Xylella fastidiosa (Xf) e Xanthomonas axonopodis pv. citri (Xac) são gama proteobactérias gram negativas, responsáveis por grandes perdas econômicas no setor citrícola brasileiro. Com seus genomas seqüenciados e anotados, fizemos uma análise comparativa entre suas composições gênicas e seus ambientes de vida. Xac apresenta um genoma de 5.2Mb contra 2.7Mb de Xf Isto reflete no número de genes (4432 contra 2838) que acabam refletindo em uma maior complexidade metabólica de Xac, caracterizada por: uma extensa gama de genes de degradação de parede celular (44), biossíntese de proteases (92), genes de funções regulatórias (296), um completo metabolismo energético (209), quimiotático (inexistente em Xf) e secretório (presença dos tipos I, II, III e IV , sendo o II em duplicata), além de um grande número de genes envolvidos com captação de ferro (65), fazem de Xac um patógeno de alto poder invasivo e de rápida propagação e virulência Em contrapartida, Xf por não possuir a complexidade supracitada, parece ter seus recursos adaptados ao ambiente em que vive, como por exemplo um alto número de genes envolvidos com biossíntese de pili, que associado à biossíntese de goma, favorecem sua adesão nas glândulas salivares do vetor (cigarrinha) e a formação de aglomerados celulares responsáveis pelo entupimento dos vasos que levam às patologias decorrentes do evento. / Xylella fastidiosa (Xf) and Xanthomonas axonopodis pv. citri Xac are gram negative gamma proteobacteria, responsible for great economical losses in the Brazilian citrus sector. With their sequenced and annotated genomes, we have done a comparative analysis between their genetic composition and life habitat. Xac displays a genome of 5.2Mb against 2. 7Mb of Xf. This reflects the number of genes (4432 against 2838) which results in a greater metabolic complexity of Xac, characterized by: a wide range of genes of cell wall degradation (44), biosynthesis of proteases (92), many genes of regulatory functions (296), a complex energy metabolism (209), chemotatic (absent in Xf) and secretory systerns (presence of types I, II, III and IV, type II in duplicate), besides a great number of genes involved in iron acquisition (65), make of Xac a pathogen of high invasive power and of quick spreading and virulence. In the other hand Xf, due to the lack of the complexity just cited, seems to have its resources adapted to the habitat in which it lives, as for example a large number of genes involved in pili biosynthesis, that associated with gum biosynthesis, favor its adhesion to the salivary glands of the vector (sharpshooter) and the formation of cellular agglomerations responsible for the blockage of the vessels which leads to the pathologies resulted from this event.

O fungo Pleurotus ostreatus pertence ao grupo de basidiomicetos que degradam madeira. Este cogumelo cultivado em todo mundo apresenta grande rusticidade e produtividade, e pode ainda ser usado em processos de biorremediação e biopolpação. Devido a seu potencial biotecnológico, torna-se interessante a compreensão da interação deste com outros microrganismos. Neste sentido, recentemente foi observada a presença de bactérias associadas a P. ostreatus em culturas in vitro, que apresentavam grande pleomorfismo. A partir desta observação foram elaborados ensaios que visaram a confirmação da presença de bactérias. Para tanto, foi utilizada a estratégia do “Ciclo Completo de Análise do rRNA” (full-cycle rRNA analysis) empregada em microrganismos não cultiváveis ou de crescimento fastidioso, além do emprego de técnicas de microbiologia básica, e de estudos de ultraestrutura. Os estudos de microbiologia básica indicaram que se tratava de um microrganismo fastidioso e que se desenvolvia melhor na presença do fungo em sistema de co-cultivo em meios contendo Tween 80 ou Tween 20. Por sua vez, a análise de ultraestrutura demonstrou a presença de estruturas pleomórficas, tanto internamente como externamente à hifa. Em relação ao “Ciclo completo de Análise do rRNA” este se iniciou pela amplificação e seqüenciamento de parte do rDNA bacteriano, que revelou a proximidade desta bactéria com o Complexo Burkholderia cepacia (CBC). A partir desta seqüência, foi realizado um estudo de bioinformática que indicou sondas específicas para este grupo de bactérias. Completando o Ciclo completo de Análise do rRNA, foram realizados ensaios de hibridização in situ fluorescente (FISH) para a confirmar a relação entre as estruturas bacterianas e a seqüência obtida. Este método comprovou a presença das bactérias no interior das hifas de P. ostreatus. Este trabalho constitui o primeiro relato de bactérias pleomórficas pertencente ao complexo B. cepacia associados a P. ostreatus. / The fungus Pleurotus ostreatus, which belongs to white rot basidiomycete group, is a widely cultivated mushroom; this species has high productivity and rusticity, besides its use in biobleaching and bioremediation processes. This biotechnological potential justifies microbial interaction studies between this fungi and others microorganisms. In P. ostreatus mycelia, it has been observed pleomorphic bacteria growing on agar media. This research describes several assays to confirm bacterial presence in this sample. Therefore, the full-cycle rRNA analysis (described for unculturable or fastidious microorganism), ultrastructure and basic microbiology approaches were employed. Basic microbiology approaches indicated slow growing bacteria, which grown faster near to fungi colonies in solid media amended with Tween 80 or Tween 20 (co-culture system). Ultrastructure studies confirm the presence of intracellular and extracellular pleomorphic bacteria. The full-cycle rRNA analysis started with 16S rDNA amplification and sequencing. This approach demonstrated a relation between these bacteria with Burkholderia cepacia complex. By bioinformatics analysis was determinate which DNA probes can be use to identified this bacterial group. The last step for full-cycle rRNA analysis was applying fluorescent in situ hybridization (FISH). This technique confirmed the relationship between 16S rDNA bacterial sequence and bacterial forms. This is the first time that a pleomorphic bacteria from B. cepacia complex is found associated with P. ostreatus.

H. pylori has a panmictic population structure due to high genetic diversity. The homoplasy index for H. pylori is 0.85 (where 0 represents a completely clonal organism and 1.0 indicates a freely recombining organism) which is much higher than homoplasy index for E. coli (0.26) or naturally competent Neisseria meningitides (0.34). It undergoes both inter as well as intra strain transformation. Intergenomic recombination is subject to strain specific restriction in H. pylori. Hence, a high homoplasy index means that competence predominates over restriction in H. pylori. Annotation of the genomes of H. pylori strains 26695 and J99 show the presence of nearly two dozen R-M systems out of which 16 were postulated to be Type II for J99. H. pylori has been described to be an ideal model system for understanding the equilibrium between competing tension of genomic integrity and diversity (42). R-M systems allow some degree of sexual isolation in a population of competent cells by acting as a barrier to transformation. The mixed colonizing population of H. pylori has a polyploidy nature where each H. pylori strain adds to ‘ploidy’ of the colonizing population. Maintenance of polyploidy nature of mixed colonizing population in a selective niche of stomach needs a barrier to free gene flow. Restriction barrier maintains a polyploidy nature of H. pylori population which is considered as yet another form of genetic diversity helping in persistence of infection. Thus, according to the model proposed by Kang and Blaser, where H. pylori are considered as perfect gases like bacterial population, transformation and restriction both add to genetic diversity of the organism. Again, restriction barriers are not completely effective, which could be due to cellular regulation of restriction system. Thus, a perfect balance between restriction and transformation in turn regulates the gene flow to equilibrate competition and cooperation between various H. pylori strains in a mixed population. RecA, DprA and DprB have been shown to be involved in the presynaptic pathway for recombination substrates brought in through the Com system. Biochemical characterization of HpDprA, during this study revealed its ability to bind to ssDNA and dsDNA. Binding of HpDprA to both ssDNA as well as dsDNA results in large nucleoprotein complex that does not enter the native PAGE. However, DNA trapped in the wells could be released by the addition of excess of competitor DNA, illustrating that the complex are formed reversibly and do not represent dead-end reaction products. Transmission electron microscopy for SpDprA interaction with ssDNA established that a large nucleoprotein complex consisting of a network of several DNA molecules bridged by DprA is formed which is retained in the well. A large DNA-protein complex that sits in the well has also been observed with other DNA binding proteins like RecA. It has been observed for ssDNA binding protein (SSB) that they bind non-specifically to dsDNA under low salt condition (20 mM NaCl) in the absence of Mg2+. The non specific binding of SSB to dsDNA was prevented under high salt conditions (200 mM NaCl) or in the presence of Mg2+. HpDprA interaction with both ssDNA and dsDNA was stable under high salt condition (200 mM NaCl) and in the presence of Mg2+ indicating that these interactions are specific. The interaction of HpDprA with dsDNA is significant since dsDNA plays an important role in natural transformation of H. pylori. The pathway of transformation by dsDNA is highly facilitated (nearly 1000 fold) as compared to ssDNA. However, dsDNA is a preferred substrate for REases which are a barrier to horizontal gene transfer. This implies that the decision of ‘restriction’ or ‘facilitation for recombination’ of incoming DNA might be taken before the conversion of dsDNA into ssDNA. The incoming DNA has been shown to be in the double-stranded form in periplasm and in single-stranded form in cytoplasm. Hence, the temporal and spatial events surrounding endonuclease cleavage remain to be understood. Taken together, these results suggest a very important role of dsDNA in natural transformation in H. pylori. Hence, binding and protection of dsDNA by HpDprA is possibly of crucial importance in the success of natural transformation process of the organism. DprA is characterized by presence of a conserved DNA binding domain. The DNA binding domain adopts a Rossman fold like topology spanning most region of the protein. Rossman fold consists of alternating alpha helix and beta strands in the topological order of β-α-β-α-β. It generally binds to a dinucleotide in a pair as a single Rossman fold can bind to a mononucleotide only. All homologous DprA proteins characterized till date show that in addition of the prominent Rossman fold domain they consist one or more smaller domains. RpDprA consists two more domains other than the Rossman fold domain i.e., N- terminal SAM (sterile alpha motif) domain and a C-terminal DML-1 like domain. SpDprA consist of an N-terminal SAM domain other than Rossman fold domain. While the main function of Rossman fold is to bind DNA, the supplementary domains are highly variable in sequences and functions. For example, the SAM domain in S. pneumoniae plays a key role in shut-off of competence by directly interacting with ComE~P. HpDprA consist of an N-terminal Rossman fold domain and a C-terminal DML-1 like domain. Both these domains are found to be prominently α-helical in nature. Amino acid sequence analysis of the protein suggests that NTD is basic and CTD is acidic in nature. NTD is sufficient for binding with ssDNA and dsDNA, while CTD plays an important role in formation of higher order polymeric complex with DNA. For HpDprA and SpDprA, dimerization site was mapped in Rossman fold domain. Gel filtration data revealed an important observation that HpDprA can exist as a monomer (dominant species at lower concentration) as well as a dimer (dominant species at higher concentration) in solution. However, the exchange between these two forms is very fast resulting in a single peak of elution. Since, HpDprA binds to DNA in dimeric form, the dimer species will be favoured in presence of DNA. Hence, even at lower concentrations HpDprA will be mainly a dimer in presence of DNA. Interestingly, both domains of HpDprA i.e., NTD and CTD were able to form dimers but no higher oligomeric form. On the other hand, HpDprA was seen to form oligomeric forms higher than dimer in gluteraldehyde cross linking assay. The strength of CTD dimer was much lower that NTD dimer, therefore it could be proposed that there are two sites of interaction present in HpDprA - a primary interaction site (N-N interaction) and a secondary interaction site (C-C interaction). The N-N interaction is responsible for dimer formation but further oligomerization of HpDprA necessitates the interaction of two dimers using C-C interaction site. It was shown that NTD binds to ssDNA but forms lower molecular weight complex. SPR analysis of DprA and NTD – DNA interaction pointed out that deletion of CTD leads to faster dissociation of the protein from DNA. Concomitantly, reduction in binding affinity was observed for both ss and ds DNA upon deletion of CTD from full length protein. These results suggest that CTD does play an important role in interaction of full length HpDprA with DNA. Two possible roles of CTD were proposed by Wang et al (2014) group to explain their observation of formation of lower molecular weight complex in absence of CTD. (i) CTD possesses a second DNA binding site but much weaker than site present in NTD. (ii) CTD is not involved in DNA binding but mediates nucleoprotein complex formation through protein – protein interaction. EMSA and SPR analysis with purified CTD protein confirmed that there is no secondary DNA binding site present in CTD. As discussed above, it was observed that CTD can mediate interaction between two HpDprA through C-C interaction. Since the interaction is weaker it is lesser likely to be responsible for dimer formation but in trimer or higher oligomeric form of HpDprA, the presence of N-N interaction will facilitate and stabilize C-C interaction. These observations together bring forward an interesting model for HpDprA – DNA interaction. HpDprA forms dimer through N-N interaction (favourably in presence of DNA) and many HpDprA dimers bind to DNA owing to their high affinity and sequence independent nature of binding. These dimers interact with each other through C-C interaction resulting in higher molecular weight nucleoprotein complex. HpDprA - DNA complex formation is slower than NTD – DNA complex but the former one is more stable (Fig. 2). According to the above proposed model there are two binding events (DNA – protein and protein – protein) in case of HpDprA – DNA complex formation and hence it would take longer time than NTD-DNA complex formation which involves only one binding event. But the resulting higher order complex with HpDprA – DNA would be much more stable. NTD is able to offer equally efficient protection from nuclease to ssDNA and dsDNA (Fig. 7). This shows that NTD alone is sufficient to completely coat single molecule DNA. AFM images confirm the difference in binding pattern of HpDprA full length protein and NTD. As can be seen in Fig. 8F, NTD binds a DNA molecule by entirely occupying all the available space but forms nucleoprotein filaments isolated from each other. In contrast to full length HpDprA, which forms tightly packed, condensed, extensively cross linked polynucleoprotein complexes, NTD forms much thinner complexes with DNA. In the electron micrographs of SpDprA – DNA complex, extensive cross filament interaction was observed resulting in a dense molecular aggregate. Similar kinds of complexes with DNA were also observed for Bacillus subtilis DprA in atomic force microscope images. Thus, it could be proposed that HpDprA binds to a single DNA molecule (single strand or double strand) mainly as a dimer formed through N-N interaction. Such multiple individual nucleoprotein filaments come together and interact with each other through C- C interaction resulting in dense and intricate poly – nucleoprotein complex. HpDprA is proposed to undergo conformational changes from closed state to open state in presence of ssDNA. In agreement with this, structural transition (resulting in reduction of α-helicity of the protein) was observed in presence of ssDNA. Similar structural transitions were observed for dsDNA indicating possibly a common mode of interaction for both forms of DNA. Further, mutation of the residues shown to be involved in binding ssDNA from crystallographic data, resulted in decrease of binding affinity with dsDNA as well. The fold reduction in binding affinity of dsDNA was lower than that for ssDNA despite that it is obvious that the same positively charged pocket which is primarily involved in ssDNA interaction is also responsible (atleast partially) for binding with dsDNA. However, the residues crucial for interaction with these two forms of DNA may be different. Both DprA and R-M systems have been shown to have presynaptic role in natural transformation process. While DprA has a protective role, R-M systems have an inhibitory role for incoming DNA suggesting a functional interaction between them. Results of this study show that HpDprA interacts with dsDNA, inhibits Type II restriction enzymes from acting on it and at the same time stimulates the activity of MTases resulting in increased methylation of bound DNA. This observation is of significance from the view of genetic diversity as the only way a bacterial cell discriminates between self and nonself DNA is through the pattern of methylation. Binding of HpDprA to incoming DNA inhibits its access to restriction endonucleases but not to methyltransferases. As a result DNA will be methylated with the same pattern as that of the host cell. Hence, it no longer remains a substrate for restriction enzymes. HpDprA thus, effectively alleviates the restriction barrier. However, it remains to be understood as to how DNA in complex with HpDprA, while not accessible to REases or other cellular nucleases, is accessible to a MTase? A possible explanation could be that HpDprA interacts with MTase and recruits it on DNA. It has been shown that there is a overlap between DprA dimerization and RecA interaction interfaces and in presence of RecA, DprA-DprA homodimer is replaced with DprA-RecA heterodimer allowing RecA nucleation and polymerization on DNA followed by homology search and synapsis with the chromosome. A similar scenario can be thought for interaction of HpDprA with the MTase. R-M systems play an important role in protection of genomic DNA from bacteriophage DNA. Hence, downregulation of restriction barrier by HpDprA may not be desirable by host during the entire life cycle. Therefore, the expression of HpDprA, which is ComK dependent and that which takes place only when competence is achieved is noteworthy. In H. pylori, DNA damage induces genetic exchange via natural competence. Direct DNA damage leads to significant increase in intergenomic recombination. Taken together it can be proposed that when genetic competence is induced, R-M systems are down regulated to allow increased genetic exchange and thus, increasing adaptive capacity in a selective environment of stomach. There is an evolutionary arms race between bacterial genomes and invading DNA molecules. R-M systems and anti-restriction systems have co-evolved to maintain an evolutionary balance between prey and predator. Phages and plasmids employ anti-restriction strategies to avoid restriction barrier by a) DNA sequence alteration, b) transient occlusion of restriction sites and c) subversion of restriction-modification activities. DNA binding proteins have been shown to bind and occlude restriction sites. On the other hand, λ Ral protein alleviates restriction by stimulating the activity of Type IA MTases. The observations of MTase stimulation and site occlusion of restriction sites by HpDprA appears to be analogous to anti restriction strategies, otherwise employed by bacteriophages. Thus, DprA could be a unique bacterial anti-restriction protein used by H. pylori for downregulating its own R-M systems to maintain the balance between fidelity and diversity. In conclusion, HpDprA has unique ability to bind to dsDNA in addition ssDNA but displays higher affinity towards ssDNA. Binding of HpDprA to DNA results in a compact complex that is inert to the activity of nucleases. A novel site of oligomerization for HpDprA was observed which suggests the role of C-C interaction in inter-nucleoprotein filament interaction. It would be interesting to further study the effects of CTD deletion on the transformation efficiency of H. pylori, to understand these mechanisms better. It has been well demonstrated that R-M systems offer a barrier to incoming DNA, but our understanding of the regulation of R-M systems has been poor. While other factors like regulation of cellular concentration of restriction enzymes and conversion of dsDNA into ssDNA might play crucial roles in striking the perfect balance between genome diversity and integrity, one of the factors that regulate R-M systems could be DprA.

Cupriavidus metallidurans CH34 est la bactérie Gram négative considérée comme organisme-modèle pour l’étude de la résistance aux métaux lourds. Notre travail a porté sur sa résistance au cuivre, codée par les gènes cop du plasmide pMOL30. Ces gènes, responsables des différentes étapes de la résistance (compartimentation des systèmes d’efflux entre périplasme et cytoplasme, modification de valence, et d’autres fonctions totalement inconnues) ont suscité notre intérêt.<p><p>On distingue dans l’îlot cop des gènes codant pour des fonctions de résistance proprement dite (essentiellement par détoxication active du cytoplasme et du périplasme). En effet, les mutants de copSRABCD, copF, et dans une moindre mesure copJ et copE deviennent sensibles. Les phénotypes des mutants divergent toutefois suivant que la mutation soit sur un cosmide qui ne porte que l’îlot (pMOL1024) ou dans son plasmide d’origine (pMOL30). Un second groupe de mutants (copVTMK, copG, copL, copQ) se distingue par un phénotype plus résistant ou identique à la souche parente, sauf autour de la CMI. Ces gènes interviendraient donc à la CMI pour assurer la résistance la plus élevée et le maintien d'un état viable latent.<p><p>La présence de l’îlot cop permet de contenir le taux d’oxygène radicalaire qui reste à un taux basal lorsque les cellules sont adaptées au cuivre environnent. Après un choc de Cu (ou stress aigu), l’îlot cop répond de façon « explosive » au stress, en consommant l’énergie du potentiel membranaire et en augmentant fortement l’activité de la chaîne respiratoire.<p><p>La résistance au cuivre est inductible, mais de façon différenciée pour la souche sauvage (CH34) et celle qui ne porte qu l’îlot cop (AE1744) :la CMI de CH34 triple après adaptation au cuivre, alors que celle d’AE1744 est inchangée. Après un choc de Cu, la résistance au cuivre est plus fortement induite pour AE1744 que pour CH34. Ces observations suggèrent que l’îlot cop ait été sélectionné pour sa capacité à répondre à un stress aigu puis intégré dans un ensemble de gènes plus vaste qui répond à des impératifs de stress chronique.<p><p>L’analyse biochimique de CopI, une petite protéine bleue à cuivre, montre qu’elle porte un site analogue à celui des oxydases multicuivre. Son rôle pourrait dès lors être celui d’une réductase multicuivre. La protéine CopK lie de façon très spécifique le Cu(I) et il semble que la liaison du cuivre modifie sa structure. L’analyse écologique a montré que des homologues de copK pourraient être présents dans l’ADN extrait de la terre de biotopes chargés en cuivre, et dans les souches cuprorésistantes qu’on y trouve. <p><p>La contribution majeure de cette thèse est de montrer que l’effet d’un stress métallique ne se résume pas à deux états physiologiques « mort ou vif ». Il y a lieu de considérer des états transitoires (choc de Cu, adaptation au métal, survie autour de la CMI, persistance) où interviennent des gènes spécifiques dans un ou plusieurs états donnés. Les résultats biochimiques et physiologiques ne nous éclairent pas encore assez sur les interconversions Cu(I)/Cu(II) ni sur les flux de cations notamment vers l'espace extracellulaire. Cette thèse ouvre des perspectives sur des mécanismes (protection à la CMI, phénotype persistant) assurant la survie des bactéries ou leur potentiel de recolonisation lors d'une diminution de la pression toxique :les gènes copT, copV, copK, copM, copB, copG, copL et copQ semblent impliqués dans ces fonctions. <p> / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

La perméabilité membranaire joue un rôle important dans la résistance aux antibiotiques chez lesbactéries à Gram négatif.L’objectif de notre travail était de caractériser la fonction tenue par les deux régulateurs globaux dela perméabilité membranaire chez Enterobacer aerogenes: mar et ram. L’objectif initial futd’identifier le répresseur spécifique de RamA qui manquait en tant qu’élément de la cascade derégulation actuellement définie. La sélection de 60 souches nous a permis de confirmer le rôlecentral joué par RamA dans la régulation, ainsi qu’identifier des mutations pouvant être critiques, auniveau de RamR. Ainsi, l’absence variations observées dans le régulon marRAB et l’expressionmodérée des transcrits montrée par qRT-PCR laisse penser, que RamA a un rôle clef dans larégulation de l’expression des porines et des pompes d’efflux chez E. aerogenes.L’autre partie de notre travail reposait sur l’étude de la translocation des antibiotiques au traversdes porines. L’étude des interactions porine-carbapénèmes s’est faite sur la porine sauvage OmpFd'Escherichia coli et deux mutations. Les résultats indiquent également l'importance de l'aspartateen position 113 dans la sélectivité de translocation des carbapénèmes au sein de la porine OmpF.Ce travail montre ainsi que la translocation des pénicillines est aussi sous la dépendance desinteractions qui se créent entre le substrat et le résidu en position 113 de OmpF et limitent alorsleur passage au niveau du canal porine. Nous avons recherché la contribution attribuée à la porineOmp36 d'E. aerogenes dans la translocation de certaines béta-lactamines. Les mesures ont permisde conclure que les deux beta-lactamin / Genetic permeability plays an important role in antibiotic resistance of Gram-negative bacteria.Our work was to characterize and better understand of the genetic regulation of membranepermeability in E. aerogenes. We focused on two global regulators, mar and ram, in about 60clinical isolates. Alterations in the upstream region of ramA and in ramR but no mutations in marAnor marR were observed. Overexpression of ramA or ramR led to an altered antibiotic susceptibilityassociated to decrease of porins expression and over-expression of efflux-pumps. qRT-PCR pointedout the estimated importance of the ram-regulon in the regulation cascade.Another part of this work was to characterize the translocation of compounds through porins andthe role of porins in drug uptake in general. Measurement of the rate of antibiotic action of threecarbapenems in an E. coli strain solely expressing OmpF as porin clearly indicated the importanceof the aspartate at position 113 in antibiotic translocation. A multi-disciplinary three way approachof computer modeling, black-lipid-bilayer assays and measurement of antibiotic action, suggestedthat interactions with residue D113 of E. coli porin OmpF are rate-limiting for transport throughthe porin channel. Combination of biological and biophysical measurements with E. aerogenesporin Omp36 denoted that interactions between the porin channel and the antibiotic facilitate andaccelerate transport.

Le but de ce travail est d’étudier le lien suspecté entre l’inflammation buccale et la survenue des AVC ischémiques. Dans les pays occidentaux, l’incidence des infarctus cérébraux est en augmentation malgré les campagnes de prévention visant à limiter l’exposition aux facteurs de risque classiques des pathologies ischémiques. Près de 9% des accidents vasculaires cérébraux sont sans étiologie connue. Le facteur déclenchant de l’AVC ischémique ou le « key trigger » reste inconnu. Des études récentes montrent qu’un AVC ischémique est plus susceptible de se déclencher dans la semaine qui suit un événement infectieux. De ce fait, l’inflammation buccale entraînant une élévation de différents biomarqueurs inflammatoires est susceptible de favoriser la survenue des AVC. Dans un premier temps, une méta-analyse a été effectuée afin de faire la synthèse des données étudiant la relation entre l’inflammation buccale et la survenue des AVC. Elle a montré que le risque d’avoir un AVC ischémique fatal augmente de 38% chez les sujets atteints de parodontite sévère. Dans un deuxième temps, deux études cliniques observationnelles ont été mises en place afin de renforcer la validité des liens épidémiologiques supposés et d’apporter de nouveaux éléments dans la compréhension des mécanismes physiopathologiques liant l’inflammation buccale et la survenue des AVCI. Les résultats montrent une relation entre le degré d’inflammation buccale et les marqueurs biologiques athéromateux et inflammatoires. En effet, nos résultats montrent une augmentation des taux de CRP, de VLDL, de triglycérides et une diminution des taux de HDL lors d’une atteinte parodontale sévère. C’est la perte osseuse parmi les marqueurs cliniques de l’inflammation buccale aisément évaluable sur un panoramique dentaire, qui est la plus significativement liée au risque de la survenue des AVC ischémiques. Les résultats de cette thèse suggèrent que la présence d’un contexte buccal inflammatoire favoriserait la survenue le développement d’un AVCI. En outre, nos résultats confirment la nécessité d’une coopération entre l’odontologue et le neurologue afin d’améliorer la prise en charge du risque vasculaire chez un patient ayant un AVCI avec une inflammation buccale. / The aim of this work is to investigate the suspected link between oral inflammation and the occurrence of ischemic stroke. In Western countries, the incidence of ischemic stroke is rising despite prevention campaigns aiming at limiting the exposure to common risk factors for ischemic diseases. Nearly 9% of strokes are of unknown etiology. The triggering factor for ischemic stroke or "trigger key" remains unknown. Recent studies have shown that ischemic stroke is more likely to occur in the week following an infectious event. Therefore, oral inflammation, that causes a rise in various inflammatory biomarkers is studied as potentially increasing the risk of stroke. Firstly, a meta-analysis was performed to synthesize data about the relationship between oral inflammation and the occurrence of stroke. It has shown that the risk of fatal ischemic stroke increases by 38% in patients with severe periodontitis. Secondly, two observational clinical studies have been implemented to strengthen the validity of the supposed epidemiological links and bring new elements in our understanding about the pathophysiological mechanisms linking oral inflammation and the occurrence of ischemic stroke. The results show a proportional relationship between the degree of oral-inflammation and biological assessments that demonstrate pro-atherosclerotic and pro-inflammatory state. Indeed, we observe an increase in CRP levels, VLDL triglycerides and a decrease in HDL in patients with severe periodontal disease. Bone loss, that is easily measurable on a dental panoramic radiograph, appears to be the main risk factor of the occurrence of ischemic stroke.The results of this thesis show that the presence of an inflammatory oral environment is an additional marker for the discovery of a cardiovascular risk in patients combining other conventional risk factors of ischemic stroke. In addition, our results suggest the need for cooperation between the neurologists and odontologists to improve the management of cardiovascular risk in patients with ischemic stroke and oral inflammation.

Orientador: Carlos Emílio Levy / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T01:21:38Z (GMT). No. of bitstreams: 1 CarvalhoFilho_ElioBarreto_M.pdf: 1484872 bytes, checksum: da445ed854a5a8f11143d06dd28aab5c (MD5) Previous issue date: 2015 / Resumo: Introdução: Bacilos gram-negativos não fermentadores (BGN-NFs) são microrganismos que se caracterizam pela incapacidade de utilizar a glicose como fonte de energia pela fermentação, degradando-a pela via oxidativa. A identificação dos BGN-NFs continua sendo um desafio para os laboratórios de rotina em microbiologia pela dificuldade de identificação, em virtude, da baixa ocorrência em amostras ambulatoriais, assim como, pela falta de recursos rápidos, eficientes e pela complexidade e alto custo dos testes de identificação. Estes microrganismos têm importância clínica para pacientes imunocomprometidos com infecções sistêmicas e pacientes com Fibrose Cística, com infecções oportunistas pulmonares. Nosso objetivo foi utilizar técnicas fenotípicas e de espectrometria de massas para identificar BGN-NFs pouco frequentes isolados em amostras clínicas de pacientes com FC e de pacientes internados no Hospital de Clínicas-Unicamp. Método: Foram analisados 71 isolados de amostras clínicas recuperadas do Banco de bactérias do Laboratório de Microbiologia do HC-Unicamp, envolvendo Chryseobacterium indologenes, Elizabethkingia meningoseptica, Cupriavidus pauculus, Ochrobactrum anthropi, Ralstonia pickettii, Ralstonia mannitolilytica, Ralstonia insidiosa, identificados por técnicas fenotípicas (provas bioquímicas manuais padronizadas pelo Laboratório de Microbiologia [método fenotípico manual] e automatizadas pelo Vitek®2, Phoenix¿) e por espectrometria de massa MALDI-TOF MS (BioMerieux®) e MALDI BD (Becton& Dickson®). Para 41 isolados de Ralstonia spp e Ochrobactrum anthropi estes métodos também foram comparados com PCR, pois não foram encontrados oligonucleotídeos específicos para os demais BGN-NFs estudados. Resultados: pelo método Fenotípico Manual foi possível identificar ao nível de espécie 54,9% dos isolados, apenas gênero 19,7% e 25,4% não puderam ser identificados. Houve uma baixa concordância entre as técnicas Fenotípica Manual e a Automatizada Vitek®2, sendo de 50,7%, quando considerada a concordância pelo menos ao nível de gênero. A maior concordância verificou-se entre os dois equipamentos de MALDI-TOF, de 87,3%, quando considerada a concordância pelo menos ao nível de gênero. Discussão: Quando comparamos todos os métodos utilizados para identificação dos 71 isolados encontramos uma concordância simultânea de 23,9% (17/71), quando considerado pelo menos ao nível de gênero e apenas 2 (2,8%) ao nível de espécie. Quando comparamos os métodos Fenotípico Manual, Vitek®2, Phoenix®, MALDI MS, MALDI BD e incluindo PCR para a identificação de 41 amostras de Ralstonia spp e Ochrobactrum anthropi, encontramos uma concordância simultânea de 19,5% (8/41) quando considerado pelo menos ao nível de gênero e 2 (2,8%) de espécie. Possíveis justificativas para a baixa concordância entre as metodologias seriam a diversidade de princípios, de acurácia e de bancos de dados dos métodos. Conclusão: É muito baixa a concordância entre as diferentes metodologias utilizadas, sendo que os equipamentos de MALDI-TOF possuem entre si uma correlação muito boa para identificação de BGN-NFs, porém mostram-se discordantes aos níveis de confiança dos resultados. Foi encontrada uma importante limitação na PCR apresentando reação cruzada com gêneros testados, sugerindo não ser confiável para este fim. Para os gêneros estudados e as metodologias empregadas, a discordância dos resultados sugerem cautela pela possibilidade de erro de identificação / Abstract: Introduction: Gram-negative non-fermenting (GN-NFB) are microorganisms that are characterized by the inability to use glucose by fermentation as an energy source, degrading it by the oxidative pathway. The identification of BGN-NFS remains a challenge for the routine of microbiology laboratories, due to the low occurrence in outpatient samples, the lack of fast and efficient resources and the complexity and high cost of the tests. These microorganisms are clinically important for immunocompromised patients with systemic infections and patients with Cystic Fibrosis, with opportunistic pulmonary infections. Our goal was to use phenotypic and mass spectrometry techniques to identify uncommon GN-NFB isolated in samples from CF patients and patients admitted to the Hospital de Clinicas Unicamp. Method: 71 isolates recovered from clinical specimens were selected from the from the Microbiology Laboratory bacteria collection, previously identified by fenotipic methods as Chryseobacterium indologenes, Elizabethkingia meningoseptica, Cupriavidus pauculus, Ochrobactrum anthropi, Ralstonia pickettii, Ralstonia mannitolilytica, Ralstonia insidiosa. The samples were identified by in house Phenotypic manual method, automated by Vitek®2, by Phoenix¿ and MALDI-TOF mass spectrometry MS (BioMerieux®) and MALDI BD (Becton Dickson®). These methods were also compared to PCR for only 41 isolates of Ralstonia spp and Ochrobactrum anthropi, because they were not found specific primers for the other GN-NFB analised. Results: the phenotypic Manual method identified to species level 54.9% of the isolates, 19.7% only to genera and 25.4% were not identified. There was a poor correlation between the techniques phenotypic Manual method and Automated Vitek®2, with 50.7% of agreement, when considering at least in terms of gender. The best agreement of 87.3% occurred between the results of the two MALDI-TOF equipment, when considering the correlation of at least at the level of genus. Discussion: When comparing all the methods used for the identification of 71 isolates were found simultaneous agreement of 23.9% (17/71) when considered at least to genus level and only 2 (2.8%) to species level. When analyzed the phenotypic Manual method, Vitek®2, Phoenix®, MALDI MS, MALDI BD and PCR applied to 41-GN-NFB samples, simultaneous agreement were found for 19.5% (8/41) when considered at least to genus level and 2 (2.8%) to species. Possible reasons for the low agreement between the methodologies could be the diversity of principles, accuracy and databases of the methods. Conclusion: The two methods of MALDI-TOF have a very good correlation for GN-NFB identification, however showed discordance for the confidence levels of results. With the primers tested one important limitation of the PCR is the cross-reaction among many genera, suggesting not to be safe for the discrimination of the analyzed genera. For the genera and methods analised the discrepancy of results suggests caution because of the possibility of wrong identification / Mestrado / Saude da Criança e do Adolescente / Mestre em Ciências

Fímbrias do tipo IV estão associadas a diversos fenótipos em bactérias gram-negativas, e o presente estudo consistiu na caracterização da fímbria Pil e investigação de seu papel na adesão bacteriana de isolados de EPEC atípica. Por PCR e RT-PCR foram investigadas a presença e a funcionalidade do operon Pil e os resultados demonstraram que este está sendo transcrito somente nos isolados BA558 e BA956. Os genes pilS e pilV foram clonados em vetor de expressão para obtenção das proteínas Pil recombinantes e produção de anticorpos policlonais. A análise qualitativa dos testes de inibição da adesão utilizando os soros anti-PilS e anti-PilV juntos demonstraram que o isolado BA558 apresentou mudança de fenótipo de adesão. Esses resultados nos permitem concluir que o operon Pil está funcional em BA558 e BA956, e a expressão da fímbria Pil nessas cepas não está relacionada à formação de biofilme e autoagregação, porém a proteína fimbrial PilS juntamente com a adesina PilV parecem exercer uma função acessória importante na interação de BA558 às células HEp-2. / Type IV fimbriae are associated with several phenotypes in gram-negative bacteria. The aim of this study was the characterization of the Pil fimbria and its role in the interaction of atypical EPEC isolates in bacterial adhesion. Using PCR and RT-PCR, we investigated the presence and functionality of the pil operon genes. The results showed that these genes are transcribed only in the BA558 and BA956 isolates. The pilS and pilV genes were cloned into an expression vector for recombinant proteins and polyclonal antibodies production. Qualitative analysis of the adherence inhibition assays using both rabbit sera changed to localized-like the phenotype of BA558 isolate adhesion. Together, these results allow us to conclude that the Pil operon is functional only in the BA558 and BA956 isolates and that the expression of Pil fimbriae in aEPEC is not related to biofilm formation and autoaggregation but, the fimbrial PilS protein together with PilV adhesin seem to play an important accessory function in the interaction between the BA558 and epithelial cells in vitro.

Infecção do Trato Urinário (ITU) é o segundo tipo de infecção bacteriana mais comum em crianças. Nesse estudo amostras de urina de 6012 pacientes pediátricos foram analisadas, a prevalência de ITU foi determinada, os uropatógenos foram identificados e o perfil antimicrobiano dos mesmos foi determinado. Os resultados mostraram que a prevalência de ITU varia de acordo com o sexo e a idade do paciente. Bactérias Gram-negativas foram responsáveis por 89 % de todos os casos de ITU e Escherichia coli foi a espécie mais prevalente. Os uropatógenos foram resistentes a ampicilina 63 %, a nitrofurantoina 37 % e ao trimethoprim-sulfamethoxazole 28 %. Todavia, 99 % deles foram sensíveis a cefalexina e 96 % ao cloranfenicol. Resultados obtidos com a caracterização de 90 isolados de E. coli, mostraram que todas as amostras foram positivas para os marcadores fimA e fimH, 53 % para pap, 32 % para sfa, 10 % para o marcador genético da toxina pic e 29 % foram capazes de produzir hemolisina-a. Esses isolados se distribuíram entre os grupos filogenéticos da seguinte maneira: B2 42 %, D 25 %, A 21 % e B1 11 %. Dessas amostras 19 % não foram tipáveis (ONT), 15,56 % pertenceram ao sorogrupo O2 e 12,22 % aos sorogrupos O6 e OR. A maioria dos isolados de E. coli aderiu às células epiteliais, poliestireno e PVC. / Urinary Tract Infection (UTI) is the second most common type of bacterial infection in children. In this study, 6012 urine samples from pediatric patients were analyzed, the prevalence of UTI was determined, the uropathogens were identified and their antimicrobial profile was determined. The results have shown that the prevalence of UTI varies according to the sex and age of the patient. Gram negative bacteria were responsible for 89 % of all cases of UTI and E. coli was the most prevalent species. The uropathogens were resistant to: ampicillin 63 %, nitrofurantoin 37 % and trimethoprim-sulfamethoxazole 28 %. However, 99 % of them were sensitive to cephalexin and 96 % to chloramphenicol. Results obtained with the characterization of 90 isolates of E. coli showed that all of them were positive for fimA and fimH, 53 % were positive for pap, 32 % were positive for sfa, 10 % were positive for the genetic marker of pic and 29 % were able to produce hemolysin-a. These isolates were distributed between the phylogenetic groups as follows: B2 42 %, D 25 %, A 21 % and B1 11 %. Nineteen percent of these samples were untypeable (ONT), 15.56 % belonged to O2 serogroup and 12,22 % belonged to the O6 and OR serogroups. Most E. coli isolates were able to adhere to epithelial cells, polystyrene and PVC.

Bordetella pertussis e Bordetella parapertussis são os principais agentes causadores da coqueluche no homem. O óxido nítrico é fundamental para o controle de diversos processos fisiopatológicos. Neste trabalho analisamos sinais moleculares envolvidos na produção de NO em macrófagos derivados de medula óssea murina (BMDMO) infectadas por Bpertussis e Bparapertussis. Nossos resultados mostraram que BMDMO de C57BL/6 estimulados com Bpertussis não sintetizaram níveis significativos de nitrito, ao contrário da infecção com Bparapertussis. BMDMO de C57BL/6 infectados por Bpertussis e Bparapertussis produziram níveis elevados de arginase e de TGFb e esta produção foi dependente de TLR4, porém a produção de NO pelos BMDMO de C3H/HeJ infectados com Bparapertussis foi independente deste receptor. A adição exógena de PT em BMDMO infectados com Bparapertussis reduziu a quantidade de NO sintetizada. Concluímos que TGFb e arginase contribuem para o controle da produção de NO durante a infecção in vitro de BMDMO com Bpertussis e este mecanismo depende de LPS envolvendo TLR4 e PT. / Bordetella pertussis and Bordetella parapertussis are the main etiologic causes of human whooping cough. Nitric oxide (NO) is crucial for several physiopathologic events. Herein we analyzed the molecular signals required for NO production by murine bone marrow-derived macrophages (BMDM) infected with Bpertussis or Bparapertussis. Our data show that BMDM obtained from C57Bl/6 mice was not able to produce measurable levels of nitrite when stimulated with Bpertussis while infection of these cells with Bparapertussis induced high levels of nitrite. Arginase and TLR4-dependent TGF-b were produced in response to infection with either Bpertussis or Bparapertussis. NO production by BMDM obtained from C3H/HeJ mice occurred after Bparapertussis infection in the absence of TLR4. Addition of pertussis toxin to the C57Bl/6 BMDM cultures infected with Bparapertussis decreased NO levels. In conclusion, TGF-b and arginase play a role controlling NO production by BMDM during in vitro infection by Bpertussis. This effect depends on the presence of LPS-TLR4 and PT signaling pathways.