Cryptococcus neoformans e C. gattii são as principais espécies do gênero que causam infecção no homem, C. albidus e C. laurentii são espécies menos envolvidas. O presente trabalho teve por objetivos avaliar a patogenicidade in vivo, os fatores e os genes relacionados à virulência, e verificar o perfil de suscetibilidade ao fluconazol de 10 isolados ambientais de cada uma das espécies: C. neoformans, C. albidus e C. laurentii, antes e após a inoculação em camundongos BALB/c imunocompetentes; pesquisar os sorotipos, mating types e realizar a tipagem molecular. Proteinase, fosfolipase, urease, produção de melanina e crescimento à 37ºC foram pesquisados utilizando metodologias clássicas, e a pesquisa dos genes e determinação dos sorotipos e mating types foram feitas por PCR. A tipagem molecular foi realizada por PCR-fingerprinting, com os iniciadores (GACA)4 e M13. A determinação da CIM do fluconazol foi realizada pelo método da microdiluição em caldo. Todos os isolados de C. neoformans foram sorotipos A e MAT-alfa. A inoculação em animais mostrou que 9 isolados de C. neoformans mataram 100% deles em até 33 dias, e 1 levou os animais à morte num período entre 40 e 82 dias; 9 isolados foram recuperados dos pulmões e cérebro dos animais em 7 e 14 dias, e um deles levou todos os animais à morte em 12 dias, sendo possível recuperá-lo somente no 7º dia. Os animais inoculados com C. albidus e C. laurentii permaneceram vivos até negativação das culturas dos órgãos avaliados. C. albidus foi isolado principalmente do fígado e dos pulmões até 10 dias após a inoculação, C. laurentii dos pulmões e do cérebro até 120 dias. Todos os isolados das 3 espécies produziram cápsula antes e após a inoculação. Todos C. neoformans, 6 C. albidus e 6 C. laurentii cresceram à 37ºC antes e depois da inoculação. Melanina foi produzida por todos os isolados de C. neoformans e nenhum C. albidus nas duas ocasiões; e por 6 e 9 isolados de C. laurentii, antes e depois da inoculação, respectivamente. Seis isolados de C. neoformans e 1 de C. laurentii produziram proteinase nas duas ocasiões. Sete isolados de C. albidus produziram proteinase antes e todos depois da inoculação. Fosfolipase foi produzida por todos C. neoformans e C. albidus, e por 6 C. laurentii nas duas ocasiões. A avaliação da atividade da urease realizada em meio líquido foi positiva em 24 a 48 horas pelos isolados de C. neoformans e C. laurentii, e em 24 a 96 horas por C. albidus. A CIM de fluconazol variou de 2 a 8 ug/mL para C. neoformans, de 8 a >= 64 ug/mL para C. albidus, e de 1 a 64 ug/mL para C. laurentii, nas duas ocasiões. Todos os isolados de C. neoformans apresentaram os genes lacase (Lac1), fosfolipase (PLB1), proteinase (cnap1), calcineurina (CNA1), urease (URE1), e ERG11, com os oligonucleotídeos utilizados. A PCR com ERG11 mostrou uma banda no gel de agarose para todos C. albidus, porém nenhum dos outros genes pesquisados foram amplificados em C. albidus e C. laurentii. A tipagem molecular por PCR-fingerprinting dos isolados de C. neoformans revelou 2 tipos moleculares: VNI (7 isolados) e VNII (3 isolados). A maioria dos isolados de C. albidus apresentou homogeneidade nos padrões de bandas gerados, e C. laurentii foi a espécie que demonstrou maior diversidade genética por esta metodologia. Concluímos que a passagem dos isolados pelos animais não alterou os fenótipos estudados e nenhuma alteração foi detectada pela análise molecular. No entanto, verificamos a grande heterogeneidade molecular dos isolados de C. laurentii estudados. / Species of Cryptococcus neoformans and C. gattii are the main ones in the genus causing infection in man while C. albidus and C. laurentii are less involved. This study evaluated the in vivo pathogenicity, factors and genes related to virulence and the susceptibility to fluconazole before and after inoculation in immunocompetent BALB/c mice of ten environmental isolates of C. neoformans, C. albidus and C. laurentii. Serotypes, mating types and molecular typing were also determined to complete the evaluation. Enzymes like proteinases, phospholipase, urease, production of melanin and growth at 37oC were investigated by classical methods, but gene characterization and determination of serotypes and mating types were investigated by PCR. Molecular typing was done by PCR-fingerprinting with primers (GACA)4 and M13. The microdilution method was used to determine the minimum inhibitory concentration (MIC) of flucozanole. All C. neoformans isolates were serotype A and MAT-alfa and 9 of them when inoculated in animals killed 100% in up to 33 days. One isolate inoculated killed the animals in 40 to 82 days. Nine isolates were recovered from the animal lungs and brain in 7 and 14 days and the one which killed all animals in 12 days was only recovered on the 7th day. Animals inoculated with C. albidus and C. laurentii were alive until the tissue cultures of evaluated organs were negative. C. albidus was isolated mainly from the liver and lungs in up to 10 days after inoculation and strains of C. laurentii from the lungs and brain in up to 120 days. All isolates in the 3 species were capsule producers before and after inoculation. All strains of C. neoformans, 6 C. albidus and 6 C. laurentii grew at 37oC both before and after inoculation. All C. neoformans produced melanin and 6 C. laurentii produced it before inoculation and nine after. None was produced by C. albidus. Six isolates of C. neoformans and one of C. laurentii produced proteinases in both situations, before and after inoculation. Seven C. albidus isolates produced the protein hydrolyzing enzyme before inoculation and all after. Phospholipase enzyme was produced by all C. neoformans, and C. albidus and by 6 C. laurentii in both conditions, before and after inoculation. Urease activity was detected between 24 and 48 hours after incubation in a liquid medium for C. neoformans and C. laurentii cultures and after 24 to 96 hours for C. albidus. Fluconazole MICs ranged from 2 to 8 ug/ mL for C. neoformans isolates, from 8 to >= 64 ug/mL for C. albidus and from 1 to 64 ug/mL for C. laurentii in both conditions. Genes laccase (Lac1), phopholipase (PLB1) proteinase (cnap1), calcineurine (CNA1), urease (URE1) and ERG11, detected with the primers used were present in all C. neoformans. With exception of ERG11, which showed a band in agarose electrophoresis by all C. albidus, the other genes were not amplified in C. albidus and C. laurentii. Molecular typing by PCR-fingerprinting showed two molecular types in C. neoformans: VNI in 7 isolates and VNII in 3 isolates. Most C. albidus showed homogenous patterns in the bands generated and C. laurentii was the species with the higher genetic diversity by this methodology. It is concluded that isolate inoculations in animals does not alter phenotypes and no alteration is detected by molecular analysis. However, the high molecular heterogeneity of C. laurentii was detected.

<p><a>The hemotrophic mycoplasma<i>, Mycoplasma ovis</i>, is found in sheep and goats throughout the world. This pathogenic bacterium is capable of causing an acute, life-threatening infection as well as chronic or subclinical infections in these animals. The purposes of the present studies were to develop <i>M. ovis</i>-specific assays for detection of this hemoplasma, and to better understand infection dynamics within pregnant ewes and lambs. </a>The first study describes the development and validation of a SYBR^® Green quantitative PCR (qPCR) assay, which was subsequently used to determine the prevalence of <i>M. ovis</i> infection within a population of goats and to evaluate risk factors for infection. This highly sensitive and specific assay consistently detected as few as 10 copies of plasmid/reaction. Convenience-based sampling of 362 goats from 61 farms located in Indiana revealed a prevalence of infection of 18% (95% confidence interval (CI), 14% to 22%). Bacterial loads of <i>M. ovis</i> ranged from 1.05 x 10³ to 1.85 x 10⁵copies/mL of blood with a mean of 1.31 x 10⁴copies/mL of blood. The only risk factor associated with hemoplasma infection was the production use of the goat; dairy goats had a 3.3 fold increase compared with the prevalence in goats used for meat. This study not only demonstrates that <i>M. ovis</i> infection is common in goats in Indiana, but shows the variability of bacterial loads that can be found in chronically-infected animals. While sub-clinically infected goats may have a bacteremia, levels are characteristically less than 2.0 x 10⁵copies/mL.</p><p> The second project utilized a combination of cross-sectional and longitudinal studies to estimate the prevalence of <i>M. ovis</i> infection from a cohort of naturally-infected pregnant ewes, assess changes in their bacterial loads, and determine the incidence of <i>M. ovis</i> in lambs pre- and post-weaning. The prevalence of <i>M. ovis</i> infection in ewes was not found to be significantly different during pregnancy, and before and after weaning of the lambs, with prevalence estimates of 45% (95% CI, 23.1 – 68.5), 36% (95% CI, 17.9 – 57.4), and 44%, (95% CI, 24.4 – 65.1), respectively. Bacterial loads of the ewes from the cross-sectional study ranged from 10⁴to 10⁹copies/mL of blood, with the median bacterial load at 10⁵ copies/mL of blood. While higher bacterial loads are typical of an acute infection, none of the ewes in this study had overt clinical signs. The data suggest that <i>M. ovis</i> loads may be higher in pregnant sheep, particularly in ewes half-way through pregnancy. Most of the <i>M. ovis</i> infections in the study lambs were detected post-weaning which suggests that transplacental or transmammary infection of <i>M. ovis</i> are unlikely routes.</p><p> In the third study, a subset of <i>M. ovis</i> genes for use in a multi-locus sequence typing assay (MLST) were evaluated. Next-generation sequencing was performed to generate data from pooled DNA amplicons in order to identify single nucleotide polymorphisms (SNPs) of <i>M. ovis </i>from five genes. Evaluation of the quality and depths of coverage for the reads and SNPs indicated that the pooled DNA amplicons produced reads and SNPs having high quality and sufficient depth. This pooling technique is a cost-effective alternative to whole-genome sequencing. While the MLST has good discriminatory power and may be used to identify genetically distant and divergent clusters of <i>M. ovis</i> from different geographical origins, within a herd the discrimination power is low, which may hamper its usefulness in transmission studies. </p><p> The fourth and final study was the development of a loop-mediated isothermal amplification (LAMP) assay targeting the dnaK gene of <i>M. ovis</i>, with comparison of the assay to conventional PCR (cPCR). The metal ion indicator hydroxynaphthol blue (HNB) was added prior to the reaction, which allowed for visual detection of LAMP-positive samples as indicated by a color change from violet to sky blue. <i>Mycoplasma ovis</i> was consistently detected in 45 minutes with the LAMP assay at a reaction temperature of 64°C, with more infected sheep being detected than by cPCR. Therefore, the LAMP assay is fast and reliable in the detection of <i>M. ovis</i>. The developed LAMP assay may have applications in diagnostics, surveillance and disease management as well as prevalence studies. However, a more robust molecular technique is necessary for <i>M. ovis</i> isolate or stain discrimination to investigate transmission or disease spread in an outbreak.</p><p> </p><p> In conclusion, three new molecular tools for the detection of <i>M. ovis</i> in goats and sheep were developed as results of these studies. We have shown that the qPCR assay is an efficient tool for detection and quantification of <i>M. ovis</i> loads in blood from both of these species. On the other hand, the value of the LAMP assay is for reliable detection of infection (not quantification), especially in resource-limited situations. The five-locus MLST protocol developed herein, a typing assay based on the polymorphism of five gene sequences, is a laborious technique requiring DNA extraction, PCR amplification, purification and sequencing of target loci. The value of this technique is not as a routine diagnostic, but rather it may be used to better understand the genetic diversity of <i>M. ovis</i> and investigate strain variations. Most importantly, the scheme is sufficiently robust to allow direct genotyping of <i>M. ovis</i> in total blood DNA extracts without culture isolation. The MLST approach may prove useful as a tool for future investigations of transmission and disease spread. These studies have also expanded our understanding of the infection dynamics of <i>M. ovis</i> in pregnant sheep and lambs. It is shown herein that despite the high prevalence and sometimes high bacterial loads in pregnant ewes, <i>M. ovis</i> does not appear to be transmitted to the lambs in utero or during the perinatal period. The lambs become infected mostly after weaning; this may suggest a protective effect during the pre-weaning period and/or subsequent exposure/infection from their environment. </p><br>

O gênero Stenotrophomonas inclui doze espécies de bactérias Gram- negativas, não fermentadoras, que podem ser encontradas no ambiente. Até o presente, S. maltophilia é a única espécie com linhagens que são patógenos oportunistas em seres humanos. Essa espécie tem ganhado importância clínica por estar envolvida em um crescente número de infecções em pacientes imunodeprimidos e em UTI neonatais, apresentando altas taxas de morbimortalidade. Isolados ambientais e clínicos de S. maltophilia compartilham 85% de homologia genômica, podendo ser a diferença uma consequência da adaptação da bactéria aos diferentes nichos ecológicos em que é encontrada. Embora infecções e/ou colonizações por S. maltophilia aconteçam principalmente em ambiente nosocomial, diversos estudos têm mostrado um aumento do número de infecções de origem comunitária. Neste estudo, isolados nosocomiais e linhagens clínicas de S. maltophilia foram fenotipados e comparados quanto ao seu perfil filogenético por Multilocus Sequence Typing (MLST). Na análise por MLST utilizaram-se os genes atpD, gapA, guaA, nuoD, ppsA, recA e rpoA, por serem genes constitutivos. O perfil filogenético mostrou alta variabilidade clonal, provavelmente refletindo o processo de adaptação de S. maltophilia ambientais a outros habitats. Verificou-se que dois subgrupos de isolados clínicos de S. maltophilia com grande homogeneidade filogenética apresentam recombinação intergrupos, indicando alta permissividade à transferência horizontal de informação genética, envolvida na resistência a antibióticos e na expressão de fatores de virulência. Mais ainda, para a maioria das amostras clínicas aqui estudadas, inferências filogenéticas podem ser feitas apenas com o uso do gene ppsA. Portanto, o sequenciamento de apenas um fragmento específico para esse gene seria suficiente, em muitos casos, para determinar se a infecção por S. maltophilia foi causada por cepa já presente no ambiente nosocomial ou por bactérias de origem comunitária introduzidas nesse ambiente pela circulação de pessoas e materiais. Finalmente, foi possível mostrar que até mesmo isolados e linhagens clínicas filogeneticamente próximos não compartilham similaridade de perfil metabólico, o que indica sua origem comunitária / The genus Stenotrophomonas comprises twelve species of Gram- negative and non-fermentative bacteria, which can be found in the environment. The species S. maltophilia is the only one, until the present, that includes opportunistic pathogenic strains in humans. S. maltophilia gained clinical importance by being involved in an increasing number of infections in immunocompromissed patients and in NICUs, with high rates of morbidity and mortality. Environmental and clinical isolates of S. maltophilia share around 85% of genomic homology and this difference may be a consequence of adaptation to the different ecological niches where S. maltophilia can be found. Although infection and/or colonization by S. maltophilia occur mainly in the nosocomial environment, several studies have shown a growing number of community-acquired infections. In this study, nosocomial isolates and clinical strains of S. maltophilia were phenotyped and their phylogenetic profiles compared by using Multilocus Sequence Typing (MLST). In order to accomplish the MLST analysis the constitutive genes atpD, gapA, guaA, nuoD, ppsA, recA and rpoA were used. The resultant global phylogenetic profile showed high clonal variability, what correlates with the adaptability process of environmental S. maltophilia to other habitats. It was found that two clinical isolates subgroups of S. maltophilia with great phylogenetic homogeneity present intergroup recombination indicating the high permittivity to horizontal gene transfer, a mechanism involved in the acquisition of antibiotic resistance and expression of virulence factors. Moreover, for most of the clinical samples studied here, phylogenetic inferences can be made with the use of the gene ppsA only. Therefore, the sequencing of just one specific fragment of this gene would allow, in many cases, to determine whether the infection with S. maltophilia ? ? was caused by a strain already present in the nosocomial environment, or by bacteria introduced from the community in this environmental through the movement of people and materials. Finally, phenotyping data showed that even closely phylogenetic related nosocomial isolates and clinical strains do not share the same metabolic profile, thus indicating their community- acquired origin

Listeria monocytogenes (L. monocytogenes) est une bactérie ubiquitaire Gram-positive aux niches écologiques et porteurs divers. L'infection humaine, par ce micro-organisme, liée à la consommation d’aliments contaminés peut provoquer une infection rare, mais grave, la listériose. L'identification et le typage de cette bactérie sont des étapes importantes pour la maîtrise des dangers à chaque étape de la chaîne alimentaire. Les méthodes de typage disponibles que sont le sérotypage, le sérotypage moléculaire, la PFGE (Electrophorèse en Champs pulsé) et la MLST (Multiloci Type de séquence), ont permis de comprendre la diversité de L. monocytogenes et d’endiguer des épidémies. Ces dernières années, les NGS (Next Generation Sequencing) et le développement de méthodes de calcul ont rendu possible l'application du séquençage du génome entier (WGS) comme outils de typage. Dans cette étude, nous avons profité de la vaste collection de souches de L. monocytogenes isolées de l'alimentation et gérée par l'ANSES. Les outils de typage standards comme les protocoles WGS ont été appliqués et comparés sur cette collection. Nous avons observé que la population de L. monocytogenes est très structurée, quel que soit l’outil de typage utilisé, de plus, les résultats sont concordants. Chaque groupe de souches, désigné comme "séquence type " (ST) ou "complexe clonal" (CC) dans ce travail, montre des caractéristiques spécifiques sur leur fréquence, les résultats de typage, l'association à l'aliment ou au cas clinique et le profil de virulence. Ces données permettent de classer plus finement les souches de L. monocytogenes et de prédire le potentiel pathogénique de souches. De plus, les WGS permettent la surveillance en routine, la détection d'épidémies, l’identification des sources de contamination et des risques, de par leur forte résolution. Avec le programme « BlastP », une base de données de gènes associés à la virulence et le panel de souches de l'ANSES, nous avons pu distinguer un gène de virulence, InlF (internalin F), lequel, tronqué, pourrait expliquer la fréquence extrêmement faible de cas cliniques dans le groupe de souches ST121.Les WGS représentent une nouvelle étape vers une meilleure appréciation et gestion des risques de santé liés à L. monocytogenes. Cependant, des améliorations comme une nomenclature commune, des protocoles standardisés pour les WGS et des outils simplifiés pour partager des données et ainsi renforcer l'efficacité de la surveillance de L. monocytogenes seraient nécessaires / Listeria monocytogenes (L. monocytogenes) is a gram-positive bacterium present in diverse ecological environments and hosts. The microorganism may infect humans and these infections can often be traced back to contaminated foods. The bacterium can sometimes lead to rare but serious infection and in particular a lethal infection known as listeriosis. The bacterium can enter the food chain at all production stages and therefore identification and characterisation of this bacterium are critical steps in the control of potential hazards to the food chain. The current available characterisation methods, which include serotyping, molecular serotyping, PFGE (Pulsed-Field Gel Electrophoresis) and MLST (Multilocus Sequence Type), have provided understanding about the diversity of L. monocytogenes. Further the characterisation methods can facilitate the investigation of outbreaks. During the last few years, (NGS) Next Generation Sequencing and development of computational method for genome wide studies have made it possible to apply WGS (whole genome sequencing) as a typing tool. In this study, we took advantage of the large and the well-characterized collection of L. monocytogenes strains isolated from foods, which is available at Anses. Standard typing tools as well as recent WGS protocols were applied and compared. We observed that L. monocytogenes population could be distinctly separated and structured when analysed by diverse typing tools. Moreover, the investigation displayed consistency among the typing tools. Each cluster of strains, commonly referred to as Sequence Type (ST) or Clonal Complex (CC), shows specific features regarding prevalence, typing results, association to food or the clinical and virulence profile. It opens the possibility for a detailed classification of L. monocytogenes and the possibility to predict the potential pathogenicity of strains based on knowledge of those specific features. By utilising the BlastP program, a virulence associated genes database and the panel of strains housed by Anses, we identified one gene, internalin F (InlF), truncated specifically in ST121. It could therefore explain the observed low frequency of clinical case among strains from ST121.WGS represents a step towards even better identification and management of health risks related to L. Monocytogenes. However, in order to enhance efficiency of foodborne pathogen surveys it would be necessary to implement improvements such as common nomenclature, standard WGS protocols and uniform standards for data sharing

Qi Wei. / "November 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 158-185). / 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.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Streptococcus pneumoniae é um importante agente etiológico de infecções invasivas e não invasivas, incluindo meningite, pneumonia e otite média. A cápsula polissacarídica é o principal fator de virulência desse microrganismo, sendo também considerada um importante marcador em estudos epidemiológicos. Dentre os mais de 90 tipos capsulares conhecidos, o sorotipo 14 se destaca pela prevalência elevada em várias regiões, inclusive no Brasil. A avaliação da diversidade genética desse microrganismo também inclui a aplicação de métodos moleculares, como PFGE e MLST. Entretanto, essas metodologias são relativamente onerosas, consomem muito tempo e os resultados obtidos com a técnica de PFGE são de difícil comparação entre diferentes laboratórios. A técnica de análise do polimorfismo numérico de segmentos repetitivos em múltiplos loci [MLVA, do inglês Multiple Loci VTNR (Variable-Number Tandem Repeat) Analysis] se apresenta como uma alternativa, embora ainda necessite de padronização e avaliação mais ampla para a espécie em questão. No presente estudo, 60 amostras de Streptococcus pneumoniae pertencentes ao sorotipo 14, isoladas de diversas fontes clínicas, em diferentes locais e períodos de tempo, foram caracterizadas pelas técnicas de MLVA (baseada na análise de 18 loci distintos), MLST, PFGE e tipagem do gene pspA. O gene pspA2 predominou entre as amostras analisadas, seguido pelo gene pspA1. Os tipos de MLVA, perfis de PFGE, e STs encontrados apresentaram resultados, em geral, concordantes, indicando o elevado poder discriminatório da versão da técnica de MLVA empregada. Cinco complexos clonais (CC) de MLVA e cinco singletons puderam ser definidos. O CC de MLVA denominado de L7 foi o predominante, compreendendo 36,7% da amostragem estudada. O CC L7 mostrou-se relacionado com genes pspA da família 2, com o CC1 de MLST, com o CC Pen14-H de PFGE, e com a não susceptibilidade à penicilina, Entre os complexos clonais de MLST, o CC1 foi o prevalente e incluiu predominantemente o ST156, pertencente ao clone internacional Spain9V-3. O CC L3 e o singleton L17 de MLVA apresentaram-se associados ao CC de PFGE Eri14-A, a família 1 de PspA e ao CC2 de MLST, que por sua vez também estava relacionado com o clone internacional England14-9. O CC L15 de MLVA esteve associado ao CC de PFGE Pen14-A, ao gene pspA2, aos CC3 e CC4 de MLST e ao clone internacional do PMEN Tennessee14-18. A técnica de MLVA revelou-se significativamente mais discriminatória que as técnicas de PFGE e MLST, conforme exemplificado pela detecção de 21 perfis de MLVA, 13 perfis de PFGE e cinco STs, entre as 22 amostras pertencentes ao CC de MLVA L7. Uma versão de MLVA, compreendendo um painel com os oito loci de maior poder discriminatório, pôde ser proposta a partir da análise dos resultados obtidos. Estes aspectos, aliados ao menor tempo e custo de execução, indicam que a técnica de MLVA constitui uma alternativa importante e satisfatória para uso em estudos sobre a diversidade genética de S. pneumoniae. / Streptococcus pneumoniae is a major pathogen causing invasive and non-invasive diseases in humans, including meningitis, pneumonia and otitis media. The polysaccharide capsule of this microorganism is considered a major virulence factor and an important marker for epidemiological studies. More than 90 pneumococcal capsular serotypes are recognized, and serotype 14 is highly prevalent in many regions, including Brazil. Genotyping methods, such as PFGE and MLST, are essential to evaluate genetic diversity of this bacterium. However, these methods are expensive, time-consuming and results from different laboratories are difficult to compare. Multiple Loci VTNR (Variable-Number Tandem Repeat) Analysis (MLVA) appears as an alternative, despite the fact that standardization and wide evaluation for application to this species is still required. In the present study, a total of 60 S. pneumoniae isolates belonging to serotype 14, isolated from different sources, regions and periods of time, were analyzed by MLVA (based on the analysis of 18 distinct loci), MLST, PFGE and pspA typing methods. Gene pspA2 was the predominant, followed by pspA1. Overall, the results of PFGE, MLST and MLVA typing were congruent, and indicated the discriminatory power of the MLVA method used. Five clonal complexes (CC) and five singletons were identified by MLVA. CC L7 was the predominant MLVA CC, comprising 36.7% of all the isolates. L7 was associated with pspA2 gene and non-susceptibility to penicillin, and it was related to MLST CC1, and to PFGE Pen14-H. CC1 was the prevalent MLST CC and included mostly ST156 that belongs to international clone (IC) Spain9V-3. Another MLVA CC, named L3, and the singleton L17 were related to PFGE CC Eri14-A, MLST CC2, and IC England14-9. MLVA CC L15 was related to PFGE Pen14-A, MLST CC3 and CC4, and IC Tennessee14-18. MLVA was found to be more discriminatory than PFGE and MLST, as exemplified by detection of 21 MLVA types, 13 PFGE profiles and 5 STs among the 22 strains belonging to L7, the predominant MLVA CC. A modified version of the MLVA method, based on the analysis of 8 loci only, is proposed. This aspect, in conjunction with reducing time and costs, indicate that MLVA represents an important and satisfactory alternative method to evaluate the genetic diversity of S. pneumoniae.

Submitted by Liliane Ferreira (ljuvencia30@gmail.com) on 2018-03-21T12:04:52Z No. of bitstreams: 2 Dissertação - Thaynara Gonzaga Santos - 2018.pdf: 3745794 bytes, checksum: db8d5a41bb72d586b2468abd5e6ad7b6 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-03-22T10:33:07Z (GMT) No. of bitstreams: 2 Dissertação - Thaynara Gonzaga Santos - 2018.pdf: 3745794 bytes, checksum: db8d5a41bb72d586b2468abd5e6ad7b6 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-03-22T10:33:07Z (GMT). No. of bitstreams: 2 Dissertação - Thaynara Gonzaga Santos - 2018.pdf: 3745794 bytes, checksum: db8d5a41bb72d586b2468abd5e6ad7b6 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-02-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Urinary tract infections (UTI) are the most prevalent bacterial infections and, in this context, E. coli is the primary an etiological agent. Virulence mechanisms participating in the process of colonization, invasion and tissue damage observed in the UTI. Phylogenetic analyses contribute to the knowledge about the ancestry of a pathogen, as well as allow for the differentiation of various microbial isolates according to the genetic similarity between them. Thus, this study aims to phylogenetic characterization of central UPEC strains isolated from patients in the municipality of Jataí-GO, as well as the comparison between the profile of virulence and antimicrobial resistance. With the completion of the study was possible to verify that one of the samples analysed, 74.42% presented to the virulence gene iucD as well as 69.77% to 44.19% for fimH, papC 20.93% and hlyA. With the phylogenetic typing showed that 3.22% of the samples analysed are proving to be belonging to the groups A and D, 4.84% of the samples analysed, belong to the filogrupo B1, filogrupo B2, 61.29% 6.45% to filogrupo F, and 20.48% not classified in any of the filogrupos searched. Between-group comparisons showed that the isolates belonging to the filogrupo, presented 100% of amplification to iucD and 50% and fimH to papC, referring to group B1 presented 66.67% virulence gene amplification iucD and 33.33% hlyA, that the filogrupo B2 presented 63.16% of fimH, 68.42% amplification to iucD 36.84% to papC, 34.21% hlyA, and that in D, 50% was observed for fimH, papC amplification and iucD and finally filogrupo F 25% presented to fimH, papC hly and 50% and to iucD. As regards culture, the samples analysed presented, resistance to gentamicin (38%), cephalothin (38%), streptomycin (51%), ciprofloxacin (51%), Chloramphenicol (29%), ceftriaxone (22%), amikacin (31%), and tetracycline (40%) and 100% the samples demonstrated susceptibility to Imipenem. In short, except for the antibiotics gentamicin and ciprofloxacin, no increase of resistance of isolates analyzed and not that there was a pattern of resistance in UPEC according to filogrupos research. / As infecções do trato urinário (ITUs) são as infecções bacterianas mais prevalentes e, nesse contexto, Escherichia coli é o principal agente etiológico. Diversos mecanismos de virulência participam no processo de colonização, invasão e lesão tecidual observados nas ITUs. As análises filogenéticas contribuem para o conhecimento a respeito da ancestralidade de um patógeno, assim como permitem a diferenciação de diversos isolados microbianos de acordo com a similaridade genética entre eles. Assim, este estudo tem por objetivo central a caracterização filogenética de cepas UPEC isoladas de pacientes no município de Jataí-GO, bem como a comparação entre o perfil de virulência e resistência aos antimicrobianos. Com a realização do estudo foi possível verificar que dentre as amostras analisadas, 74,42% apresentaram amplificação para o gene de virulência iucD, bem como 69,77% para fimH, 44,19% para papC e 20,93% para hlyA. Com a tipagem filogenética observou-se que 3,22% das amostras analisadas demonstram ser pertencentes aos grupos A e D, 4,84% das amostras analisadas, pertencem ao filogrupo B1, 61,29% ao filogrupo B2, 6,45% ao filogrupo F, e 20,48% não se classificaram em nenhum dos filogrupos pesquisados. As comparações entre grupos demonstraram que os isolados pertencentes ao filogrupo A, apresentaram 100% de amplificação para fimH e iucD e 50% para papC, os referentes ao grupo B1 apresentaram 66,67% amplificação o gene de virulência iucD e 33,33% hlyA, que o filogrupo B2 apresentou 63,16% de amplificação para fimH, 68,42% para iucD, 36,84% para papC e 34,21% para hlyA, que em D, observou-se 50% de amplificação para fimH, papC e iucD e por fim filogrupo F apresentou 25% de amplificação para fimH, papC e hly e 50% para iucD. No que se refere ao antibiograma, as amostras analisadas apresentaram, resistência à gentamicina (38%), a cefalotina (38%), estreptomicina (51%), a ciprofloxacina (51%), a cloranfenicol (29%), a ceftriaxona (22%), a amicacina (31%) e tetraciclina (40%) e 100 % das amostras demostraram susceptibilidade ao Imipenem. Em resumo, exceto para os antibióticos gentamicina e ciprofloxacina, não houve aumento da resistência dos isolados analisados e não que houve um padrão de resistência em UPEC de acordo com os filogrupos pesquisa.

O gênero Stenotrophomonas inclui doze espécies de bactérias Gram- negativas, não fermentadoras, que podem ser encontradas no ambiente. Até o presente, S. maltophilia é a única espécie com linhagens que são patógenos oportunistas em seres humanos. Essa espécie tem ganhado importância clínica por estar envolvida em um crescente número de infecções em pacientes imunodeprimidos e em UTI neonatais, apresentando altas taxas de morbimortalidade. Isolados ambientais e clínicos de S. maltophilia compartilham 85% de homologia genômica, podendo ser a diferença uma consequência da adaptação da bactéria aos diferentes nichos ecológicos em que é encontrada. Embora infecções e/ou colonizações por S. maltophilia aconteçam principalmente em ambiente nosocomial, diversos estudos têm mostrado um aumento do número de infecções de origem comunitária. Neste estudo, isolados nosocomiais e linhagens clínicas de S. maltophilia foram fenotipados e comparados quanto ao seu perfil filogenético por Multilocus Sequence Typing (MLST). Na análise por MLST utilizaram-se os genes atpD, gapA, guaA, nuoD, ppsA, recA e rpoA, por serem genes constitutivos. O perfil filogenético mostrou alta variabilidade clonal, provavelmente refletindo o processo de adaptação de S. maltophilia ambientais a outros habitats. Verificou-se que dois subgrupos de isolados clínicos de S. maltophilia com grande homogeneidade filogenética apresentam recombinação intergrupos, indicando alta permissividade à transferência horizontal de informação genética, envolvida na resistência a antibióticos e na expressão de fatores de virulência. Mais ainda, para a maioria das amostras clínicas aqui estudadas, inferências filogenéticas podem ser feitas apenas com o uso do gene ppsA. Portanto, o sequenciamento de apenas um fragmento específico para esse gene seria suficiente, em muitos casos, para determinar se a infecção por S. maltophilia foi causada por cepa já presente no ambiente nosocomial ou por bactérias de origem comunitária introduzidas nesse ambiente pela circulação de pessoas e materiais. Finalmente, foi possível mostrar que até mesmo isolados e linhagens clínicas filogeneticamente próximos não compartilham similaridade de perfil metabólico, o que indica sua origem comunitária / The genus Stenotrophomonas comprises twelve species of Gram- negative and non-fermentative bacteria, which can be found in the environment. The species S. maltophilia is the only one, until the present, that includes opportunistic pathogenic strains in humans. S. maltophilia gained clinical importance by being involved in an increasing number of infections in immunocompromissed patients and in NICUs, with high rates of morbidity and mortality. Environmental and clinical isolates of S. maltophilia share around 85% of genomic homology and this difference may be a consequence of adaptation to the different ecological niches where S. maltophilia can be found. Although infection and/or colonization by S. maltophilia occur mainly in the nosocomial environment, several studies have shown a growing number of community-acquired infections. In this study, nosocomial isolates and clinical strains of S. maltophilia were phenotyped and their phylogenetic profiles compared by using Multilocus Sequence Typing (MLST). In order to accomplish the MLST analysis the constitutive genes atpD, gapA, guaA, nuoD, ppsA, recA and rpoA were used. The resultant global phylogenetic profile showed high clonal variability, what correlates with the adaptability process of environmental S. maltophilia to other habitats. It was found that two clinical isolates subgroups of S. maltophilia with great phylogenetic homogeneity present intergroup recombination indicating the high permittivity to horizontal gene transfer, a mechanism involved in the acquisition of antibiotic resistance and expression of virulence factors. Moreover, for most of the clinical samples studied here, phylogenetic inferences can be made with the use of the gene ppsA only. Therefore, the sequencing of just one specific fragment of this gene would allow, in many cases, to determine whether the infection with S. maltophilia ? ? was caused by a strain already present in the nosocomial environment, or by bacteria introduced from the community in this environmental through the movement of people and materials. Finally, phenotyping data showed that even closely phylogenetic related nosocomial isolates and clinical strains do not share the same metabolic profile, thus indicating their community- acquired origin

<p>Campylobacter jejuni is the main cause of bacterial diarrhoeal illness in developed countries, with ~7000 cases being reported each year in Sweden. C. jejuni has received growing attention since it’s recognition as a human pathogen in the 1970s, but its epidemiology is complex and much still remains unknown. There are several potential reservoirs for C. jejuni, including environmental sources as water and soil, wild and domesticated animals, particularly poultry, but also other livestock and pets. In this study 348 Swedish C. jejuni isolates from the year 2000 from humans (n = 164) and broilers (n = 184) were characterized with multilocus sequence typing (MLST) with the aim of comparing the population structures and diversity of C. jejuni between isolates from the two hosts. MLST is a method for characterization of bacterial isolates that indexes the variation in DNA sequence of multiple protein encoding housekeeping genes. A secondary aim in this study was to compare populations of C. jejuni from 11 subgroups of isolates based on location of the sampling. The overlap between the populations was analyzed numerically based on genotypes detected and with analysis of phylogeny, gene flow and molecular variation. It was shown that the population structure of C. jejuni isolates from broilers and humans show a high degree of similarity, supporting broilers as an important source of human infection. However, even though the population structure of human and broiler C. jejuni were almost genetically indistinguishable other sources of C. jejuni infections in humans cannot be ruled out since the same genotypes can be found in other sources as well. Analysis of the 11 subgroups suggested that there may be a difference in populations infecting humans in different Swedish regions, and between populations of C. jejuni in broilers from different slaughterhouses. But this could be a result of chance since most of the subgroups were small. Future studies to improve the understanding of C. jejuni epidemiology, for which MLST has proven itself as a valid method, is important to develop control strategies to prevent infection with this common cause of diarrhoeal illness.</p>

Identical domain concepts reified in different (meta)modelling projects may be named, represented and connected differently. It turns out that a transformation defined for a particular metamodel cannot be directly used for another metamodel; that is, the reuse of transformations is restricted. To tackle this problem, in this dissertation, we propose a solution for automatically migrating legacy transformations. Such a transformation is adapted to the new metamodel that has a slightly different representation in comparison with the original one, while preserving the original semantics of the transformation. To this end, we first introduce MetaModMap, a Domain Specific Language that allows the description of the correspondences of intended semantics between the elements of two metamodels that model the same domain. Then we provide a rewriting mechanism using these user-defined correspondences to migrate the transformation automatically. The proposed solution uses a graph-based model typing relation that enables safe adaptations. Our approach has been prototyped with MOMENT2 and can be used with any framework based on the same graph transformation paradigm.