Depuis une dizaine d'année, le rôle des biofilms dans la résistance aux antibiotiques et à la réponse immunitaire a été mis en évidence dans les infections chroniques. Les plaies chroniques possèdent ces mêmes particularités de résistance aux divers traitements. La formation de biofilm dans les plaies induit une protection des bactéries qui participe potentiellement à la chronicité et est responsable de l'inefficacité des soins. Le but de ma thèse est de concevoir un biogel actif contre les biofilms pouvant être présents dans les plaies chroniques.Nous avons développé un gel de gélatine innovant contenant divers agents anti-biofilms et permettant l'éradication des bactéries pathogènes. Ces gels sont des solides mous composés d'une phase liquide emprisonnée dans un réseau de biopolymères. Biocompatibles et biorésorbables, ils présentent des avantages pour une utilisation en tant que biomatériaux pour la santé. Il est possible d'y inclure des molécules qui vont ensuite diffuser vers le milieu extérieur. Pour mieux appréhender cette cinétique de diffusion, nous avons étudié le relargage de molécules modèles de différents poids moléculaires et de différentes charges.La stratégie anti-biofilm adoptée consiste à prévenir la colonisation bactérienne, déstabiliser le biofilm et éradiquer les bactéries pathogènes de la plaie (Diminution de la biomasse d'au moins 5 logarithmes). Pour développer ce système, 3 types de molécules ont été combinées : un antiseptique commercial, utilisé dans le cadre des soins des plaies chroniques, un chélateur d'ions actifs contre les MMPs surexprimées dans les plaies et une protéase capable de dégrader la matrice du biofilm. Cette étude a été réalisée sur des biofilms mono-espèce de P. aeruginosa et S. aureus, bactéries pathogènes fréquemment retrouvées dans les plaies chroniques. La combinaison des principes actifs a été testée en solution, en contact direct avec le biofilm. Puis, les agents ont été encapsulés dans un biogel. Nous avons étudié à la fois les propriétés viscoélastiques de ces gels mais aussi leur efficacité contre un biofilm, comparé au traitement non encapsulé. Lors de ce travail, nous avons élaboré un gel manipulable capable d'éradiquer un biofilm mono espèce ou multi espèces, constitué de souches de laboratoire ou de souches cliniques issues de plaie chronique. / Over the past 10 years, researchers have highlighted the role of biofilms on resistance to immune response and antibiotics treatments of chronic infections.Chronic wounds share these characteristics as they are resistant to care treatments. The possible biofilm formation in wound can protect bacteria participating to the chronicity and the resistance to wound care treatment. The aim of this thesis is to conceive a biogel against biofilms in chronic wounds.We have developed an innovative gelatin gel containing various anti-biofilm agents able to eradicate pathogenic bacteria biofilm. This gel is soft matter composed of a liquid phase entrapped in a biopolymer network. It is biocompatible and bioresorbable; these properties are necessary for a biomaterial. The gel has the capacity to deliver molecules with controlled release. We have studied the release of model molecules with different charges and weights.The anti-biofilm strategy consisted in preventing bacterial colonization, disrupting the biofilm and eradicating pathogen bacteria in wound (a decrease biomass of at least 5 log reductions). The system developed consisted in the combination of three different types of molecules: a commercial antiseptic usually applied in chronic wound care, an ion chelator active against MMPs which are over-expressed in chronic wounds and a protease which can disrupt the matrix of the biofilm.The study has been carried out on mono-species biofilms synthesized in vitro, with P. aeruginosa and S. aureus, two pathogenic bacteria frequently encountered in chronic wounds. The combination of the active agents was tested in solution or in directly contact with the biofilm. Then molecules were entrapped in the biogel. The viscoelastic properties of the gel were studied and the efficacy of the entrapped treatment compared to that of the solution. A handeable and efficient biomaterial has been elaborated during this study. It is able to eradicate mono – and multi- species biofilms from both laboratory and clinical bacterial strains.

Dans un contexte mondial d’émergence de bactéries résistantes aux antibiotiques, il est nécessaire d’explorer de nouvelles voies de recherche pour trouver de nouveaux traitements. Cet état de fait est particulièrement marqué dans le cadre d’infections chroniques associées à une colonisation des tissus par des biofilms bactériens. L’endocrinologie microbienne est un champ de recherche axé sur l’étude des mécanismes de communication inter-règnes qui peut s’établir entre des bactéries et leurs hôtes. Les molécules humaines qui permettent ce dialogue constituent de potentiels outils capables de moduler la physiologie des bactéries pour empêcher leur développement. Dans cette optique, l’objectif de ma thèse était d’approfondir nosconnaissances sur l’effet des peptides natriurétiques, une famille d’hormones humaines, sur la physiologie du pathogène opportuniste P. aeruginosa. Les travaux que j’ai menés ont permis de préciser les mécanismes de l’action anti-biofilm du peptide natriurétique de type C ou CNP. J’ai également montré qu’un autre peptide, le peptide natriurétique atrial (ANP), est capable de disperser un biofilm établis de P. aeruginosa. Dans un second temps, j’ai pu identifier que l’opéron ami, dans son intégralité, est indispensable aux effets des peptides natriurétiques et qu’en plus les protéines codées par les gènes de l’opéron ami ont un rôle important dans la régulation de la virulence bactérienne et dans la formation des biofilms. Ainsi, j’ai pu mettre en évidence que les protéines AmiE et AmiR, en plus de leur rôle dans le métabolisme secondaire, sont impliquées dans la régulation de la virulence et de la formation de biofilm de P. aeruginosa. / In a global context of emergence of antibiotic-resistant bacteria, it is necessary to explore new paths of research to find new treatments. This state of affairs is particularly marked in the context of chronic infections associated with tissue colonization by bacterial biofilms. Microbial endocrinology is a field of research focused on the study of inter-kingdom communication that can be established between bacteria and their hosts. The human molecules that allow this dialogue are potential tools capable of modulating bacterial physiology to prevent their development. In this perspective, the aim of my thesis was to deepen our knowledge about the effect of natriuretic peptides, a family of human hormones, on the physiology of the opportunistic pathogen P. aeruginosa. The work that I carried out allowed us to characterize the mechanisms of the anti-biofilm action of the natriuretic peptide type C or CNP. I have also shown that another peptide, the atrial natriuretic peptide (ANP), is able to disperse an established biofilm of P. aeruginosa. In a second step, I was able to identify that the entire ami operon is essential for the effects of the natriuretic peptides and that the proteins encoded by the genes of the ami operon have an important role in bacterial virulence regulation and in the formation of biofilms. Thus, I was able to demonstrate that the AmiE and AmiR proteins, in addition to their role in secondary metabolism, are involved in the regulation of virulence and biofilm formation of P. aeruginosa.

La contamination microbienne des surfaces est l’une des préoccupations majeures des secteurs d’activités comme l’industrie agro-alimentaire, la santé publique et les milieux hospitaliers. Face aux problèmes de santé publique liés à contamination bactérienne sur les surfaces, la préparation de surfaces aux propriétés antibactériennes est devenue un intérêt de recherche majeur pour de nombreux scientifiques et ce, dans de nombreux domaines de recherches. Du point de vue de la chimie, des matériaux et de la microbiologie, la fonctionnalisation des surfaces de matériaux polymères préexistants sans altérer leur propriété initiale est une solution séduisante. Pour cela, développer des nouveaux matériaux antibactériens/antifouling où la surface serait fonctionnalisée par des polymères antimicrobiens, greffés de manière robuste i.e. de façon covalente représente une solution idéale. Afin de faciliter et d’accélérer le processus de criblage, il est proposé dans ce travail une nouvelle approche pour obtenir des polymères ayant des propriétés antimicrobiennes à la fois en solution et à partir de la surface. Ce travail comprend une étude de (co)-polymérisations contrôlées d'esters actifs servant d’intermédiaires pouvant être post-modifiés pour synthétiser des polymères d'intérêts présentant les caractéristiques antimicrobiennes attendues.Ce travail démontre que la polymérisation radicalaire contrôlée en présence de Cu(0)/Cu(II) est une technique appropriée qui permet de préparer facilement des (co)-polymères réactifs, en solution mais aussi à partir de surface de poly (téréphtalate d’éthylène), communément appelé PET. Dans un premier temps, nous aborderons l'étude de la polymérisation contrôlée du méthacrylate de pentafluorophényle (PFPMA), avec son optimisation en solution, puis à partir de surface du PET porteuse de groupement d’amorçage. De plus, la polymérisation du méthacrylate de p-nitrophényle (NPMA) sera également examinée, ainsi que la copolymérisation des deux esters actifs là-encore par polymérisation de type contrôlée en présence de Cu(0)/Cu(II). La post-modification des polymères activés est ensuite présentée. La post-modification s’est révélée efficace et facile à mettre en œuvre. La structure et les caractéristiques des polymères obtenus ont été analysées et confirmées. Il est à noter que la post-modification a pu être effectuée par un processus séquentiel avec une fonctionnalisation simple ou avec plusieurs huiles essentielles, qui possèdent des propriétés naturelles antibactériennes ou antioxydantes. Différents films de PET ont été modifiés, des polymères aux propriétés anti-adhérentes ont été greffés par cette même méthodologie. Ces surfaces modifiées ont été testées contre deux bactéries modèles telles que Staphylococcus aureus et Pseudomonas aeruginosa afin de déterminer si les modifications de surface ont conféré au film de PET les propriétés biologiques attendues. / Microbial contamination on surfaces has become major concern in various areas including industrial process as well as public health and hospitalization. Being aware of several problems causing by adherence and attachment of bacteria on a surface, preparation of antibacterial surface has become a global research interest for researchers in many domains. From the chemistry integrated with material science and microbiology point of view, functionalization of existing polymeric material surfaces is an attractive solution. In this domain, the surface functionalized with covalently grafted antimicrobial polymers represents an ideal solution. In order to facilitate the screening process, it is proposed in this particular research a new approach to obtain polymers with antimicrobial properties both in solution and from surface. The present approach includes a study in controlled (co)polymerization of active ester(s) serving as intermediate templates that can be eventually modified by polymer post-modification process to fabricate polymer of interest with expected antimicrobial characteristics.In general, it is demonstrated herein that the use of Cu(0)-mediated reversible deactivation radical polymerization (RDRP) is a suitable technique that allows facile preparation of reactive (co)polymers in solution and from surface of poly(ethylene terephthalate). First of all, this thesis focused on the study of controlled polymerization of pentafluorophenyl methacrylate (PFPMA) which appeared to be challenging. Furthermore, along with the optimization of polymerization in solution was the investigation of surface-initiated polymerization of this monomer from PET surface. Besides, polymerization of p-nitrophenyl methacrylate (NPMA) and copolymerization of the two active esters by Cu(0)-mediated RDRP were also examined. In addition, polymer post-modification of obtained (co)polymers with various compounds had been proven to be efficient, easy to perform. The structure and characteristics of obtained products were confirmed to match with expectations. It is remarkable that the post-modification can be done as sequential process, single or dual functionalization with several different essential oils, which are natural antibacterial or antioxidant compounds. On the other hand, the success in polymerization and post-modification of polymer of active esters in solution allowed the fabrication of different PET film grafted with polymers that are envisaged to have antiadhesion properties. Attempts to test such properties were also done against two model bacteria including Staphylococcus aureus and Pseudomonas aeruginosa to investigate if expectations are valid.

Muitas espécies de pimentas vermelhas do gênero Capsicum são utilizadas em práticas medicinais tradicionais. Essas plantas são empregadas em algumas preparações para tratar uma variedade de doenças, incluindo infecções. Algumas bactérias produzem biofilme como um importante fator de virulência, pois a estrutura do biofilme intermedia a adesão bacteriana a superfícies, como em dispositivos implantados, sondas e cateteres além de promover proteção física contra os antibióticos ou as respostas do sistema imunológico. Dessa maneira, este estudo investigou a capacidade do extrato e de produtos isolados das sementes de Capsicum baccatum como agentes antibiofilme. Este estudo demonstra, pela primeira vez, que um extrato de C. baccatum apresentou importante atividade antibiofilme contra Staphylococcus epidermidis e Pseudomonas aeruginosa. A fração ativa foi obtida através de ensaios bioguiados e analisada por HPLC-DAD-MS, MALDI-TOF MS e MALDI-MS/MS, identificando-a como peptídeos da proteína 2S sulfur-rich seed storage protein 2-like. Estes peptídeos (2mg/ml) foram potentes no controle da formação de biofilme de S. epidermidis (>96%) em solução e adsorvidos em lâminas de Permanox® recobertas. De modo interessante, não inibiram o crescimento bacteriano, indicando que a inibição do biofilme é independente da morte celular bacteriana. Ainda, esses peptídeos foram capazes de preservar eritrócitos, bem como a integridade de linfócitos humanos após 24 e 48 horas de exposição, demonstrando que o fracionamento do extrato de C. baccatum potencializou a sua atividade antibiofilme e reduziu significativamente a sua citotoxicidade. Nossos resultados corroboram com a pesquisa de novas estratégias não antibióticas para combater microrganismos com reduzida possibilidade para o desenvolvimento de resistência. / Many species of Capsicum red peppers are used in traditional medicinal practices. These plants are utilized in a number of preparations to treat a variety of illnesses including infections. Some bacteria produce biofilm as an important virulence factor, due to this its structure mediates the adhesion to surfaces as implanted devices, probes, catheters and also promotes physical protection against the antibiotics or the immune system response. Accordingly, this study investigated the ability of the extract and isolated products from seeds of Capsicum baccatum as anti-biofilm agent. This study demonstrates by the first time that an extract from C. baccatum presented relevant anti-biofilm activity against Staphylococcus epidermidis and Pseudomonas aeruginosa. The active fraction was obtained by bio-guided assays and analyzed by HPLC-DAD-MS, MALDI-TOF MS and MALDI-MS/MS, identifying it as peptides from 2S sulfur-rich seed storage protein 2-like. It strongly controlled (2mg/ml) the S. epidermidis biofilm formation (>96%) when the compound was in solution and adsorbed on Permanox™ slides. Interestingly, it did not inhibit the growth of this bacterium, indicating the inhibition of biofilm is independent of bacterial cell death. Moreover, this peptides preserved human erythrocytes and lymphocytes integrity after 24-48 h of exposure, suggesting the fractionation potentiated the anti-biofilm activity of the C. baccatum crude extract while absolutely reduced its cytotoxicity. Our results corroborate to the search of new non-antibiotic strategies to combat microorganisms with a reduced pressure for resistance development.

Muitas espécies de pimentas vermelhas do gênero Capsicum são utilizadas em práticas medicinais tradicionais. Essas plantas são empregadas em algumas preparações para tratar uma variedade de doenças, incluindo infecções. Algumas bactérias produzem biofilme como um importante fator de virulência, pois a estrutura do biofilme intermedia a adesão bacteriana a superfícies, como em dispositivos implantados, sondas e cateteres além de promover proteção física contra os antibióticos ou as respostas do sistema imunológico. Dessa maneira, este estudo investigou a capacidade do extrato e de produtos isolados das sementes de Capsicum baccatum como agentes antibiofilme. Este estudo demonstra, pela primeira vez, que um extrato de C. baccatum apresentou importante atividade antibiofilme contra Staphylococcus epidermidis e Pseudomonas aeruginosa. A fração ativa foi obtida através de ensaios bioguiados e analisada por HPLC-DAD-MS, MALDI-TOF MS e MALDI-MS/MS, identificando-a como peptídeos da proteína 2S sulfur-rich seed storage protein 2-like. Estes peptídeos (2mg/ml) foram potentes no controle da formação de biofilme de S. epidermidis (>96%) em solução e adsorvidos em lâminas de Permanox® recobertas. De modo interessante, não inibiram o crescimento bacteriano, indicando que a inibição do biofilme é independente da morte celular bacteriana. Ainda, esses peptídeos foram capazes de preservar eritrócitos, bem como a integridade de linfócitos humanos após 24 e 48 horas de exposição, demonstrando que o fracionamento do extrato de C. baccatum potencializou a sua atividade antibiofilme e reduziu significativamente a sua citotoxicidade. Nossos resultados corroboram com a pesquisa de novas estratégias não antibióticas para combater microrganismos com reduzida possibilidade para o desenvolvimento de resistência. / Many species of Capsicum red peppers are used in traditional medicinal practices. These plants are utilized in a number of preparations to treat a variety of illnesses including infections. Some bacteria produce biofilm as an important virulence factor, due to this its structure mediates the adhesion to surfaces as implanted devices, probes, catheters and also promotes physical protection against the antibiotics or the immune system response. Accordingly, this study investigated the ability of the extract and isolated products from seeds of Capsicum baccatum as anti-biofilm agent. This study demonstrates by the first time that an extract from C. baccatum presented relevant anti-biofilm activity against Staphylococcus epidermidis and Pseudomonas aeruginosa. The active fraction was obtained by bio-guided assays and analyzed by HPLC-DAD-MS, MALDI-TOF MS and MALDI-MS/MS, identifying it as peptides from 2S sulfur-rich seed storage protein 2-like. It strongly controlled (2mg/ml) the S. epidermidis biofilm formation (>96%) when the compound was in solution and adsorbed on Permanox™ slides. Interestingly, it did not inhibit the growth of this bacterium, indicating the inhibition of biofilm is independent of bacterial cell death. Moreover, this peptides preserved human erythrocytes and lymphocytes integrity after 24-48 h of exposure, suggesting the fractionation potentiated the anti-biofilm activity of the C. baccatum crude extract while absolutely reduced its cytotoxicity. Our results corroborate to the search of new non-antibiotic strategies to combat microorganisms with a reduced pressure for resistance development.

Muitas espécies de pimentas vermelhas do gênero Capsicum são utilizadas em práticas medicinais tradicionais. Essas plantas são empregadas em algumas preparações para tratar uma variedade de doenças, incluindo infecções. Algumas bactérias produzem biofilme como um importante fator de virulência, pois a estrutura do biofilme intermedia a adesão bacteriana a superfícies, como em dispositivos implantados, sondas e cateteres além de promover proteção física contra os antibióticos ou as respostas do sistema imunológico. Dessa maneira, este estudo investigou a capacidade do extrato e de produtos isolados das sementes de Capsicum baccatum como agentes antibiofilme. Este estudo demonstra, pela primeira vez, que um extrato de C. baccatum apresentou importante atividade antibiofilme contra Staphylococcus epidermidis e Pseudomonas aeruginosa. A fração ativa foi obtida através de ensaios bioguiados e analisada por HPLC-DAD-MS, MALDI-TOF MS e MALDI-MS/MS, identificando-a como peptídeos da proteína 2S sulfur-rich seed storage protein 2-like. Estes peptídeos (2mg/ml) foram potentes no controle da formação de biofilme de S. epidermidis (>96%) em solução e adsorvidos em lâminas de Permanox® recobertas. De modo interessante, não inibiram o crescimento bacteriano, indicando que a inibição do biofilme é independente da morte celular bacteriana. Ainda, esses peptídeos foram capazes de preservar eritrócitos, bem como a integridade de linfócitos humanos após 24 e 48 horas de exposição, demonstrando que o fracionamento do extrato de C. baccatum potencializou a sua atividade antibiofilme e reduziu significativamente a sua citotoxicidade. Nossos resultados corroboram com a pesquisa de novas estratégias não antibióticas para combater microrganismos com reduzida possibilidade para o desenvolvimento de resistência. / Many species of Capsicum red peppers are used in traditional medicinal practices. These plants are utilized in a number of preparations to treat a variety of illnesses including infections. Some bacteria produce biofilm as an important virulence factor, due to this its structure mediates the adhesion to surfaces as implanted devices, probes, catheters and also promotes physical protection against the antibiotics or the immune system response. Accordingly, this study investigated the ability of the extract and isolated products from seeds of Capsicum baccatum as anti-biofilm agent. This study demonstrates by the first time that an extract from C. baccatum presented relevant anti-biofilm activity against Staphylococcus epidermidis and Pseudomonas aeruginosa. The active fraction was obtained by bio-guided assays and analyzed by HPLC-DAD-MS, MALDI-TOF MS and MALDI-MS/MS, identifying it as peptides from 2S sulfur-rich seed storage protein 2-like. It strongly controlled (2mg/ml) the S. epidermidis biofilm formation (>96%) when the compound was in solution and adsorbed on Permanox™ slides. Interestingly, it did not inhibit the growth of this bacterium, indicating the inhibition of biofilm is independent of bacterial cell death. Moreover, this peptides preserved human erythrocytes and lymphocytes integrity after 24-48 h of exposure, suggesting the fractionation potentiated the anti-biofilm activity of the C. baccatum crude extract while absolutely reduced its cytotoxicity. Our results corroborate to the search of new non-antibiotic strategies to combat microorganisms with a reduced pressure for resistance development.

Charles University Faculty of Pharmacy in Hradec Králové Study program: Healthcare Bioanalytics Author: Bc. Adéla Diepoltová Supervisor: RNDr. Klára Konečná, Ph.D. Title of thesis: Optimization of the method for sensitivity evaluation of biofilm- forming staphylococci against candidate antimicrobial compounds Background: The aim of this thesis was to optimize approach for in vitro formation of staphylococcal biofilms on the pegs and on the wells of the 96-well panel as an analogous approach to commercially available Calgary Biofilm Device system. The aim of the Experiment 1 was to evaluate incubation conditions (such as impact of a growth medium, incubation mode, optical density of the starting bacterial inoculum and type of surface) leading to maximal biofilm formation of two biofilm producer strains with unknown biofilm phenotype and one staphylococcal strain known as strong biofilm producer. The most advisable conditions were used in incubation process of Experiment 2. This work should propose the approach leading to in vitro formation of the most voluminous staphylococcal biofilms exploitable for candidate drug antimicrobial activity testing. Methods: Spectrophotometric measurement of the crystal violet colour extracted from wells with fixed and stained Staphylococci to evaluate the ability to...

Les bactéries buccales n'infectent pas seulement la bouche mais y resident. Elles peuvent également passer dans la voie sanguine et atteindre des organes secondaires. S’il n'est pas traité, le biofilm dentaire peut provoquer une inflammation destructrice dans la cavité buccale, entrainant de graves complications médicales. Dans ce biofilm, Streptococcus gordonii, colonisateur oral primaire, constitue la plate-forme sur laquelle des colonisateurs pathogènes tardifs comme Porphyromonas gingivalis, l'agent causal des maladies parodontales, se lieront. L'objectif de la première partie de la thèse était de déterminer l'activité antibactérienne de onze composés de lichens appartenant à différentes familles chimiques, pour découvrir de nouveaux antibiotiques pouvant combattre ces bactéries buccales. Nous avons montré que trois composés avaient des activités antibactériennes prometteuses. L'acide psoromique enregistrait les CMIs le plus faibles. De nouveaux analogues de butyrolactone ont ensuite été conçus et synthétisés sur la base des composés antibactériens licheniques connus, les acides lichesteriniques, en substituant différents groupes fonctionnels sur le cycle butyrolactone pour améliorer son activité sur S. gordonii et P. gingivalis. Parmi les dérivés, B-12 et B-13 avaient la plus faible CMI où ils se sont révélés être des bactéricides plus forts, 2 à 3 fois plus, que l'antibiotique, doxycycline. B-12 et B-13 étaient également les plus efficaces vis-à-vis de P. gingivalis. La cytotoxicité de ces 2 composés a ensuite été vérifiée contre les cellulaires épithéliales gingivales humaines et les macrophages. Ils ne présentaient pas de toxicité contre les cellules testées. Une étude préliminaire de relation structure-activité a révélé le double rôle important apporté par deux substituants, chaîne alkyle en C5 et groupe carboxyle en C4 positions, dans leur mécanisme d'action. Ceci a été suivi par l'étude de l’activité antibiofilmique de B-12 et B-13 contre les deux souches orales en utilisant un test de cristal violet et microscopie confocale. Les deux dérivés ont montré, à une concentration plus faible, une inhibition maximale de la formation du biofilm, LCMI, de 9.38 μg/mL contre S. gordonii et 1.17 μg/mL contre P. gingivalis. Cependant, lorsque des concentrations sous-inhibitrices de B-12 et B-13 ont été utilisées, nous avons démontré que les deux souches étudiées pouvaient former des biofilms in vitro, accompagné d’une diminution de l'expression des gènes impliqués dans l'adhésion et la formation de biofilm. Pour mieux comprendre les mécanismes d'action des butyrolactones, nous avons étudié la localisation bactérienne du composé B-13 en synthétisant un B-13 marqué au NBD (4-nitro-benzo [1,2,5] oxadiazole) fluorescent conservant son activité antibactérienne. Par microscopie confocale et HPLC, nous avons montré que ce composé se lie à la surface cellulaire de S. gordonii. Ensuite, B-13 induit une rupture de la paroi cellulaire conduisant à la libération des constituants bactériens et par conséquent, à la mort de S. gordonii, une bactérie Gram-positive. L'expression de deux gènes, murA et alr, impliqués dans la synthèse de la paroi cellulaire, a été modifiée en présence de cette butyrolactone. Les bactéries Gram négatives telles que P. gingivalis ont également montré des cellules abimées présentant une rupture de la paroi en présence de B-13, ce qui suggère que cette butyrolactone agit sur des Gram-positives et Gram-négatives avec une plus grande efficacité contre les Gram-négatives. En outre, nous avons également démontré que l'analogue de B-13, B-12, induit une perturbation de la morphologie de P. gingivalis et S. gordonii. Toutes ces études ont démontré que les butyrolactones dérivées de lichen peuvent être proposés comme des composés antibactériens puissants contre les agents pathogènes oraux qui causent des complications médicales graves. / The oral bacteria do not only infect the mouth and reside there, but also travel via the blood and reach distant body organs. If left untreated, the dental biofilm that can cause destructive inflammation in the oral cavity may result in serious systemic medical complications. In dental biofilm, Streptococcus gordonii, a primary oral colonizer, constitutes the platform on which late pathogenic colonizers like Porphyromonas gingivalis, the causative agent of periodontal diseases, will bind. The aim of the first study was to determine the antibacterial activity of eleven natural lichen compounds belonging to different chemical families to uncover new antibiotics which can fight against the oral bacteria. Three compounds were shown to have promising antibacterial activities where psoromic acid had the lowest MICs of 11.72 and 5.86 µg/mL against S. gordonii and P. gingivalis, respectively. Novel butyrolactone analogues were then designed and synthesized based on the known lichen antibacterial compounds, lichesterinic acids (B-10 and B-11), by substituting different functional groups on the butyrolactone ring trying to enhance its activity on S. gordonii and P. gingivalis.. Among the derivatives, B-12 and B-13 had the lowest MIC of 9.38 µg/mL where they have shown to be stronger bactericidals, by 2-3 times, than the reference antibiotic, doxycycline. B-12 and B-13 were also the most efficient on P. gingivalis exhibiting MIC of 0.037 and 0.293 µg/mL and MBC of 1.17 and 0.586 µg/mL, respectively. These 2 compounds were then checked for their cytotoxicity against human gingival epithelial cells and macrophages by MTT and LDH assays which confirmed their safety against the tested cell lines. A preliminary study of the structure-activity relationships unveiled the important dual role contributed by two substituents, alkyl chain at C4 and carboxyl group at C5 positions, in their mechanism of action. This was followed by the investigation of B-12 and B-13 for their antibiofilm activity against both oral strains using crystal violet assay and confocal microscopy. Both derivatives displayed a lowest concentration with maximal biofilm inhibition, LCMI, of 9.38 µg/mL against S. gordonii and 1.17 µg/mL against P. gingivalis. However, when sub-inhibitory concentrations of B-12 and B-13 were used, we demonstrated that the two investigated strains were able to form biofilms in vitro. Indeed, this antibiofilm activity decreased as indicated by the expression of the genes implicated in adhesion and biofilm formation. To better understand the mechanism of action of butyrolactones, we have investigated B-13 bacterial localization by synthesizing a fluorescently labeled B-13 with NBD (4-nitro-benzo[1,2,5]oxadiazole) conserving its antibacterial activity. By confocal microscope, we showed that this compound binds to S. gordonii cell surface and this was also demonstrated by HPLC analysis. By adhering to cell surface, B-13 induced cell wall disruption leading to the release of bacterial constituents and consequently, the death of S. gordonii, a Gram-positive bacterium. The expression of two genes, murA and alr, implicated in cell wall synthesis, was modified in the presence of this butyrolactone. Gram-negative bacteria such as P. gingivalis showed also cracked and ruptured cells in the presence of B-13, suggesting that this butyrolactone acts on Gram-positive and Gram-negative strains, but with greater efficacy against the Gram-negatives. Besides, we also demonstrated that the analogue of B-13, B-12, has also induced disruption of P. gingivalis and S. gordonii. All these studies demonstrated that butyrolactones derived from a lichen metabolite can be proposed as potent antibacterial agents against oral pathogens causing serious medical complications.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-09-27T14:21:32Z No. of bitstreams: 1 osmarnascimentosilva.pdf: 5346948 bytes, checksum: c6c4bb9eae4172e04c4553f57d4e27d0 (MD5) / Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2016-09-27T15:15:36Z (GMT) No. of bitstreams: 1 osmarnascimentosilva.pdf: 5346948 bytes, checksum: c6c4bb9eae4172e04c4553f57d4e27d0 (MD5) / Made available in DSpace on 2016-09-27T15:15:36Z (GMT). No. of bitstreams: 1 osmarnascimentosilva.pdf: 5346948 bytes, checksum: c6c4bb9eae4172e04c4553f57d4e27d0 (MD5) Previous issue date: 2015-04-28 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Com o aumento na incidência de infecções resistentes a múltiplos antibióticos, existe hoje um grande interesse pelos peptídeos antimicrobianos (PAMs) como modelos para a produção de novos antibióticos. Os PAMs são mediadores multifuncionais da resposta imune inata, com atividade antibacteriana direta. O uso de PAMs como agentes terapêuticos tem algumas limitações, como a estabilidade, a toxicidade e alta massa molecular. Apesar dessas limitações, eles apresentam propriedades compensatórias, como imunomodulatória e antitumoral bem como a capacidade de inibir β-lactamases. O desenho racional de PAMs tem sido usado para gerar análogos com atividade melhorada. No presente trabalho avaliamos a atividade antibacteriana in vitro e in vivo da clavanina A e através da modificação incremental criamos dois análogos dos peptídeos mostoparano-L e clavanina A (clavanina-MO e mastoparanoMO),além disso, utilizamos o desenho racional de peptídeos para a criação de dois inibidores de β-lactamase (dBLIPs 1 e 2). A clavanina A mostrou se eficiente na eliminação de S. aureus em um modelo de infecção de ferida e impediu o início da sepse e, assim, reduziu a mortalidade de camundongos infectados em um modelo de infecção bacteriana sistêmica. A clavanina-MO e mastoparano-MO impediram o crescimento de bactérias planctônicas e levaram à erradicação de biofilmes bacterianos maduros. Os peptídeos modificados mostram-se promissores como agentes terapêuticos contra infecções bacterianas sistêmicas e biofilmes causadas por uma variedade de bactérias. dBLIP-1 e dBLIP-2 em combinação com antibióticos convencionais foram eficazes na eliminação de Escherichia coli e Staphylococcus aureus que expressam β-lactamases em um modelo murino de infecção sistêmica. dBLIPs 1 e 2 fornecem pistas para superar a resistência à base de β-lactamase. / With the increased incidence of multiple antibiotic resistant infections, there is huge interesting in antimicrobial peptides (AMPs) as templates to produce novel antibiotics. The AMPs are multifunctional mediators of innate immune response with direct antibacterial activities. Nevertheless, the use of AMPs as therapeutic agents has certain limitations such as stability, toxicity and high molecular mass. Despite such limitations, they show additional properties such as antitumor and immunomodulatory as well as the ability to inhibit β-lactamases. Furthermore, the rational AMPs design has been used to produce analogues with improved activity. In the present study, we utilized the rational AMPs design for generation of two β-lactamase inhibitors (dBLIPs 1 and 2) and through two incremental modification created analogues of clavanin A and mostoparan-L (clavanin-MO and mastoparan-MO respectively) peptides. Both inhibitors in combination with conventional antibiotics were effective for control of Staphylococcus aureus and Escherichia coli expressing β-lactamase in a murine model of systemic infection. dBLIPs 1 and 2 provide clues to overcome resistance to β-lactamase base. The clavanin-MO and mastoparan-MO prevented the growth of planktonic bacteria, leading to the mature biofilm eradication of pathogenic Gram-negative and -positive. The clavanina-MO and mastoparan-MO are promising therapeutic agents against systemic infections and bacterial biofilms caused by a wide bacterial variety.