Thesis--Catholic Univ. of America. / Description based on print version record.

Leipzig, Universiẗat, Diss., 2003.

Made available in DSpace on 2014-06-12T15:53:17Z (GMT). No. of bitstreams: 2 arquivo5207_1.pdf: 1038836 bytes, checksum: fefc6684baa0c15fe33f48b0a036c0b6 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2006 / A Yersinia pestis, bactéria Gram-negativa da família Enterobacteriaceae, é uma espécie muito homogênea quando observada pelos métodos fenotípicos: apresenta apenas um sorotipo, um fagotipo e um biotipo subdividido em três biovars ou variedades geográficas. A peste é uma doença primária dos roedores, geralmente transmitida por pulgas e que ocasionalmente pode infectar outros mamíferos, inclusive o homem, que é atingido acidentalmente ao penetrar no ecossistema da doença. Ela ainda persiste nos dias atuais entre diversos hospedeiros/reservatórios em numerosos focos silvestres em vários países do mundo e atualmente é considerada uma doença reemergente. Devido à alta taxa de letalidade e ao seu potencial de epidemização, a peste é classificada como doença de notificação Classe I de acordo com o Regulamento Sanitário Internacional (RSI) vigente, o que exige vigilância permanente dos focos, sobretudo porque a Y. pestis pode ser usada como agente de bioterrorismo. A vigilância da peste no Brasil baseia-se na pesquisa da bactéria em roedores e pulgas e de anticorpos antipestosos em animais sentinela (algumas espécies de roedores resistentes à infecção e carnívoros domésticos, como os cães e gatos). O conhecimento das características dos isolados de cada foco permitirá detectar a introdução de uma nova cepa e, consequentemente, a sua origem, se de outro foco ou por ato deliberado. Os estudos de tipagem molecular de cepas brasileiras de Y. pestis por diferentes técnicas, como a RAPD-PCR, PCR-ribotipagem e RFLP-IS100, revelaram, na maioria das vezes, padrões genômicos idênticos entre as cepas, independentemente das fontes, procedência e ano. Recentemente, um estudo utilizando MLVA (análise de múltiplos locos do número variável de repetições em tandem), mostrou que as cepas de Y. pestis do Brasil apresentavam polimorfismo. Este trabalho teve como objetivo caracterizar isolados de Y. pestis em um mesmo evento epidemiológico através de locos VNTR (número variável de repetições em tandem). Um conjunto de vinte cepas de Y. pestis isoladas durante a investigação de uma epizootia em um foco da Chapada do Araripe, município de Exu, Pernambuco, Brasil, em agosto de 1967, foi analisado. As cepas conservadas na bacterioteca do SRP (Serviço de Referência em Peste) foram reativadas e a identificação bacteriológica confirmada pela suscetibilidade ao bacteriófago antipestoso. Alterações no genoma foram pesquisadas pela presença ou ausência de genes de virulência nos plasmídeos prototípicos da Y. pestis e na Ilha de Patogenicidade (HPI) das yersínias, através da multiplex-PCR. Foram analisados seis locos VNTR pela reação de PCR. A ausência de alguns genes de virulência foi evidenciada em dez cepas, sugerindo que modificações genômicas ocorreram nesses isolados. Apesar disso, dos seis VNTR analisados cinco se revelaram monomórficos e apenas um apresentou polimorfismo, gerando três alelos. Colônias morfologicamente diferentes, grandes e pequenas, em algumas cepas, apresentaram padrão VNTR atípico, com duas bandas amplificadas. Por MLVA as cepas revelaram-se geneticamente relacionadas, o que reflete a relação epidemiológica desses isolados. Ao mesmo tempo, é necessário a análise de um maior número de VNTRs para confirmar a relação genética dessas cepas, em comparação com cepas de outros focos

Trois bactéries du genre Yersinia sont pathogènes pour l’homme : Yersinia pestis (bacille de la peste), et les bactéries entéropathogènes Yersinia pseudotuberculosis et Yersinia enterocolitica. Yersinia pestis est responsable de plus de 20 000 cas humains de peste déclarés à l’Organisation Mondiale de la Santé (OMS) ces dix dernières années dans différents foyers en Afrique, Asie et Amériques. Considérée aujourd’hui à tort comme une maladie du passé, elle est au contraire classée parmi les maladies réémergentes Même si elle ne se présente plus sous la forme d’épidémies massives, elle pose encore au monde actuel d’importants défis de par son extrême gravité, sa rapidité de dissémination, une apparition de résistances aux antibiotiques et une éventuelle utilisation terroriste du bacille. Dans ce contexte, l’immunothérapie contre Y. pestis pourrait être une bonne alternative pour traiter la peste bubonique et pulmonaire. Un des objectifs de cette thèse était de produire des anticorps monoclonaux murins contre trois protéines de l’injectisome (YscF, YscC et LcrV), un facteur de virulence clé des Yersinia. Les anticorps obtenus ont été caractérisés et pour certains leurs épitopes identifiés. Par la suite, en collaboration avec Elisabeth Carniel à l’Institut Pasteur, leur pouvoir neutralisant a été évalué in vivo dans un modèle murin de peste bubonique. Ces mêmes anticorps monoclonaux, produits contre les protéines de l’injectisome sont en cours d’évaluation pour la mise au point d’un test de diagnostic rapide de Y. pestis dans différents fluides et échantillons biologiques. Yersinia pseudotuberculosis et Yersinia enterocolitica sont présentes dans le monde entier et sont transmises par contamination à partir de viande de porc mal cuite, de lait ou produits laitiers et de végétaux, ou par contact avec des animaux porteurs sauvages ou domestiques. Une transmission interhumaine par voie fécale-orale est également possible. Ces bactéries sont responsables très fréquemment d’infections entériques. Cependant leur recherche dans les coprocultures n’est pas réalisée de façon systématique en laboratoires d’analyses médicales du fait de leur croissance lente et difficile sur les milieux usuels, ce qui rend leur isolement à partir de fèces difficile. De plus, les procédures de routine sont coûteuses et longues. Cela entraine probablement une sous-estimation de l’incidence des infections à Yersinia entéropathogènes, la prescription de traitements non adaptés et la réalisation d’appendicectomies non nécessaires, d’où la nécessité de développer des tests de diagnostic rapides, spécifiques, sensibles et faciles à utiliser. Un des objectifs de cette thèse était de produire un panel d‘anticorps monoclonaux murins contre les principaux biotypes et sérotypes pathogènes de Y. pseudotuberculosis et Y. enterocolitica pour le développement de tests de diagnostic immunologiques (ELISA et tests bandelettes) répondant aux caractéristiques recherchées et utilisables directement avec des échantillons biologiques humains. / Three bacteria of the genus Yersinia are pathogenic for the human: Yersinia pestis (the plague bacillus) and the enteropathogenic bacteria: Yersinia pseudotuberculosis and Yersinia enterocolitica. Yersinia pestis is responsible for more than 20,000 human cases of plague declared to the World Health Organization (WHO) during the ten last years in different areas from Africa, Asia and America. Mistakenly considered today as a disease from the past, on the contrary, the plague is re-emerging. Even if it doesn’t occur as a massive epidemic, it still lays down a challenge to the world for its important severity, its quick spreading, the appearance of antimicrobial resistance and a potential use for terrorism. Under the circumstances, the immunotherapy against Y. pestis could be a good option to treat bubonic and pneumonic plague. One the aims of this thesis was to produce murine monoclonal antibodies against the three proteins of the injectisom (YscF, YscC, LcrV), a key virulence factor of Yersinia. The obtained antibodies were characterized and for certain, the epitopes were identified. Then, in collaboration with Elisabeth Carniel from Institut Pasteur, their therapeutic effect was evaluated in vivo with a bubonic plague model in mice. The antibodies generated against the proteins from the injectisom are now evaluated in a diagnosis test for a fast detection of Y. pestis in different biological samples. Yersinia enterocolitica and Yersinia pseudotuberculosis, the two enteropathogenic Yersinia species for humans, have a worldwide distribution and are among the most frequent agents of human diarrhea in temperate and cold countries. However, research of enteropathogenic Yersinia is not consistently performed in medical laboratories because of their specific growth characteristics, which makes their isolation from the stool samples difficult. Moreover, current procedures for isolation are expensive and time consuming, which leads to underestimation of the incidence of yersiniosis and prescriptions of inappropriate antibiotic treatments. One the aims of this thesis was to produce different murine monoclonal antibodies against the main pathogenic biotypes and serotypes of Y. pseudotuberculosis and Y. enterocolitica for the development of fast, sensitive, specific and easy-to-use immunoassays (ELISA and dipsticks), useful for both human and veterinary diagnosis.

Listeria monocytogenes e Yersinia enterocolitica são agentes zoonóticos e têm capacidade de transmissão através dos alimentos, inclusive carne suína. O presente estudo avaliou, mensalmente, de maio de 2007 a abril de 2008, alguns pontos da cadeia produtiva da carne suína em abatedouros e açougues do Estado de São Paulo. Foram avaliados ambientes dos estabelecimentos visitados e amostras de línguas, tonsilas e cortes de carne suína. Listeria monocytogenes foi isolada de todos os tipos de amostra, com presença dos sorotipos 4b, 1/2b, 1/2a e 1/2c. Estes isolados demonstraram grande similaridade, sugerindo até que haja persistência do agente em ambiente, de acordo com a PFGE, reforçando seu potencial de transmissão para humanos. Yersinia enterocolitica 4/ O:3 foi detectada exclusivamente em abatedouros, principalmente nos animais, apresentando, portanto, menor potencial de transmissão para humanos. Entretanto, Yersinia enterocolitica 1A, considerada não patogênica, foi isolada de todos os tipos de amostras, e a maioria apresentou fatores de virulência, devendo este fato ser melhor investigado. Os resultados apresentados indicam a necessidade de se tomar medidas para controle e prevenção da disseminação dos agentes, principalmente da Listeria monocytogenes. / Listeria monocytogenes and Yersinia enterocolitica are zoonotic agents with capacity of transmission through foods, including pork. The present study evaluated, monthly, from May 2007 to April 2008, several points of swine meat productive chain in slaughterhouses and butcheries from Sao Paulo state. It were evaluated the environments of visited establishments and samples of tongues, tonsils and pork cuts. Listeria monocytogenes was isolated from all kinds of samples and the serotypes 4b, 1/2b, 1/2a and 1/2c were present. These isolates showed great similarity, even suggesting that there was persistence of the agent in the environment, according to the PFGE, reinforcing its potential of transmission to humans. Yersinia enterocolitica 4/ O:3 was exclusively detected in slaughterhouses, mainly in the animals, presenting therefore, low potential of transmission to humans. However, Yersinia enterocolitica 1A, considered no pathogenic, was isolated from all kinds of samples, and the majority presented virulence factors, suggesting that this observation should be better investigated. The results presented indicate the necessity of measures of control and prevention of agents dissemination, mainly of Listeria monocytogenes.

RfaH is a unique bacterial protein that enhances transcription of a select group of long operons in many Gram-negative bacteria. Operons regulated by RfaH possess an upstream operon polarity suppressor sequence, which recruits the RfaH protein to the RNA polymerase during transcription of genes, most of which are involved in the synthesis of cell-surface features. These include synthesis of the lipopolysaccharide (LPS) core and O-antigen in Salmonella and Escherichia coli, as well as F-plasmid conjugation pilus and capsule in E. coli. LPS is an important virulence factor in many Gram-negative bacteria, and protects Y. pseudotuberculosis against host antimicrobial chemokines. Recently published high-throughput transposon mutant screens have also suggested a role for RfaH in the ability of Y. pseudotuberculosis to colonize mice. However, the role of RfaH in Y. pseudotuberculosis and its descendent Yersinia pestis has not been carefully examined. In these studies we investigated the effect RfaH has on the structure of the LPS in both species at different temperatures. We also identified LPS-synthesis related genes that are regulated by RfaH. We determined the effect of RfaH on bacterial resistance to host defense peptides, and the ability of Y. pseudotuberculosis to colonize mice. We found that the loss of the rfaH gene had different effects in Y. pseudotuberculosis and Y. pestis. Loss of rfaH caused a truncation in the core region in Y. pseudotuberculosis strain IP32953 at both 21°C and 37°C, but only at 37°C in Y. pestis strain KIM6+. Similarly, we found that transcription of individual genes that are predicted to function in core or O-antigen synthesis were downregulated in the rfaH mutant strains in both species, but the impact of rfaH deletion was greater in Y. pseudotuberculosis. When tested for their ability to survive in the presence of antimicrobial peptides, the Y. pseudotuberculosis rfaH deficient bacteria were much more susceptible than wild-type to killing by polymyxin and by the antimicrobial chemokine CCL28. However, the Y. pestis rfaH mutant strain was equally susceptible to CCL28 as the wild-type strain. Infection of mice with Y. pseudotuberculosis show that rfaH deficient bacteria were able to survive as effectively as the wild-type following oral or intravenous inoculation, with or without the pYV virulence plasmid. Overall, our results show that while RfaH controls LPS gene expression in both Y. pseudotuberculosis and Y. pestis, its impact is much greater in Y. pseudotuberculosis. Furthermore, although loss of rfaH greatly reduces the ability of Y. pseudotuberculosis to resist antimicrobial peptides, it is not required for virulence in this species.

Yersinia enterocolitica es un patógeno Gram-negativo que provoca diversos síndromes gastrointestinales. La cadena O es el componente más expuesto al exterior del lipopolisacárido y se ha descrito como un importante factor de virulencia. En Y. enterocolitica O:8 (YeO8) la expresión de la cadena O está coordinada con la de otros factores de virulencia de Yersinia, como inv, importante en el proceso de colonización intestinal de la bacteria, y flhDC, el principal operón de regulación del flagelo. En esta Tesis Doctoral se ha demostrado como la ausencia de la cadena O también altera la expresión del sistema de secreción tipo III (SSTT) Ysc, otro importante factor de virulencia de Y. enterocolitica, en un proceso dependiente de flhDC. Además, se han descrito las bases moleculares que explican como la ausencia de cadena O determina la disminución de la expresión de inv, la disminución de la secreción de Yops por el SSTT Ysc y un aumento de la motilidad de la bacteria en un circuito de regulación en el que están implicados el operón sspAB, la proteasa ClpXP, el regulador transcripcional H-NS y flhDC.

Listeria monocytogenes e Yersinia enterocolitica são agentes zoonóticos e têm capacidade de transmissão através dos alimentos, inclusive carne suína. O presente estudo avaliou, mensalmente, de maio de 2007 a abril de 2008, alguns pontos da cadeia produtiva da carne suína em abatedouros e açougues do Estado de São Paulo. Foram avaliados ambientes dos estabelecimentos visitados e amostras de línguas, tonsilas e cortes de carne suína. Listeria monocytogenes foi isolada de todos os tipos de amostra, com presença dos sorotipos 4b, 1/2b, 1/2a e 1/2c. Estes isolados demonstraram grande similaridade, sugerindo até que haja persistência do agente em ambiente, de acordo com a PFGE, reforçando seu potencial de transmissão para humanos. Yersinia enterocolitica 4/ O:3 foi detectada exclusivamente em abatedouros, principalmente nos animais, apresentando, portanto, menor potencial de transmissão para humanos. Entretanto, Yersinia enterocolitica 1A, considerada não patogênica, foi isolada de todos os tipos de amostras, e a maioria apresentou fatores de virulência, devendo este fato ser melhor investigado. Os resultados apresentados indicam a necessidade de se tomar medidas para controle e prevenção da disseminação dos agentes, principalmente da Listeria monocytogenes. / Listeria monocytogenes and Yersinia enterocolitica are zoonotic agents with capacity of transmission through foods, including pork. The present study evaluated, monthly, from May 2007 to April 2008, several points of swine meat productive chain in slaughterhouses and butcheries from Sao Paulo state. It were evaluated the environments of visited establishments and samples of tongues, tonsils and pork cuts. Listeria monocytogenes was isolated from all kinds of samples and the serotypes 4b, 1/2b, 1/2a and 1/2c were present. These isolates showed great similarity, even suggesting that there was persistence of the agent in the environment, according to the PFGE, reinforcing its potential of transmission to humans. Yersinia enterocolitica 4/ O:3 was exclusively detected in slaughterhouses, mainly in the animals, presenting therefore, low potential of transmission to humans. However, Yersinia enterocolitica 1A, considered no pathogenic, was isolated from all kinds of samples, and the majority presented virulence factors, suggesting that this observation should be better investigated. The results presented indicate the necessity of measures of control and prevention of agents dissemination, mainly of Listeria monocytogenes.

Yersinia enterocolitica subsp. palearctica Serobiotyp O:3/4 ist verantwortlich für 80-90 % aller Yersiniosen beim Menschen in Deutschland und Europa. Y. enterocolitica Infektionen zeigen vielfältige Krankheitsbilder wie Gastroenteritis, Lymphadenitis und verschiedene Spätkomplikationen wie reaktive Arthritis. Das wichtigste Tierreservoir stellt das Hausschwein dar. Rohes Schweinefleisch in Metzgereien in Deutschland und anderen Regionen in Nord-Ost Europa ist häufig mit Yersinien kontaminiert (Bayern: 25 %). Da sich Serobiotyp O:3/4-Stämme geografisch und phylogenetisch deutlich von dem bisher sequenzierten Serobiotyp O:8/1B Stamm 8081 unterscheiden, wurde eine komplette Genomsequenzierung des europäischen Serobiotyp O:3/4 DSMZ Referenzstammes Y11 (aus Patientenstuhl isoliert) durchgeführt. Um einen genaueren Einblick in die Y. enterocolitica subsp. palearctica Gruppe zu erhalten, wurden zusätzlich zwei weitere Serobiotyp O:3/4 Isolate (Stamm Y8265, Patientenisolat, und Stamm Y5307, mit reaktiver Arthritis assoziiertes Patientenisolat), sowie ein eng verwandtes Y. enterocolitica subsp. palearctica Serobiotyp O:5,27/3 Isolat, Stamm Y527P, und zwei Biotyp 1A Isolate (ein Isolat nosokomialer Herkunft (Serogruppe O:5) und ein Umwelt-Isolat (O:36)) unvollständig sequenziert. Die nicht mausvirulenten Stämme wurden mit dem mausvirulenten Y. enterocolitica subsp. enterocolitica Serobiotyp O:8/1B Stamm 8081 verglichen, um genetische Besonderheiten von Stamm Y11 und der Y. enterocolitica subsp. palearctica Gruppe zu identifizieren. Besonderer Fokus lag hierbei auf dem pathogenen Potential von Stamm Y11, um neue potentielle Virulenz Faktoren und Fitnessfaktoren zu identifizieren, darunter vor allem solche, die eine Rolle bei der Wirtsspezifität von Serobiotyp O:3/4 spielen könnten. Y. enterocolitica subsp. palearctica Serobiotyp O:3/4 Stämmen fehlen einige der Charakteristika der mausvirulenten Gruppe Y. enterocolitica subsp. enterocolitica, beispielsweise die Yersiniabactin kodierende‚ High-Pathogenicity Island (HPI), das Yts1 Typ 2 Sekretionssystem und das Ysa Typ 3 Sekretionssystem. Die Serobiotyp O:3/4-Stämme haben ein anderes Repertoir von Virulenz Faktoren erworben, darunter Gene bzw. genomische Inseln für das Ysp Typ 3 Sekretionssystem, Rtx-ähnliches putatives Toxin, Insektizid-Toxine und ein funktionelles PTS System für die Aufnahme von N-acetyl-galactosamin, dem aga-Operon. Nach dem Transfer des aga-Operons in Y. enterocolitica subsp. enterocolitica O:8/1B konnte Wachstum auf N-acetyl-galactosamin festgestellt werden. Neben diesen Genen können möglicherweise auch zwei Prophagen (PhiYep-2 und PhiYep-3) und eine asn tRNA assoziierte genomische Insel (GIYep-01) zur Pathoadaptation von Y. enterocolitica subsp. palearctica Serobiotyp O:3/4 beitragen. Der PhiYep-3 Prophage und die GIYep-01 Insel weisen Rekombinationsaktivität auf, und PhiYep-3 wurde nicht in allen untersuchten Serobiotyp O:3/4 Stämmen gefunden. Y. enterocolitica subsp. palearctica Serobiotyp O:5,27/3 Stamm Y527P ist genetisch eng verwandt zu allen Serobiotyp O:3/4 Isolaten, wohingegen die Biotyp 1A Isolate ein mehr Mosaik-artiges Genom aufweisen und potentielle Virulenzgene sowohl mit Serobiotyp O:8/1B als auch O:3/4 gemeinsam haben, was einen gemeinsamen Vorfahren impliziert. Neben dem pYV Virulenz-Plasmid fehlen den Biotyp 1A Isolaten klassische Virulenzmarker wie das Ail Adhesin, das YstA Enterotoxin und das Virulenz-assoziierte Protein C (VapC). Interessanterweise gibt es keine beträchtlichen Unterschiede zwischen den bekannten Virulenzfaktoren des nosokomialen Isolats und dem Umweltisolat der Biotyp 1A-Gruppe, abgesehen von einem verkürzten Rtx Toxin-ähnlichem Genkluster und Überresten eines P2-ähnlichen Phagen im Krankenhausisolat der Serogruppe O:5. / Yersinia enterocolitica subsp. palearctica serobiotype O:3/4 comprises about 80-90 % of all human patient isolates in Germany and Europe and is responsible for sporadic cases worldwide. Even though this serobiotype is low pathogenic, Y. enterocolitica subsp. palearctica serobiotype O:3/4 is involved in gastroenteritis, lymphadenitis and various extraintestinal sequelae as reactive arthritis. The main animal reservoir of this serobiotype are pigs, causing a high rate of O:3/4 contaminations of raw pork in butcher shops in Germany (e.g. Bavaria 25 %) and countries in north-east Europe. As Y. enterocolitica O:3/4 is geographically and phylogenetically distinct from the so far sequenced mouse-virulent O:8/1B strain, complete genome sequencing has been performed for the European serobiotype O:3/4 DSMZ reference strain Y11, which has been isolated from a patient stool. To gain greater insight into the Y. enterocolitica subspecies palearctica group, also draft genome sequences of two other human O:3/4 isolates (strains Y8265, patient isolate, and Y5307, patient isolate associated with reactive arthritis), a closely related Y. enterocolitica palearctica serobiotype O:5,27/3 (strain Y527P), and two biotype 1A strains (a nosocomial strain of serogroup O:5 and an environmental serogroup O:36 isolate) have been performed. Those strains were compared to the high-pathogenic Y. enterocolitica subsp. enterocolitica serobiotype O:8/1B strain 8081 to address the peculiarities of the strain Y11 and the Y. enterocolitica subspecies palearctica group. The main focus was to unravel the pathogenic potential of strain Y11 and thus to identify novel putative virulence genes and fitness factors, especially those that may constitute host specificity of serobiotype O:3/4. Y. enterocolitica subspecies palearctica serobiotype O:3/4 strains lack most of the mouse-virulence-associated determinants of Y. enterocolitica subsp. enterocolitica serotype O:8, for example the HPI, Yts1 type 2 and Ysa type three secretion systems. In comparison, serobiotype O:3/4 strains obviously acquired a different set of genes and genomic islands for virulence and fitness such as the Ysp type three secretion system, an RtxA-like putative toxin, insecticidal toxins and a functional PTS system for N-acetyl-galactosamine uptake, named aga-operon. The aga-operon is able to support the growth of the Y. enterocolitica subsp. enterocolitica O:8/1B on N-acetyl-galactosamine after transformation with the aga operon. Besides these genes, also two prophages, PhiYep-2 and PhiYep-3, and a asn tRNA-associated GIYep-01 genomic island might influence the Y. enterocolitica subsp. palearctica serobiotype O:3/4 pathoadaptation. The PhiYep-3 prophage and the GIYep-01 island show recombination activity and PhiYep-3 was not found in all O:3/4 strains of a small strain collection tested. Y. enterocolitica subsp. palearctica serobiotype O:5,27/3 strain Y527P was found to be closely related to all serobiotype O:3/4 strains, whereas the biotype 1A isolates have more mosaic-segmented genomes and share putative virulence genes both with serobiotypes O:8/1B and O:3/4, which implies their common descent. Besides the pYV virulence plasmid, biotype 1A strains lack classical virulence markers as the Ail adhesin, the YstA enterotoxin, and the virulence-associated protein C. Interestingly, there are no notable differences between the known virulence factors present in nosocomial and environmental strains, except the presence of a truncated Rtx toxin-like gene cluster and remnants of a P2-like prophage in the hospital serogroup O:5 isolate.

Dissertation (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: pp. 221-242.