli CS5, Salmonella sp. CCS1 and CCS2 in pork homogenate was inhibited in the presence of sodium chloride 3,5%. Sodium nitrite 0,015% in pork homogenate inhibited the growth of the isolates E. coli CS3, Y. enterocolitica CS6 and Salmonella sp. CCS1. When both sodium chloride 3,5% and sodium nitrite 0,015% were added to the growth medium E. coli CS1, Salmonella sp. FS3, FS7 and CCS3 were inhibited. The growth of other isolates was not affected by addition of those salts to homogenate. The survival of six isolates of Salmonella spp in pork maintained at - 15°C for 20 weeks was also evaluated. After the storage period, the average reduction in population was 1,4 log 10 CFU ml -1 . Salmonella sp. CCS4 presented the smallest reduction, approximately 1,16 log 10 CFU ml -1 . Salmonella sp. FS10 lost more than 99% of its culturability after incubation in NaCl saline solution 0,85% and 3,5% at 4 °C for 30 days. The susceptibility of the isolates xto ten antimicrobials was investigated. Fourteen isolates were resistant to the antimicrobial tetracycline. Seven isolates (50%) were resistant to at least two antimicrobials. Six isolates (42,8%) exhibited resistance to three antimicrobials: one isolate of E. coli from pork (E. coli CS4) and five of Salmonella spp., one obtained from swine feces (Salmonella sp. FS7) and the others from swine carcasses (Salmonella sp. CCS1, CCS2, CCS3 and CCS4). The multidrug-resistance was observed for the antimicrobials tetracycline, sulfametoxazol/trimetoprim, chloranfenicol and nalidixic acid. The minimum inhibitory concentration (MIC) of the growth promoter olaquindox was determined for each isolate and varied from 15 to 60 μg.ml -1 . The highest value of MIC was 60 μg ml -1 for Y. enterocolitica CS6 and 50 μg ml -1 for Salmonella sp. FS7 and CCS3.

Purified DNA from a nalidixic acid resistant derivative of a virulent serotype 0:3 clinical isolate of Yersinia enterocolitica was subjected to transpositional mutagenesis in an effort to construct avirulent mutants. The resulting transpositional mutants, as well as the wild-type virulent strain and its isogenic derivative that had been cured of the virulence plasmid, were analyzed for plasmid DNA content. The plasmid DNA content of each strain was further characterized by restriction endonuclease digestion and the transposon insertion sites for the mutants were located. All of the strains were then tested for pathogenicity by the following assays: calcium dependence, colonization of the mouse gastrointestinal tract, HEp-2 cell adherence and invasion, HEp-2 cell monolayer detachment, autoagglutination, serum resistance, outer membrane protein production and production of V antigen. In addition, the hydrophobic properties of each strain were examined by a rapid polystyrene plate method and hydrophobic interaction chromatography. The results of the tests were compared to plasmid DNA analyses for each strain in an attempt to identify any plasmid-associated genes that are related to virulence. The wild-type strain was virulent, or positive, by all of the assays employed for evaluation of pathogenicity. In contrast, its isogenic derivative that had been cured of the virulence plasmid was negative, or avirulent, for the same assays with one exception. The avirulent plasmidless strain still retained the ability to adhere to and invade HEp-2 cells, supporting the belief that these properties are probably encoded by the bacterial chromosome. In addition, three transpositional mutants were constructed that were no longer calcium dependent, capable of detaching HEp-2 cell monolayers or able to produce three unique outer membrane proteins. Restriction endonuclease analysis confirmed the presence of the transposon on the Hind III "A" fragment of the virulence plasmid and located the region responsible for the lost virulence properties. The gene or set of genes identified were designated cal and the respective calcium independent mutants Cal⁻. Furthermore, the assays for hydrophobicity indicated that the virulence plasmid, specifically the cal gene(s), codes for hydrophobic properties on the surface of the bacterium. The study demonstrated that a virulence-associated region, cal, is located on the virulence plasmid of Y. entrocolitica and responsible for calcium dependence, HEp-2 cell monolayer detachment, the production of the three plasmid-specified outer membrane proteins and cell-surface hydrophobicity.

In dieser Arbeit wurden zwei Aspekte der Yersinia β-Laktamasen bearbeitet: (1) Charakterisierung der β-Laktamasen hinsichtlich β-Laktam-Antibiotikaresistenz, Sekretion und Thermostabilität. (2) Untersuchung der Sekretionsfähigkeit von verschiedenen thermostabilen β Laktamasen über das Yersinia T3SS. Im ersten Teil wurden β Laktamase-Deletionsmutanten im Y. enterocolitica Serotyp O:8 Stamm WA-314 hergestellt, um den Einfluss der chromosomalen β Laktamasen auf die in vitro-Resistenz zu untersuchen. Es konnte gezeigt werden, dass WA-314 konstitutiv BlaA produziert und BlaA somit – unter nicht-induzierbaren Bedingungen – der dominante Faktor in der in vitro-Resistenz gegenüber Penicillinen mit erweitertem Wirkungsspektrum (z.B. Ampicillin) und Cephalosporinen der 1. Generation (z.B. Cefazolin) ist. Weiterhin konnte gezeigt werden, dass die zweite chromosomale β Laktamase AmpC (BlaB) unter Zugabe von subinhibitorischen Konzentrationen von Imipenem stark induziert wird. Keine der β Laktamasen ist in der Lage, in vitro-Resistenz gegenüber Carbapenemen und Monobactamen zu vermitteln. Die Konstruktion und Bestimmung der in vitro Antibiotika-Empfindlichkeit der β Laktamase-Deletionsmutanten dient als Grundlage für nachfolgende Untersuchungen im Mausinfektionsmodell. Weiterhin wurden die Transporteigenschaften beider β Laktamasen untersucht. In Gram-negativen Bakterien sind reife β Laktamasen im Periplasma lokalisiert und müssen somit nach der Synthese im Cytosol über die Cytoplasmamembran transportiert werden. Bis auf drei Ausnahmen (β Laktamasen aus Mycobacterium smegmatis, M. tuberculosis und Stenotrophomonas maltophila) sind bisher nur Sec-abhängige β Laktamasen beschrieben worden. Mittels Fusionsproteinen bestehend aus β Laktamase-Signalpeptiden und GFP konnte in dieser Arbeit eindeutig gezeigt werden, dass es sich bei Yersinia BlaA um ein Tat-Substrat handelt, bei Yersinia AmpC hingegen um ein Sec-Substrat. Somit konnte im Rahmen dieser Arbeit zum ersten Mal eine Tat-abhängige β Laktamase bei einer Bakterienart aus der Familie der Enterobacteriaceae nachgewiesen werden. Außerdem konnte gezeigt werden, dass die β Laktamase BlaA nicht diffus im Periplasma, sondern auf bestimmte Bereiche im Periplasma lokalisiert verteilt ist. Allerdings konnte die Art der Lokalisierung bisher nicht genau spezifiziert werden. Die cytosolische Faltung und die Tat-abhängige Translokation von BlaA lassen vermuten, dass eine besondere Thermostabilität von BlaA vorliegt. Deshalb wurde das BlaA-Enzym hinsichtlich seiner Thermostabilität und temperaturabhängigen enzymatischen Aktivität untersucht. Im Vergleich zur E. coli β Laktamase TEM-1 und der hitzestabilen TEM-1-Variante MEGA zeigte BlaA eine erhöhte Thermostabilität und einen starken Anstieg der Aktivität in einem Temperaturbereich zwischen 30 °C und 45 °C. Im zweiten Teil dieser Arbeit wurde geprüft, ob die charakterisierten Yersinia β Laktamasen als Reporterkonstrukte zur Untersuchung des Typ III Sekretionssystems (T3SS) geeignet sind. Y. enterocolitica besitzt ein pYV Virulenzplasmid, auf dem der vollständige Satz der Gene für das Ysc-T3SS und die Effektor-Yops (Yersinia outer protein) lokalisiert sind. Injektion der Yops in eukaryotische Zielzellen ermöglicht das extrazelluläre Überleben der Yersinien im Wirtsorganismus. Bei YopE handelt es sich um ein gut charakterisiertes Effektor-Yop, dessen N Terminus fusioniert an den reifen Teil der β Laktamase TEM-1 bereits vielfach als Reporterkonstrukt eingesetzt wurde. Unter Verwendung des fluoreszierenden β Laktamase-Substrats CCF4-AM kann die Translokation von YopEi-TEM-1 in Zielzellen in Zellkultur-Experimenten und im Mausinfektionsmodell visualisiert werden. In dieser Arbeit sollte deshalb die T3SS-Sekretionsfähigkeit von YopE-β Laktamase-Fusionsproteinen in Abhängigkeit von der „Schmelztemperatur“ (temperaturabhängige Stabilität, TM) untersucht werden. Yop-Substrate werden im ungefalteten Zustand (YscN wirkt dabei vermutlich als ATP-abhängige „Unfoldase“) über das Ysc-„Injektisom“ transloziert. YopEi-TEM-1 wird effizient sekretiert und transloziert (TM (TEM-1) = 50,8 °C). YopE-Fusionsproteine mit thermostabilen TEM-1 Varianten, YopEi-RLT bzw. YopEi-MEGA (TM (RLT) = 60,4 °C; TM (MEGA) = 69,2 °C) werden hingegen nur schwach bzw. nicht sekretiert. Weiterhin konnte gezeigt werden, dass die Sec-abhängige β Laktamase AmpC als YopE-Fusionsprotein (YopEi-AmpC) effizient T3SS-abhängig sekretiert und transloziert werden kann; das native Tat-Substrat BlaA (YopEi-BlaA) kann jedoch weder sekretiert noch transloziert wird. Eine mögliche Erklärung wäre, dass die ATPase YscN nicht in der Lage ist, BlaA und die thermostabilen TEM-1-Varianten zu entfalten und über das T3SS zu sekretieren und zu translozieren. RLT und MEGA können hingegen mithilfe ihrer nativen Signalsequenz über das Sec-System (und somit im ungefalteten Zustand) transloziert werden. / In this work, two aspects concerning the Yersinia β lactamases have been processed: (1) Characterization of the β lactamases in regard of β lactam antibiotic resistance, secretion and thermostability. (2) Analysis of the secretability of different thermostable β lactamases via the Yersinia T3SS. In the first part of this work we described the β lactam sensitivity pattern of the highly mouse pathogenic Y. enterocolitica strain WA 314 by creating a set of β lactamase deletion mutants and their subsequent characterization in vitro. In accordance to previous studies on biovar 1B, serovar O:8 strains, WA 314 produces both enzymes BlaA and AmpC (BlaB) and latter one is inducible using subinhibitory concentrations of imipenem. BlaA is dominant in providing in vitro-resistance towards extended-spectrum β lactams (e.g. ampicillin) and 1st generation cephalosporins (e.g. cefazolin). None of the β lactamases is able to mediate in vitro-resistance towards carbapenems and monobactams. The construction of β lactamase deletion mutants and their in vitro-characterization is the basis for their subsequent analysis within a murine infection model. Furthermore, we investigated the export mechanism of both β lactamases. In Gram-negative bacteria β lactamases are localized in the periplasmic space to be able to cleave the amide bond of the β lactam ring resulting in inactivation of the antibiotic. Proteins are translocated across the inner membrane either via the general secretory pathway (Sec system) or via the twin arginine pathway (Tat system). To date, only three β lactamases have been described as Tat-dependently translocated namely BlaC from Mycobacterium tuberculosis, BlaS from M. smegmatis and L2 from the plant pathogen Stenotrophomonas maltophila. In silico studies and experimental approaches lead to the assumption that the majority of β lactamases is translocated in a Sec-dependent manner. Analysis of β lactamase signal sequence-GFP fusion proteins revealed that Yersinia AmpC is a Sec-substrate whereas Yersinia BlaA is a Tat-substrate. Thus, BlaA of Y. enterocolitica is the first reported β lactamase within the family of Enterobacteriaceae which is secreted by the Tat system. Interestingly, closely related blaA genes are widely distributed within all Y. enterocolitica biovars and several environmental Yersinia species. It is generally accepted that Tat-dependent substrates rapidly fold in the cytoplasma. To compare the BlaA protein stability with that of the Sec-dependent β-lactamases of the TEM-1 family we determined thermal unfolding transitions and temperature dependent enzymatic activities. In contrast to TEM-1 β lactamase, BlaA shows an entire different activity profile with steep increase of activity in the temperature range of 30 °C to 45 °C and steep decrease between 50 °C to 60 °C. In the second part, the Yersinia β lactamases AmpC and BlaA characterized in this work were tested for suitablility as reporter constructs for analyzing the Ysc-T3SS. Y. enterocolitica harbors a pYV virulence plasmid which encodes genes for components of the Ysc-T3SS secretion machinery and the effector Yops (Yersinia outer protein). Injection of Yops into the eukaryotic host cell enables the extracellular survival of Yersinia within the host organism. YopE is a well described effector Yop and YopE-β lactamase (TEM-1) fusion proteins have been successfully applied to analyze translocation of YopE in cell culture experiments and in the murine mouse infection model by using the fluorescent β lactamase substrate CCF4-AM. In this work, we aimed to analyze the T3SS-dependent secretability of YopE-β-lactamase fusion proteins in dependency on the melting temperature (temperature dependent stability, TM). It has been described that Yop substrates are translocated in an unfolded conformation via the Ysc-“injectisom” (YscN has been discussed as an ATP-dependent unfoldase). YopEi-TEM-1 is efficiently secreted and translocated (TM (TEM-1) = 50.8 °C). However, YopE fusion proteins with heat stable TEM-1 variants, YopEi-RLT and YopEi-MEGA (TM (RLT) = 60.4 °C; TM (MEGA) = 69.2 °C), were weakly secreted or secretion is completely abolished, respectively. Furthermore we could show that a YopE fusion protein with the Sec-dependent β lactamase AmpC (YopEi-AmpC) is efficiently secreted and translocated via the T3SS. In contrast, the Tat-dependent β lactamase BlaA (YopEi-BlaA) can neither be secreted nor translocated T3SS-dependently. A putative explanation for that observation would be that the ATPase YscN is unable to unfold BlaA and the heat stable TEM-1 variants and therefore secretion and translocation is prevented. Interestingly, native RLT and MEGA β lactamases can be translocated in an unfolded conformation via the Sec system.

Techn. Univ., Diss., 2010--München.