En esta Tesis se estudia el posible papel de las FLA como reservorio de H. pylori y otras bacterias patógenas en aguas y alimentos mediante técnicas moleculares. En primer lugar se realizó un ensayo de cocultivo entre la bacteria H. pylori y la ameba Acanthamoeba castellanii. Se comprobó, mediante técnicas moleculares específicas para la detección de células viables, PMA-qPCR y DVC-FISH, que la ameba es capaz de internalizar a la bacteria y que esta última permanece viable, demostrando que H. pylori se comporta como una bacteria ARB. Seguidamente, se analizaron un total de 120 muestras ambientales, 100 de agua y 20 de vegetales para comprobar la presencia, tanto de FLA como de H. pylori internalizado en estas FLA. En el caso de las muestras de agua, se analizaron 69 muestras de agua residual y 31 de agua potable. Un total de 55 (79,7%) muestras de agua residual y 12 (38,7%) de agua potable resultaron positivas para la presencia de FLA. Mediante la técnica PMA-qPCR se demostró la presencia de H. pylori internalizado en las FLA presentes en 28 (50,9%) y 11 (91,7%) de las muestras de agua residual y potable analizadas, respectivamente. Mediante DVC-FISH se demostró que las células de H. pylori internalizadas dentro de las FLA presentes en las muestras eran viables en 16 (29,5%) y 5 (41,7%) de las muestras de agua residual y potable analizadas, respectivamente. Además, se consiguió recuperar formas viables cultivables de H. pylori procedente del interior de FLA en 10 (18,2%) de las muestras de agua residual analizadas. Las FLA aisladas e identificadas en las aguas residuales pertenecieron a los géneros Acanthamoeba, Naegleria, Vanellidae y a la familia Vahlkampfiidae. En el caso del agua potable, las FLA aisladas e identificadas pertenecieron a los géneros Acanthamoeba, Echinamoeba y Vermamoeba. En el caso de las muestras de vegetales, concretamente lechugas, todas ellas resultaron positivas para el aislamiento de FLA (100%). Mediante la técnica PMA-qPCR se demostró la presencia de H. pylori internalizado en las FLA en 11 (55,0%) de las muestras y, mediante DVC-FISH, se demostró que las células de H. pylori internalizadas dentro de las FLA eran viables en 5 (25,0%) de las muestras. En este caso no se recuperaron formas viables cultivables de la bacteria. Finalmente, mediante metagenómica de secuenciación dirigida, se analizó el microbioma de las FLA presentes en 20 de las muestras analizadas en esta Tesis (11 de agua residual, 3 de agua potable y 6 de lechugas). Para ello, se eligieron los iniciadores, se evaluaron in silico e in vitro y, una vez comprobada su idoneidad, se emplearon para la secuenciación de las muestras. En los tres tipos de muestras, la clase bacteriana más abundante fue la Gammaproteobacteria. Para los tres tipos de muestras, los filos más abundantes de las bacterias del microbioma de las FLA fueron Proteobacteria y Bacteroidetes y, en el caso del agua residual, también lo fue el filo Planctomycetes. H. pylori se detectó mediante esta técnica en los tres tipos de muestra. Además, como parte del microbioma de FLA de muestras ambientales, se detectaron otras bacterias de interés para la salud pública, tales como Aeromonas, Legionella, Mycobaterium o Pseudomonas. Los resultados obtenidos en esta Tesis demuestran la presencia de FLA patógenas en las muestras ambientales, así como el hecho de que, en algunos casos, estas son transportadoras de bacterias patógenas. Este trabajo también confirma que H. pylori se comporta como una bacteria ARB y que se encuentra viable en el interior de FLA presentes, tanto en aguas residuales y potables como en vegetales. De esta forma, se postula que un modo de transmisión de esta bacteria podría ser a través de las FLA presentes en agua o vegetales. / In this Thesis, the possible role of FLA is studied as a reservoir of H. pylori and other pathogenic bacteria in waters and food by means of molecular techniques. Firstly, a coculture assay between the bacterium H. pylori and the amoeba Acanthamoeba castellanii was carried out. It was verified by means of molecular techniques specific for the detection of viable cells, PMA-qPCR and DVC-FISH, that the amoeba is capable of internalizing the bacterium and that the latter remains viable, demonstrating that H. pylori behaves as an ARB bacterium. Afterwards, a total of 120 environmental samples, 100 of water and 20 of vegetables, were analyzed to verify the presence FLA as well as internalized H. pylori into these FLA. In case of water samples, 69 samples of wastewater and 31 samples of drinking water were analyzed. A total of 55 (79,7 %) wastewater and 12 (38,7 %) of drinking water samples turned out to be positive for FLA's presence. By means of PMA-qPCR technique, the presence of FLA-internalized H. pylori was demonstrated in 28 (50,9 %) and 11 (91,7 %) of the wastewater and drinking water samples analyzed, respectively. By means of DVC-FISH it was demonstrated that the FLA-internalized H. pylori cells were viable in 16 (29,5 %) and 5 (41,7 %) of the wastewater and drinking water samples analyzed, respectively. In addition, viable cultivable forms of H. pylori coming from the inside of FLA were recovered from 10 (18,2 %) of the analyzed wastewater samples. The isolated and identified FLA from wastewater samples belonged to the genus Acanthamoeba, Naegleria, Vanellidae and to the family Vahlkampfiidae. In the case of drinking wáter, the isolated and identified FLA belonged to the genus Acanthamoeba, Echinamoeba and Vermamoeba. In the case of the vegetable samples, specifically lettuces, all of them turned out to be positive for FLA's isolation (100 %). By means of the PMA-qPCR technique, the presence of FLA-internalized H. pylori was demonstrated in 11 (55,0 %) of the samples and, by means of DVC-FISH, it was demonstrated that FLA-internalized H. pylori cells were viable in 5 (25,0 %) of the samples. In this case, viable cultivable forms of the bacterium could not be recovered. Finally, by means of amplicon-based metagenomics, the FLA microbiome of 20 previously analyzed samples in this Thesis (11 wastewater, 3 drinking water and 6 lettuce samples) was analyzed. To do so, a pair of primers were selected and evaluated in silco and in vitro and, once checked its suitability, they were used to perform the samples' sequencing. In the three types of samples, the most abundant bacterial class was the Gammaproteobacteria. For the three types of samples, the most abundant bacterial phylum of the FLA microbiome were Proteobacteria and Bacteroidetes and, in case of the wastewater, it was also the phylum Planctomycetes. H. pylori was detected by means of this technology in the three types of samples. In addition, as part of FLA's microbiome of environmental samples, other bacteria of public health interest were detected, such as Aeromonas, Legionella, Mycobacterium or Pseudomonas. The results obtained in this Thesis demonstrate the presence of pathogenic FLA in the environmental samples, as well as the fact that, in some cases, these they are carriers of pathogenic bacteria. This work also confirms that H. pylori behaves as an ARB bacterium and that it is viable inside the present FLA in wastewater as well as in drinking water and in vegetables. This way, it is postulated that a way of transmission of this bacterium might be through the FLA present in water or vegetables. / En esta Tesi s'estudia el possible paper de les FLA com a reservori de H. pylori i altres bacteris patògens en aigües i aliments per mitjà de tècniques moleculars. En primer lloc es va realitzar un assaig de cocultiu entre el bacteri H. pylori i l'ameba Acanthamoeba castellanii. Es va comprovar per mitjà de tècniques moleculars específiques per a la detecció de cèl¿lules viables, PMA-qPCR i DVC-FISH, que l'ameba és capaç d'internalizar al bacteri i que esta última roman viable, demostrant que H. pylori es comporta com un bacteri ARB. A continuació, es van analitzar un total de 120 mostres ambientals, 100 d'aigua i 20 de vegetals per a comprovar la presència tant de FLA com de H. pylori internalitzat en estes FLA. En el cas de les mostres d'aigua, es van analitzar 69 mostres d'aigua residual i 31 d'aigua potable. Un total de 55 (79,7%) mostres d'aigua residual i 12 (38,7%) d'aigua potable van resultar positives per a la presència de FLA. Per mitjà de la tècnica PMA-qPCR es va demostrar la presència d'H. pylori internalitzat en les FLA presents en 28 (50,9%) i 11 (91,7%) de les mostres d'aigua residual i potable analitzades, respectivament. Per mitjà de DVC-FISH es va demostrar que les cèl¿lules d'H. pylori internalitzades dins les FLA presents en les mostres eren viables en 16 (29,5%) i 5 (41,7%) de les mostres d'aigua residual i potable analitzades, respectivament. A més, es va aconseguir recuperar formes viables cultivables d'H. pylori procedent de l'interior de FLA en 10 (18,2%) de les mostres d'aigua residual analitzades. Les FLA aïllades i identificades en les aigües residuals van pertànyer als gèneres Acanthamoeba, Naegleria, Vanellidae i a la família Vahlkampfiidae. En el cas de l'aigua potable, les FLA aïllades i identificades van pertànyer als gèneres Acanthamoeba, Echinamoeba i Vermamoeba. En el cas de les mostres de vegetals, concretament encisams, totes elles van resultar positives per a l'aïllament de FLA (100%). Per mitjà de la tècnica PMA-qPCR es va demostrar la presència d'H. pylori internalitzat en les FLA en 11 (55,0%) de les mostres i, per mitjà de DVC-FISH, es va demostrar que les cèl¿lules d'H. pylori internalitzades dins les FLA eren viables en 5 (25,0%) de les mostres. En este cas no es van recuperar formes viables cultivables del bacteri. Finalment, per mitjà de metagenómica de seqüenciació dirigida, es va analitzar el microbioma de les FLA presents en 20 de les mostres analitzades en esta Tesi (11 d'aigua residual, 3 d'aigua potable i 6 d'encisams). Per tal de fer això, es van triar els iniciadors, es van avaluar in silico i in vitro i, una vegada comprovada la seua idoneïtat, es van emprar per a la seqüenciació de les mostres. En els tres tipus de mostres, la classe bacteriana més abundant va ser la Gammaproteobacteria. Per als tres tipus de mostres, els filos més abundants dels bacteris del microbioma de les FLA van ser Proteobacteria i Bacteroidetes i, en el cas de l'aigua residual, també ho va ser el filo Planctomycetes. H. pylori es va detectar per mitjà d'esta tècnica en els tres tipus de mostra. A més, com a part del microbioma de FLA de mostres ambientals, es van detectar altres bacteris d'interés per a la salut pública, com ara Aeromonas, Legionella, Mycobaterium o Pseudomonas. Els resultats obtinguts en esta Tesi demostren la presència de FLA patògenes en les mostres ambientals, així com el fet de que, en alguns casos, estes són transportadores de bacteris patògens. Este treball també confirma que H. pylori es comporta com un bacteri ARB i que es troba viable en l'interior de FLA presents, tant en aigües residuals i potables com en vegetals. D'esta manera, es postula que una manera de transmissió d'este bacteri podria ser a través de les FLA presents en aigua o vegetals. / Moreno Mesonero, L. (2018). Estudio del papel de las amebas de vida libre como reservorio de Helicobacter pylori y otras bacterias patógenas en aguas y alimentos mediante técnicas moleculares [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/111952

Les facteurs de transcription NikR et FUR sont impliqués dans l'homéostasie des métaux. Les propriétés de métallation et de liaison à l'ADN de NikR d'E.coli (Ec) et d'H.pylori (Hp) ont été comparées. EcNikR, protéine tétramérique, lie 8 nickels par sous unité dont un nickel dans un site dit de haute affinité ayant un Kd submicromolaire. Les autres sites métalliques, de plus basse affinité, sont impliqués dans le processus l'agrégation Ni- et pH-dépendant. HpNikR, partage des propriétés de métallation avec EcNikR, dont la liaison possible de différents métaux dans son site de haute affinité (Cu(II), Ni(II), Co(II)). Les 4 sites de haute affinité de cette protéine semblent égaux 2 à 2. La métallation des premiers sites faciliterait une « fermeture de la protéine » permettant la métallation des deux derniers sites. La métallation de ces 4 sites semble suffire pour qu'HpNikR puisse lier l'ADN.<br />Cependant cette liaison serait améliorée en présence d'un métal stabilisateur dans d'autres sites. Selon les séquences opératrices, le métal et la technique employée, l‘affinité d'HpNikR métallée pour l'ADN varie. En présence d'un excès de Ni(II), HpNikR se lie à pureA et à pnixA avec un Kd de 2,5 et 1,7 nM mais se lie faiblement à pnikR et pexbB. En présence d'un excès de Ni(II) puis de Mn(II), HpNikR se lie à pnikR et à pexbB avec un Kd de 38 et 24 nM. FUR d'H.pylori (HpFUR), un métallorégulateur Fe dépendant, a aussi été caractérisé dans le but d'étudier sa co-régulation avec HpNikR de la région intergénique nikR-exbB. HpFUR est dimérique et contient au moins deux sites métalliques : un site structural et un site de régulation permettant l'activation pour la liaison à l'ADN.

Earlier ecological studies on the relation between H. pylori infection and stomach cancer have considered that the relation between these two variables, as estimated by the exposure coefficient, is constant. However, there is evidence to suggest that this relation changes geographically due to differences in strains of H. pylori. Since the prevalence of H. pylori varies with socio-economic status, the association between the latter and stomach cancer mortality may also vary geographically. This thesis studies stomach cancer by taking into account the geographical variability of the exposure coefficients. The study proposes the use of regression mixtures, clustering models and spatially varying regressions for the study of varying exposure coefficients. The effect of transformations of variables in these models appears to have been little considered. We provide new necessary conditions for invariance under transformations of variables for mixed effect models in general, and for the proposed models in particular. In addition, we show that varying exposure coefficients may induce a varying baseline risk. The regression mixtures and the clustering model are applied to a data set on stomach cancer incidence and H. pylori prevalence in 57 countries worldwide. We extend the clustering model to reflect any distance measure between the geographical units, including the Euclidean distance, in the formation of clusters. We also show that the clustering model performs better than the regression mixture model when the aim is to identify connected clusters and the observations present large variance. The results obtained with the clustering model supported the existence of three clusters where the interaction between the human and H. pylori populations have similar characteristics. Spatially varying regressions are applied to a data set of areal death counts of stomach cancer and spending power in 275 counties in continental Portugal. We provide an original strategy for implementing multivectorial intrinsic autoregressions as the distribution for the random effects. The results obtained with the application of this methodology were consistent with a varying exposure coefficient of spending power.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-04-29T22:35:16Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_CaracteristicasEpidemiologicasRelativas.pdf: 753877 bytes, checksum: 9bc3af6fdd01ee36ff7a2050b8fc7b2b (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-04-30T14:41:59Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_CaracteristicasEpidemiologicasRelativas.pdf: 753877 bytes, checksum: 9bc3af6fdd01ee36ff7a2050b8fc7b2b (MD5) / Made available in DSpace on 2013-04-30T14:41:59Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_CaracteristicasEpidemiologicasRelativas.pdf: 753877 bytes, checksum: 9bc3af6fdd01ee36ff7a2050b8fc7b2b (MD5) Previous issue date: 2009 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A infecção pela Helicobacter pylori é uma das mais comuns em humanos e apesar de possuir tropismo pelo estômago, pode ser encontrada na cavidade oral, mantendo uma relação comensal com o hospedeiro, enquanto a cárie dental também é uma doença infecciosa e resulta do metabolismo da placa bacteriana. Ambas as infecções apresentam alta prevalência em países em desenvolvimento, pois estas populações estão mais expostas a fatores ambientais de risco, e normalmente são adquiridas durante a infância. A prevalência destas infecções foi investigada na cavidade oral de escolares assintomáticos para doenças gástricas, provenientes de uma população de Belém-Pa, relacionando-se a alguns parâmetros de higiene e saúde bucal, condição socioeconômica e fatores de susceptibilidade genética como os grupos sanguíneos ABO e Lewis. Foram investigados 104 indivíduos, com idade média de 17 anos. De todos os participantes foram coletadas amostras de saliva e placa dental. A saliva foi coletada para identificação do estado secretor ABO e Lewis e estimação dos parâmetros salivares, e ambas, saliva e placa dental, foram coletadas para analise molecular dos genes 16S RNAr da H. pylori e FUT2. A H. pylori foi detectada em 79,8% dos escolares, com freqüência de 66,35% na placa dental e 58,65% na saliva. A prevalência de cárie foi de 82,8% na população estudada. A avaliação clínica da saúde bucal mostrou que o CPO-D médio encontrado foi de 3,53. Observou-se que a experiência de cárie tende a aumentar à medida que acresce a idade e que a infecção por H. pylori foi maior na primeira infância. O grau de instrução e o número de visitas ao dentista mostraram diferenças significantes em relação a presença de H. pylori. A distribuição fenotípica dos grupos sanguíneos ABO e Lewis não mostrou diferenças significantes entre indivíduos infectados e não-infectados, que expliquem haver maior susceptibilidade genética para infecção por H. pylori e cárie dental. No conjunto desta analise as elevadas freqüências encontradas denotam a necessidade de cuidados e tratamento das doenças dentais, como a cárie e sugere-se que a H. pylori na cavidade oral pode contribuir para a infecção e re-infecção do estômago após tratamento. / The infection by Helicobacter pylori is one of the most common in humans and despite having tropism by stomach, can be found in the oral cavity, maintaining a commensal relationship with the host, while dental caries is also an infectious disease and results from the metabolism of the bacterial plaque. Both infections are highly prevalent in developing countries, since these populations are more exposed to environmental risk factors, and are usually acquired during childhood. The prevalence of these infections was investigated in the oral cavity of school children with no symptoms of gastric diseases, from a population of Belém-Pa, in relation to some parameters of oral hygiene and health, socioeconomic conditions and genetic susceptibility factors like the ABO and Lewis blood groups. Were investigated 104 patients, with average age of 17 years. Of all the participants were collected saliva samples and dental plaque. Saliva was collected to identify the ABO and Lewis state secretor and estimation of salivary parameters, and both, saliva and plaque samples were collected for molecular analysis of 16S rRNA genes of H. pylori and FUT2. H. pylori was detected in 79.8% of the students, with a frequency of 66.35% in dental plaque and 58.65% in saliva. The caries prevalence was 82.8% in the population studied. The clinical evaluation of oral health showed that the average CPO-D found was 3.53. It was observed that the caries experience tends to increase as in addition to age and the H. pylori infection was higher in early childhood. The education level and number of dentist visits showed significant differences in relation to the presence of H. pylori. The phenotypic distribution of ABO and Lewis blood groups did not differ significantly between infected and uninfected individuals, explaining there is greater genetic susceptibility to infection by H. pylori and dental caries. Throughout this analysis, the high frequencies found prove the need for care and treatment of dental diseases, such as caries and it is suggested that H. pylori in the oral cavity can contribute to infection and re-infection of the stomach after treatment.

Orientador: Ana Elizabete Silva / Banca: Maria Inês Moura Pardini / Banca: Fátima Pereira de Souza / Resumo: O câncer gástrico é a segunda causa de morte por câncer no mundo e o quinto tipo com maior prevalência no Brasil, sendo previstos 21.800 casos novos em 2008. Esta neoplasia apresenta etiologia bastante complexa, envolvendo fatores genéticos e ambientais. Os fatores etiológicos de maiores destaques incluem a infecção pela bactéria Helicobacter pylori, a ingestão de determinados alimentos, como defumados, enlatados e com elevada quantidade de sal, além do estilo de vida dos indivíduos, associado ao consumo de cigarro e álcool. Uma lesão pré-cancerosa importante no desenvolvimento da neoplasia gástrica é a metaplasia intestinal, podendo aumentar o seu risco em até 10 vezes. Atualmente é reconhecida a participação de alterações epigenéticas como metilação aberrante do DNA, que atua de forma igualmente relevante e complementar no processo de desenvolvimento e progressão do câncer. Vários genes com papel importante no controle do ciclo celular, reparo do DNA, apoptose, angiogênese e adesão celular podem apresentar expressão alterada devido metilação aberrante de sua região promotora, assim a investigação do padrão de metilação de genes envolvidos com o processo neoplásico pode ser uma estratégia interessante para a indicação de marcadores moleculares que possam auxiliar no diagnóstico precoce do câncer. Desta forma, no presente trabalho foi investigado o padrão de metilação dos genes WT1 e RARß em metaplasia intestinal (35 amostras) e suas respectivas mucosas gástricas normais, em comparação com o câncer gástrico (8 amostras) também com suas respectivas mucosas normais, através da técnica MS-PCR (Methylation Specific PCR). Devido à participação da infecção pela H. pylori na carcinogênese gástrica, foi investigada molecularmente a presença dessa bactéria nas amostras...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Worldwide, the gastric cancer is the second cause of death by cancer. In Brazil, it is the fifth type with more abundant, foreseen 21.800 new cases in 2008. This neoplasia presents very complex etiology involving genetic and environmental factors. The main etiologic factors include: infection by H. pylori, intake of specific foods such as curing food, canned food, and high consumption of salt wealthy food, besides people life style associated to alcohol and cigarette consumptions. An important previous-cankered lesion in development of gastric neoplasia is the intestinal metaplasia, what can increase your risk in ten times. At this moment, it is recognized the participation of epigenetic alterations like ADN aberrant methylation, which actuate in a same way considerable and complementary in development process and cancer evolution. Many genes with important role in control of cellular cycle, ADN repair, apoptosis, angiogenesis and cellular adhesion can present changed expression due aberrant metthylation of your promoter region. In this manner, the investigation of metithyation pattern of genes involved with the neoplasic process can be an interesting strategy for the indication of molecular markers that can help in cancer precocious diagnosis. Thus, in this present study were investigated the metthylation pattern of RARß and WT1genes (35 samples) and their respective normal mucous gastrics by technic MSPCR (Methylation Specific PCR). Due to participation of infection by H. pylori in gastric carcinogenesis, it was too molecular investigated the presence of this bacterium in the studied samples and the possible association with the metthylation pattern presented by both genes. The results showed high pattern of methylation in both valued lesions, that is, 97% and 100%, respectively of methylated samples in metaplasia group ...(Complete abstract click electronic access below) / Mestre

<p>Antibiotic resistance is of current concern. Bacteria have become increasingly resistant to commonly used antibiotics and we are facing a growing resistance problem. The present thesis was aimed at studying the impact of antibiotic treatment on pathogenic bacteria as well as on the normal human microbiota, with focus on resistance development.</p><p>Among the factors that affect the appearance of acquired antibiotic resistance, the mutation frequency and biological cost of resistance are of special importance. Our work shows that the mutation frequency in clinical isolates of <i>Helicobacter pylori</i> was generally higher than for other studied bacteria such as <i>Enterobacteriaceae; </i>¼ of the isolates displayed a mutation frequency higher than<i> Enterobacteriaceae </i>defective<i> </i>mismatch repair mutants and could be regarded as mutator strains.</p><p>In <i>H. pylori</i>, clarithromycin resistance confers a biological cost, as measured by decreased competitive ability of the resistant mutants in mice. In clinical isolates, this cost could be reduced, consistent with compensatory evolution stabilizing the presence of the resistant phenotype in the population. Thus, compensation is a clinically relevant phenomenon that can occur in vivo.</p><p>Furthermore, our results show that clinical use of antibiotics selects for stable resistance in the human microbiota. This is important for several reasons. First, many commensals occasionally can cause severe disease, even though they are part of the normal microbiota. Therefore, stably resistant populations increase the risk of unsuccessful treatment of such infections. Second, resistance in the normal microbiota might contribute to increased resistance development among pathogens by interspecies transfer of resistant determinants.</p>

<p>In recent years molecular biology has become an integral part of the clinical laboratory. With an ever increasing number of methodologies and applications being presented each year it has increased our knowledge of how bacteria cause disease as well as our ability to predict disease outcome. </p><p>The main focus of this thesis has been to develop methods for identifying biomarkers and prediction methods for bacterial infectious diseases by taking advantage of the ever increasing possibilities of molecular biology. We applied cutting edge techniques in order to establish novel platforms for identifying and characterizing biomarkers of disease. </p><p>In paper one we describe a novel approach to measure levels of antibiotic resistance and viability of C. trachomatis, a method that is a clear improvement over existing techniques. In the second paper we describe the development of two assays designed to type pertussis toxin subunit 1 in circulating strains, in order to facilitate multi center studies for vaccine escape surveillance. In paper three we develop a novel microarray application designed to identify a large number of bacterial traits of H. pylori simultaneously with human genetic polymorphisms in order to identify a collection of risk factors that could be used as a prediction tool for gastric cancer risk. In the last paper we define the “antigenome” of H. pylori and identified 14 promising, previously unreported antigens as well as a number of potential biomarkers.</p><p>The platform technologies described in this collection of papers will hopefully help us identifying novel ways of fighting and predicting bacterial disease in future studies. </p>

Antibiotic resistance is of current concern. Bacteria have become increasingly resistant to commonly used antibiotics and we are facing a growing resistance problem. The present thesis was aimed at studying the impact of antibiotic treatment on pathogenic bacteria as well as on the normal human microbiota, with focus on resistance development. Among the factors that affect the appearance of acquired antibiotic resistance, the mutation frequency and biological cost of resistance are of special importance. Our work shows that the mutation frequency in clinical isolates of Helicobacter pylori was generally higher than for other studied bacteria such as Enterobacteriaceae; ¼ of the isolates displayed a mutation frequency higher than Enterobacteriaceae defective mismatch repair mutants and could be regarded as mutator strains. In H. pylori, clarithromycin resistance confers a biological cost, as measured by decreased competitive ability of the resistant mutants in mice. In clinical isolates, this cost could be reduced, consistent with compensatory evolution stabilizing the presence of the resistant phenotype in the population. Thus, compensation is a clinically relevant phenomenon that can occur in vivo. Furthermore, our results show that clinical use of antibiotics selects for stable resistance in the human microbiota. This is important for several reasons. First, many commensals occasionally can cause severe disease, even though they are part of the normal microbiota. Therefore, stably resistant populations increase the risk of unsuccessful treatment of such infections. Second, resistance in the normal microbiota might contribute to increased resistance development among pathogens by interspecies transfer of resistant determinants.

In recent years molecular biology has become an integral part of the clinical laboratory. With an ever increasing number of methodologies and applications being presented each year it has increased our knowledge of how bacteria cause disease as well as our ability to predict disease outcome. The main focus of this thesis has been to develop methods for identifying biomarkers and prediction methods for bacterial infectious diseases by taking advantage of the ever increasing possibilities of molecular biology. We applied cutting edge techniques in order to establish novel platforms for identifying and characterizing biomarkers of disease. In paper one we describe a novel approach to measure levels of antibiotic resistance and viability of C. trachomatis, a method that is a clear improvement over existing techniques. In the second paper we describe the development of two assays designed to type pertussis toxin subunit 1 in circulating strains, in order to facilitate multi center studies for vaccine escape surveillance. In paper three we develop a novel microarray application designed to identify a large number of bacterial traits of H. pylori simultaneously with human genetic polymorphisms in order to identify a collection of risk factors that could be used as a prediction tool for gastric cancer risk. In the last paper we define the “antigenome” of H. pylori and identified 14 promising, previously unreported antigens as well as a number of potential biomarkers. The platform technologies described in this collection of papers will hopefully help us identifying novel ways of fighting and predicting bacterial disease in future studies.

Background: Helicobacter pylori (H. pylori) infection is a significant risk factor for peptic ulcer (PU) and gastric cancer (GC). Apart from the virulent CagA genotype of H. pylori environmental and dietary factors influence disease outcomes. There have been no studies addressing these factors in Western India. Hence, we conducted a case control study enrolling PU, GC patients and controls at Pune, India. Methods: Risk factors for PU and H. pylori infection were assessed in participant interview. H. pylori status was assessed from stool by monoclonal antigen detection. To understand treatment effect, we followed 100 H. pylori positive patients. Results: We enrolled 190 PU patients, 125 Controls and 35 GU patients. Prevalence of H. pylori was 61% among symptomatic patients and 45% among controls. H. pylori infection (OR: 1.70, 95% CI: 1.03-2.89), meat (OR: 1.10, 95% CI: 1.02-1.75), fish (OR: 1.05, 95% CI: 1.02-1.89) consumption, and family history of ulcer (OR: 1.20, 95% CI: 1.08-1.60) were risk factors for PU. Consumption of snacks with alcohol (OR: 0.32, 95% CI: 0.13-0.78) and history of anti-parasite treatment (OR: 0.51, 95% CI: 0.30-0.86) were protective factors against PU. Lower socioeconomic status (SES) (OR: 1.10, 95% CI: 1.02-1.39), meat consumption (OR: 2.35, 95% CI: 1.30-4.23), smoking (OR: 2.23, 95% CI: 1.24-4.02), eating restaurant food thrice per week (OR: 3.77, 95% CI: 1.39-10.23) and drinking non-filtered or non-boiled water (OR: 1.05, 95% CI: 1.01-1.23) were risk factors for H. pylori infection. Consumption of chili peppers (OR: 0.20, 95% CI: 0.10-0.37) and concurrent parasite infestation (OR: 0.44, 95% CI: 0.24-0.80) were protective against H. pylori infection. H. pylori infection was eradicated only in 53% (40/75) of treated patients. Conclusion: This study indicates that H. pylori infection is associated PU. Consumption of meat, fish and family history of PU are risk factors for PU. Lower SES, consumption of restaurant food, meat, non filtered water and smoking are risk factors for H. pylori infection. Consumption of chili peppers and concurrent parasite infestation are protective against H. pylori infectionwhile history of anti parasite treatment protects against PU. H. pylori were eradicated only in 53% of patients.