[pt] As infecções por bactérias Gram-negativas Resistentes aos Carbapenêmicos (CR-GNB) estão entre as maiores preocupações atuais da área da, especialmente em Unidades de Terapia Intensiva (UTI), e podem estar associadas ao aumento do tempo de hospitalização, morbidade, custos e mortalidade. Esta tese tem como objetivo desenvolver uma abordagem abrangente e sistemática aplicando técnicas de aprendizado de máquina para construir modelos para prever a aquisição de CR-GNB em UTIs de hospitais brasileiros. Propusemos modelos de triagem para detectar pacientes que não precisam ser testados e um modelo de risco que estima a probabilidade de pacientes de UTI adquirirem CR-GNB. Aplicamos métodos de seleção de características, técnicas de aprendizado de máquina e estratégias de balanceamento para construir e comparar os modelos. Os critérios de desempenho escolhidos para avaliação foram Negative Predictive Value (NPV) and Matthews Correlation Coefficient (MCC) para o modelo de triagem e Brier score e curvas de calibração para o modelo de risco de aquisição de CR-GNB. A estatística de Friedman e os testes post hoc de Nemenyi foram usados para testar a significância das diferenças entre as técnicas. O método de ganho de informações e a mineração de regras de associação avaliam a importância e a força entre os recursos. Nosso banco de dados reúne dados de pacientes, antibióticos e microbiologia de cinco hospitais brasileiros de 8 de maio de 2017 a 31 de agosto de 2019, envolvendo pacientes hospitalizados em 24 UTIs adultas. As informações do laboratório foram usadas para identificar todos os pacientes com teste positivo ou negativo para CR-GNB, A. baumannii, P. aeruginosa ou Enterobacteriaceae. Há um total de 539 testes positivos e 7.462 negativos, resultando em 3.604 pacientes com pelo menos um exame após 48 horas de hospitalização. Dois modelos de triagem foram propostos ao tomador de decisão do hospital. O modelo da floresta aleatória reduz aproximadamente 39 por cento dos testes desnecessários e prevê corretamente 92 por cento dos positivos. A rede neural evita testes desnecessários em 64 por cento dos casos, mas 24 por cento dos testes positivos são classificados incorretamente. Os resultados mostram que as estratégias de amostragem tradicional, SMOTEBagging e UnderBagging obtiveram melhores resultados. As técnicas lineares como Regressão Logística com regularização apresentam bom desempenho e são mais interpretáveis; elas não são significativamente diferentes dos classificadores mais complexos. Para o modelo de risco de aquisição, o Centroides Encolhidos Mais Próximos é o melhor modelo com um Brier score de 0,152 e um cinto de calibração aceitável. Desenvolvemos uma validação externa a partir de 624 pacientes de dois outros hospitais da mesma rede, encontrando bons valores de Brier score (0,128 and 0,079) em ambos. O uso de antibióticos e procedimentos invasivos, principalmente ventilação mecânica, são os atributos mais importantes e significativos para a colonização ou infecção de CR-GNB. Os modelos preditivos podem ajudar a evitar testes de rastreamento e tratamento inadequado em pacientes de baixo risco. Políticas de controle de infecção podem ser estabelecidas para controlar a propagação dessas bactérias. A identificação de pacientes que não precisam ser testados diminui os custos hospitalares e o tempo de espera do laboratório. Concluímos que nossos modelos apresentam bom desempenho e parecem suficientemente confiáveis para prever um paciente com esses patógenos. Esses modelos preditivos podem ser incluídos no sistema hospitalar. A metodologia proposta pode ser replicada em diferentes ambientes de saúde. / [en] Infections by Carbapenem-Resistant Gram-negative bacteria (CR-GNB) are among the most significant contemporary health concerns, especially in intensive care units (ICUs), and may be associated with increased hospitalization time, morbidity, costs, and mortality. This thesis aims to develop a comprehensive and systematic approach applying machine-learning techniques to build models to predict the CR-GNB acquisition in ICUs from Brazilian hospitals. We proposed screening models to detect ICU patients who do not need to be tested and a risk model that estimates ICU patients probability of acquiring CR-GNB. We applied feature selection methods, machine-learning techniques, and balancing strategies to build and compare the models. The performance criteria chosen to evaluate the models were Negative Predictive Value (NPV) and Matthews Correlation Coefficient (MCC) for the screening model and Brier score and calibration curves for the CR-GNB acquisition risk model. Friedman s statistic and Nemenyi post hoc tests are used to test the significance of differences among techniques. Information gain method and association rules mining assess the importance and strength among features. Our database gathers the patients, antibiotic, and microbiology data from five Brazilian hospitals from May 8th, 2017 to August 31st, 2019, involving hospitalized patients in 24 adult ICUs. Information from the laboratory was used to identify all patients with a positive or negative test for carbapenem-resistant GNB, A. baumannii, P. aeruginosa, or Enterobacteriaceae. We have a total of 539 positive and 7,462 negative tests, resulting in 3,604 patients with at least one exam after 48 hours hospitalized. We proposed to the hospital s decision-maker two screening models. The random forest s model would reduce approximately 39 percent of the unnecessary tests and correctly predict 92 percent of positives. The Neural Network model avoids unnecessary tests in 64 percent of the cases, but 24 percent of positive tests are misclassified as negatives. Our results show that the sampling, SMOTEBagging, and UnderBagging approaches obtain better results. The linear techniques such as Logistic Regression with regularization give a relatively good performance and are more interpretable; they are not significantly different from the more complex classifiers. For the acquisition risk model, the Nearest Shrunken Centroids is the best model with a Brier score of 0.152 and a calibration belt acceptable. We developed an external validation of 624 patients from two other hospitals in the same network, finding good Brier score (0.128 and 0.079) values in both. The antibiotic and invasive procedures used, especially mechanical ventilation, are the most important attributes for the colonization or infection of CR-GNB. The predictive models can help avoid screening tests and inappropriate treatment in patients at low risk. Infection control policies can be established to control these bacteria s spread. Identifying patients who do not need to be tested decreases hospital costs and laboratory waiting times. We concluded that our models present good performance and seem sufficiently reliable to predict a patient with these pathogens. These predictive models can be included in the hospital system. The proposed methodology can be replicated in different healthcare settings.

<p dir="ltr">The obligate human pathogen, <i>Neisseria gonorrhoeae </i>(Ngo), has continued to acquire widespread antibiotic resistance. Ngo is the causative agent of the sexually transmitted disease gonorrhea, and can cause additional complications such as endocarditis, septicemia, and infertility if left untreated. The Centers for Disease Control and Prevention (CDC) now recommends a treatment option of a single drug of last resort, ceftriaxone, leaving a need for novel therapeutics against this pathogen.</p><p dir="ltr">Like many bacterial pathogens, Ngo is Gram-negative consisting of both an inner membrane (IM) and outer membrane (OM). The transmembrane proteins in the IM have primarily an α-helical fold, while the transmembrane proteins in the OM have a β-barrel fold. These β-barrel outer membrane proteins (OMPs) have essential functions in regulating the homeostasis and nutrient acquisition of the cell, in addition to promoting virulence in pathogenic strains. These OMPs are folded and inserted into the outer membrane by the β-barrel assembly machinery (BAM) complex. In <i>E. coli,</i> BAM consists of five proteins: BamA, an OMP itself, and four lipoproteins, BamB, C, D, and E.</p><p dir="ltr">Here we present our work toward the structural characterization of BAM from Ngo (<i>Ng</i>BAM) using cryo-EM. Ngo lack a homolog of BamB and may function as a four component complex. To better understand the mechanism for how <i>Ng</i>BAM is able to mediate OMP biogenesis despite lacking a component that is critical in <i>E. coli</i>, we determined the cryo-EM structure of <i>Ng</i>BAM, which revealed several distinct features including that the barrel domain of BamA being observed in the inward-open conformation. We also investigated <i>Ng</i>BAM as a therapeutic target, by studying its interaction with a novel broad spectrum antibiotic darobactin. We first showed darobactin is effective against the laboratory strains of NgoFA19 and ATCC-49226. We also show it is effective against the human isolate WHOX, with a comparable MIC to ceftriaxone. To structurally characterize the mechanism of inhibition by darobactin, we used cryo-EM to determine the structures of <i>Ng</i>BAM bound to two darobactin compounds. In these structures, darobactin binding was accompanied by large conformational changes in <i>Ng</i>BamA. To further probe the effects of darobactin on the conformational plasticity of <i>Ng</i>BAM we performed experiments using double electron-electron resonance spectroscopy, which showed distance changes between the engineered site labels consistent with the conformational changes observed in our structural observation. In addition, narrowing of the peak distributions indicated that darobactin binding was reducing the overall conformational heterogeneity of the complex. Taken together, the work presented here contributes to the understanding of how <i>Ng</i>BAM functions in folding and inserting OMPs and provides a foundation for future structure based drug design of darobactin and other potential compounds.</p>

No / Mono- and disaccharide-functionalised conjugates of the fluoroquinolone antibiotic ciprofloxacin have been synthesised and used as chemical probes of the bacterial uptake of glycosylated ciprofloxacin. Their antimicrobial activities against a panel of clinically relevant bacteria were determined: the ability of these conjugates to inhibit their target DNA gyrase and to be transported into the bacteria was assessed by using in vivo and in vitro assays. The data suggest a lack of active uptake through sugar transporters and that although the addition of monosaccharides is compatible with the inhibition of DNA gyrase, the addition of a disaccharide results in a significant decrease in antimicrobial activity.

Orientador: Elisa Helena Giglio Ponsano / Resumo: O objetivo deste estudo foi investigar a composição química e as atividades antimicrobiana e antioxidante dos extratos etanólicos de Curcuma longa e Bixa orellana, na busca por substituintes aos aditivos sintéticos utilizados na indústria de alimentos. Pela espectrometria de massa (GC-MS) foram identificados bisdemetoxicurcumina, demetoxicurcumina e curcumina no extrato de C. longa e prunina e naringenina no extrato de B. orellana. C. longa apresentou atividade antimicrobiana frente a Clostridium sporogenes e Staphylococcus aureus, com concentração bactericida mínima (CBM) de 25 mg/mL e 156 µg/mL, respectivamente. O extrato de B. orellana apresentou CBM de 50 mg/mL para C. sporogenes e 625 µg/mL para S. aureus. Nenhum dos extratos apresentou atividade bactericida para Escherichia coli e Salmonella Typhimurium. A atividade antioxidante dos extratos foi evidenciada pelos métodos Poder Antioxidante por Redução Férrica (FRAP) e Capacidade de Absorção do Radical Oxigênio (ORAC). O extrato de B. orellana apresentou maior atividade antioxidante pelos métodos FRAP e ORAC (277,70 e 455,17 mM trolox equivalente/g, respectivamente) do que o extrato de C. longa (129,74 e 217,98 mM trolox equivalente/g, respectivamente). Os efeitos biológicos dos extratos etanólicos de C. longa e B. orellana revelados no presente estudo apontaram seu potencial para a utilização na indústria de alimentos como uma alternativa aos aditivos sintéticos. / Abstract: The objective of this study was to investigate the chemical composition and the antimicrobial and antioxidant activities of Curcuma longa and Bixa orellana ethanolic extracts, in the search for alternatives to the synthetic additives used in the food industry. Mass spectrometry (GC-MS), identified bisdemethoxycurcumin, demethoxycurcumin and curcumin in the extract of C. longa and prunin and naringenin in the extract of B. orellana. C. long showed antimicrobial activity against Clostridium sporogenes and Staphylococcus aureus, with a minimum bactericidal concentration (MBC) of 25 mg/mL and 156 μg/mL, respectively. MBC of B. orellana extract was 50 mg/mL for C. sporogenes and 625 μg/mL for S. aureus. None of the extracts showed bactericidal activity against Escherichia coli and Salmonella Typhimurium. The antioxidant activity of the extracts was evidenced by the methods Iron Reduction Antioxidant Power (FRAP) and Oxygen Radical Absorption Capacity (ORAC). B. orellana extract had higher antioxidant activity by FRAP and ORAC (277.70 and 455.17 mM trolox equivalent/g, respectively) than C. longa extract (129.74 and 217.98 mM trolox equivalent/g, respectively). The biological effects of C. longa and B. orellana ethanolic extracts revealed in this study indicated their potential as an alternative to synthetic additives used in the food industry. / Mestre

[Truncated abstract] Pseudomonas aeruginosa, an important opportunistic pathogen, is resistant to a wide array of functionally and structurally diverse antimicrobial agents including antibiotics, disinfectants and biocides. P. aeruginosa is more resistant than other Gram negative bacteria to tea tree oil (TTO), the essential oil steam distilled from the leaves of Melaleuca alternifolia and comprised of over 100 terpene hydrocarbon components and their oxygenated derivatives. TTO is an established topical antimicrobial agent, with antibacterial, antiviral and antifungal properties. Intrinsic antimicrobial resistance mechanisms in P. aeruginosa include the low permeability of the outer membrane and expression of multi-drug efflux pumps. A series of multi-drug efflux mutants from the resistance-nodulation-cell division family was obtained and their susceptibility to TTO and several components examined. This demonstrated that TTO and the components terpinen-4-ol, 1,8-cineole and a-terpineol were substrates of MexAB-OprM, using both pump deletion mutants and the pump inhibitor Phe-arg ß-naphthylamide dihydrochloride. In complementation studies, the addition of mexAB-oprM to deletion mutants restored susceptibility to these agents to that of the wild-type, confirming the role of MexAB-OprM in tolerance to TTO and these three components. ... An increase in susceptibility to ticarcillin and Timentin occurred in PAO1 following serial subculture in terpinen-4-ol. Susceptibility to ticarcillin has been associated with expression of the MexCD-OprJ system in P. aeruginosa. A library of transposon mutants was created to find additional mechanisms by which P. aeruginosa could tolerate TTO. The library yielded a total of 20 mutants that were more susceptible than parental strains to TTO and/or terpinen-4-ol. The insertion site of the transposon was identified in 14 mutants and, in four mutants, this was a gene related to flagellar biosynthesis. Flagella deficient mutants have previously demonstrated enhanced susceptibility to the membrane-disrupting surfactant sodium dodecyl sulfate and this echoes the increased susceptibility to TTO and terpinen-4-ol observed. Three non-sibling surA mutants were also identified. SurA is involved in the correct folding of outer membrane proteins, including porins, in Gram negative bacteria: surA mutants of Escherichia coli have phenotypes that are characteristic of a defective cell envelope, including an increased susceptibility to hydrophobic agents. The increase in susceptibility to hydrophobic TTO and terpinen-4-ol in the surA mutants is consistent with this and represents the first report linking SurA function to antimicrobial resistance in P. aeruginosa. In conclusion, several Mex efflux systems of P. aeruginosa including MexAB-OprM, MexCD-OprJ and MexEF-OprN, as well as the LPS core, outer membrane integrity and a functioning flagella biosynthetic pathway contribute to the tolerance of this organism to TTO and/or several components.

Diss. Linköping : Linköpings universitet, 2007.

No description available.

Actuellement, le traitement des pathologies infectieuses chez les bovins et les animaux de compagnie est menacé par l’accroissement de l’antibiorésistance et des bactéries multirésistantes. Il est donc primordial pour les entreprises pharmaceutiques vétérinaires de développer de nouvelles gammes d’agents antibactériens spécifiques au domaine animale. Dans ce contexte, il a été montré que certains polyaminostérols naturels tels que la squalamine possèdent un fort potentiel antimicrobien. Ainsi, l’objectif de ce travail de thèse est de répondre à la problématique présentée par la société Virbac dans le traitement des mammites (bovins) et des otites/pyodermites (chien) par le développement d’une nouvelle classe de dérivés polyaminostéroïdiens synthétiques à large spectre antimicrobien. Pour cela, nous avons constitué une chimiothèque de composés originaux préparés à partir de différents acides biliaires selon des voies de synthèse inédites. Ces composés ont été évalués in vitro pour leur cytotoxicité et leurs activités antibactériennes contre diverses bactéries à Gram positif et à Gram négatif et nous avons pu établir la preuve de concept in vitro de leur potentiel thérapeutique en tant qu’agents antibactériens ou adjuvants d’antibiotiques. De plus, nous avons montré que l’un de ces nouveaux dérivés, la claramine A1, agit sur l’intégrité physique des membranes bactériennes et sur les performances d'efflux des pompes AcrAB-TolC. Ainsi, il apparaît finalement que ces dérivés polyaminostéroïdiens au mode d’action non conventionnel pourraient constituer une nouvelle classe d’agents antibactériens pour un usage en tant que substituts d’antibiotiques en médecine vétérinaire. / Currently, the treatment of infectious pathologies in cattle and pets is threatened by the growing antimicrobial resistance and the development of multidrug-resistant bacteria. Thus it is necessary for the veterinary pharmaceutical firms to develop new lines of antibacterial agents. In this context, some natural polyaminosterols such as squalamine have gained interest due to their potent antimicrobial activities. Thus the aim of this PhD work is to provide an answer in the treatment of mastitis (cattle) and otitis/pyoderma (dog) by the development of a new class of synthetic polyaminosterols with a broad spectrum of antibacterial activity. In this purpose, a chemical library of original compounds has been prepared starting from various bile acids by using unprecedented synthesis procedures. These compounds were evaluated for their in vitro cytotoxicity on CHO cells as well as their antibacterial activities against Gram-positive and Gram-negative bacteria and we have establish the in vitro proof of concept of the therapeutic potential of this family of molecules as antibacterial agent or antibiotic adjuvant. Additional investigations were then conducted on one of these novel derivatives namely claramine A1 to deepen knowledge of its mechanism of action and showed that claramine A1 acts on the physical integrity of bacterial membranes and the efflux performance of AcrAB-TolC pumps. Based on the results of claramine A1, it finally appears that these new polyaminosterol derivatives possessing a non-classical mode of action pertain to a new class of antibacterial agents and could constitute a substitute for traditional antibiotics in veterinary medicine.

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.