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Polyurethane-Based Biosurfactant Mimics as Antibiofilm AgentsChen, Zixi 28 April 2021 (has links)
No description available.
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Interactions moléculaires entre microorganismes au sein de biofilms en milieu marin : mise en évidence de biomolécules antibiofilm / Molecular interactions between microorganisms within marine biofilms : identification of new antibiofilm moleculesDoghri, Ibtissem 15 October 2015 (has links)
En environnement marin, la colonisation des surfaces solides par les microorganismes est progressive et suit une logique taxonomique et/ou fonctionnelle des espèces. Les biofilms ainsi formés représentent des systèmes multi-cellulaires entourés d’une matrice de substances polymériques extracellulaires (SPE). L’objectif de ce travail était de comprendre comment des acteurs microbiens (bactéries et diatomées) interagissent dans deux types de biofilms marins (biofilm benthique et biofilm sur structures métalliques portuaires). Dans cette étude, des modèles bactériens isolés de ces biofilms ont été identifiés et caractérisés. Dans un premier volet, leur capacité à former des biofilms stables a été évaluée dans différentes conditions. Quatre souches ont été ainsi sélectionnées : Flavobacterium sp. II2003, Roseobacter sp. IV3009, Roseovarius sp. VA014 et Shewanella sp. IV3014. Dans un deuxième volet, les effets des sécrétomes des bactéries marines issues du même habitat ont été évalués sur ces modèles. Deux souches se distinguent par leur capacité à produire des molécules influençant négativement la formation de biofilms : Pseudoalteromonas sp. IIIA004 produit un peptide de 2224 Da présentant une activité antibiofilm vis-à-vis de Roseovarius sp. VA014 et Pseudomonas sp. IV2006 inhibe la formation de biofilm de Flavobacterium sp. II2003. Dans les deux cas, les antibiofilms sont actifs contre un large spectre de bactéries suggérant ainsi plusieurs applications potentielles dans les domaines marin et médical. Dans le dernier volet, les effets des sécrétomes de la diatomée Navicula phyllepta ont été évalués sur les modèles de bactéries benthiques. Cette diatomée s’est distinguée par sa capacité à sécréter des polysaccharides inhibant ou stimulant la formation de biofilms selon les souches cibles. / In the marine environment, solid surface colonization by microorganisms is progressive and follows a taxonomic and/or functional logic. Biofilms formed are multi-cellular systems surrounded by a matrix of extracellular polymeric substances (EPS). The objective of this work was to understand how microbial actors (bacteria and diatoms) interact in two types of marine biofilms (benthic biofilm and biofilm on metallic structures of a harbor). In this study, bacterial models isolated from these biofilms have been identified and characterized. In a first part, their ability to form stable biofilms was evaluated under various conditions. Four strains were selected: Flavobacterium sp. II2003, Roseobacter sp. IV3009, Roseovarius sp. VA014 and Shewanella sp. IV3014. In a second part, the effects of secretomes of the marine bacteria from the same habitat were evaluated on these models. Two strains are distinguished by their ability to produce molecules negatively influencing biofilm formation: Pseudoalteromonas sp. IIIA004 produces a 2224 Da peptide with an antibiofilm activity toward Roseovarius sp. VA014 and Pseudomonas sp. IV2006 inhibits the biofilm formation of Flavobacterium sp . II2003. In both cases, the antibiofilms are active against a broad spectrum of bacteria suggesting several potential applications in marine and medical fields. In the last part, the effects of secretomes of the Navicula phyllepta diatom were evaluated on benthic bacteria models. This diatom was distinguished by its ability to secrete polysaccharides stimulating or inhibiting biofilm formation by target strains.
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Évaluation des huiles essentielles en complémentation animale préventive / Evaluation of essential oils for their use in preventive animal complementationLang, Marie 03 July 2018 (has links)
L’usage de la phytothérapie pour la prévention de pathologies récurrentes dans les élevages s’est récemment développé. Les huiles essentielles (HE) sont des extraits végétaux complexes, constituant une source d’alternatives de par leur composition chimique variée et d’intéressantes activités biologiques. Ce projet de thèse a été réalisé dans un contexte industriel, avec pour objectif principal la mise en évidence des propriétés des HE et de leurs mélanges par des méthodes in vitro. Les mélanges formés doivent permettre de cumuler les activités naturelles des HE dans les sphères respiratoires, digestives et immunitaires et dans la prévention des mammites. Une sélection en entonnoir intervient d’après une trame d’essais spécifiques à chaque sphère. L’HE de girofle, intégrée dans les mélanges, développe d’après différentes méthodes biochimiques de fortes activités antioxydantes et anti-inflammatoires. Les HE de cannelle et d’origan se sont illustrées pour leur capacité à inhiber le développement des pathogènes comme les bactéries sous forme planctonique ou de biofilm, les virus ou les parasites internes. Les effets sur le biofilm bactérien ont notamment été observés par microscopie confocale à balayage laser. Enfin la capacité de l’HE de girofle à moduler la réponse inflammatoire a été démontrée sur le modèle de macrophage RAW264.7. La phagocytose, l’expression de la nitroxyde synthase et la sécrétion d’interleukine-6 ont été suivis par criblage à haut débit associé à la microscopie à épifluorescence. La caractérisation des mélanges a abouti à la formulation des noyaux Respilor®, Digelor®, Immulor® et Mastilor®, testés in vivo et commercialisés par BioArmor. / The use of phytotherapy for the prevention of livestock associated pathologies is increasing since recent years. Essential oils (EO) are complex plant extracts, forming a large source of alternatives due to their chemical diversity and interesting biological activities. This project, carried out over three years and anchored in an industrial context, aims to highlight the properties of EO and their blends using in vitro assays. The objective is to cumulate the natural properties of EO through their blending. The prevention of respiratory and digestive tract diseases, of mastitis and the promotion of the animals’ immunity are particularly targeted. A funnel selection takes place according to tests selected for their specificity to each area. Clove EO, integrated in the mixtures, develops according to different biochemical methods strong antioxidant and anti-inflammatory activities. EOs of cinnamon and oregano have been shown to inhibit the development of pathogens such as planktonic or biofilm embedded bacteria, viruses or internal parasites. The effects on the bacterial biofilm were especially measured using confocal laser scanning microscopy. Finally, the ability of clove EO to modulate the inflammatory response has been demonstrated on the RAW264.7 macrophage cells. Phagocytosis, the expression of Nitroxide synthase, and the secretion of interleukin-6 were followed by high content screening methods associated with epifluorescence microscopy. The characterization of the mixtures resulted in the formulation Respilor®, Digelor®, Immulor® and Mastilor®, four blends of EO tested in vivo and commercialized by BioArmor.
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Potentiel antimicrobien de principes actifs d’origine naturelle / Antimicrobial potential of natural bioactive compoundsJohansen, Bianca 22 December 2017 (has links)
Le développement de principe actif à partir de ressources naturelles pourrait être un moyen pour développer de nouvelles alternatives aux antibiotiques. L’huile essentielle d’arbre à thé est reconnue pour ses nombreuses propriétés et son activité antimicrobienne à spectre large, mais aussi pour sa toxicité et sa forte teneur en allergènes liée à sa composition en monoterpènes. Dans cette étude, nous cherchons de nouveaux principes actifs naturels issus de l’huile essentielle d’arbre à thé, qui auraient une activité antimicrobienne équivalente mais une toxicité et un pouvoir allergène réduit. Deux principes actifs ont ainsi été étudiés au cours de ce travail. Le Titroléane™, qui est une fraction de l’huile essentielle d’arbre à thé enrichi en monoterpène alcools mais avec un très faible taux de monoterpène, a montré des activités microbiologiques similaires à l’huile essentielle d’arbre à thé. La Synterpicine™ quant à elle, est uniquement composée des deux molécules majoritaires de l’huile essentielle, et présente une cytotoxicité réduite. La caractérisation de l’activité des principes actifs a été menée avec différentes méthodes d’études in vitro, pour définir leur activité inhibitrice et bactéricide, sur des bactéries planctoniques et en biofilm. Enfin, la recherche de nouvelles molécules actives au sein des principes actifs, s’est poursuivie à l’aide d’une méthode de chromatographie sur couche mince haute performance, couplée à une étude d’activité microbiologique par bioautographie. Cette étude a mis en évidence l’activité antimicrobienne de deux nouveaux principes actifs d’origine naturelle, pouvant être valorisés sur différents marchés notamment la santé humaine / The development of a natural bioactive compounds could be a mean of developing new alternatives to antibiotics. Tea tree essential oil is known for its numerous properties and its broad-spectrum antimicrobial activity, but also for its toxicity and its high content in allergens due to its composition in monoterpenes. In this study, we search for new natural compounds extracted from tea tree essential oil, which has equivalent antimicrobial activity but less toxicity and a reduced allergenic activity. Two bioactive compounds have been thus studied. Titroléane™ - a fraction of tea tree essential oil enriched in monoterpene alcohols but with a very low rate of monoterpenes - has shown microbiological activities similar to tea tree essential oil. As for Synterpicine™, it is only composed of the two main molecules of the essential oil, and presents a reduced cytotoxicity. The characterization of the compounds’ efficacy was carried out with different in vitro study methods, to define their inhibitory and bactericide activity, on planktonic and biofilm bacteria. Finally, the research of new active molecules within the active compounds, was pursued thanks to a high performance thin layer chromatography, paired up with a microbiological study by bioautography. This study has highlighted the antimicrobial activity of two new natural active extracts which can be valorised on different markets- especially human health
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Caractérisation de molécules antibiofilm produites par des souches de staphylocoques isolées dans des cas de mammite bovineGoetz, Coralie 07 1900 (has links)
Le biofilm bactérien est une communauté de bactéries qui s’agrègent en un amas
structuré recouvert d’une matrice polymérique et adhèrent sur une surface biotique ou
abiotique. Ce mode de vie permet à ces bactéries de survivre dans un environnement
hostile et cela entre autres grâce à une plus grande résistance aux antibiotiques que les
bactéries sous forme libre ou planctonique. Les bactéries ayant la capacité de former des
biofilms constituent donc un grave problème de santé tout aussi bien pour l’homme que
pour l’animal comme par exemple dans le cas de la mammite bovine (MB). La MB est une
inflammation de la glande mammaire de la vache induisant des dommages des tissus du pis
et de ce fait, une réduction de la production de lait ainsi qu’une altération du bien-être de
l’animal. Il est donc important de développer de nouvelles stratégies prophylactiques et
thérapeutiques contre la MB. Des résultats préliminaires obtenus dans notre laboratoire
avaient mis en évidence la capacité de certaines souches de staphylocoques à coagulase
négative (SCN) produisant peu ou pas de biofilm à inhiber la formation de biofilm par
d’autres bactéries responsables de MB. Notre hypothèse était donc que certaines souches
faibles productrices de biofilm produisaient une ou plusieurs molécule(s) capable(s)
d’inhiber de façon significative la formation de biofilm par d’autres SCN mais également
par Staphylococcus aureus et possiblement d’autres agents pathogènes de la MB. Le but de
ce projet fut donc de caractériser des molécules antibiofilm produites par des souches de
SCN. Pour cela, 30 souches de staphylocoques (cinq espèces de SCN et S. aureus) fortes
productrices de biofilm et 10 souches de SCN (Staphylococcus chromogenes et
Staphylococcus simulans) faibles productrices de biofilm ont été utilisées. Nos résultats ont
montré que certaines souches de SCN présentant un phénotype de faible production de
biofilm réduisaient significativement la formation de biofilm chez plusieurs espèces de staphylocoques dont S. aureus. De plus, quatre souches de SCN (S. chromogenes C et E et
S. simulans F et H) ont été capables d'inhiber de façon significative (p < 0,05) la formation
de biofilm de 80% des souches testées. Cette activité antibiofilm a été également visualisée
par microscopie confocale ainsi que dans des conditions dynamiques en utilisant un
système de microfluidique. En parallèle, il a été démontré que les souches de SCN
présentant un phénotype de faible production de biofilm n'inhibaient pas significativement
la croissance des souches ayant un phénotype de forte production de biofilm. Par
conséquent, l’activité antibiofilm observée ne semblait pas être due à une activité
bactéricide. De plus, nos résultats ont montré que les surnageants de ces quatre souches
présentaient également une activité antibiofilm et qu’ils possédaient un spectre d’activité
étendu suggérant ainsi que les souches excrétaient une molécule antibiofilm dans son
environnement extérieur. La caractérisation de ces surnageants a permis de mettre en
évidence que l’activité inhibitrice était notamment conservée dans la fraction des
surnageants < 3kDa et qu’une des molécules responsables de cette activité était hydrophile,
thermorésistante et sensible à l’action de la RNase A. Enfin, nous avons pu observer que
ces surnageants avaient la capacité de réduire voir même de prévenir la colonisation de la
glande mammaire par une souche de S. aureus résistante à la méthicilline dans un modèle
murin de mammite. Ces surnageants semblent être une alternative prometteuse dans le
contrôle de la MB pour prévenir et/ou traiter cette infection que ce soit seul ou en
association avec un traitement antibiotique. / Bacterial biofilms are structured communities of bacteria cells enclosed in a selfproduced
polymeric matrix which is adherent to a biotic or abiotic surface. This lifestyle
allows these bacteria to survive in hostile environments. For example, bacteria having the
ability to form biofilms are significantly less susceptible to antibiotics than bacteria in
planktonic form. Bacteria within biofilms pose a serious risk to human and animal health
such as during bovine mastitis (BM). BM is an inflammation of the mammary gland of
dairy cows which causes udder damages and reduces milk production. Therefore, it is
important to develop new prophylactic and therapeutic strategies to control and treat BM.
Preliminary results have demonstrated the ability of some coagulase-negative
staphylococci (CNS) producing a weak biofilm to inhibit biofilm formation by other
bacteria associated with BM. Our hypothesis is that some of CNS isolates having the
ability to form a weak biofilm produce one or more molecule(s) able to significantly inhibit
biofilm formation by other CNS, Staphylococcus aureus or other BM pathogens. The
purpose of this project was to characterize antibiofilm molecules produced by CNS
isolates. A total of 30 staphylococcal isolates (five species of CNS and S. aureus) with a
strong biofilm phenotype and 10 CNS (Staphylococcus chromogenes and Staphylococcus
simulans) isolates with a weak biofilm phenotype were used. Our results indicated that
some CNS isolates with a weak biofilm phenotype significantly reduced biofilm formation
of several staphylococcal species including S. aureus. Importantly, four isolates of CNS (S.
chromogenes C and E and S. simulans F and H) were able to significantly inhibit biofilm
formation (p < 0.05) of 80% of staphylococcal isolates tested. This activity was confirmed
using confocal microscopy but also in dynamic conditions using a microfluidic system.
Additionally, CNS with a weak biofilm phenotype did not significantly inhibit the growth of isolates with a strong biofilm phenotype. Therefore, the biofilm inhibition does not seem
to be due to a bactericidal activity. The results also showed that the culture supernatants
from these four bacteria still have an antibiofilm activity and own a broad spectrum of
activity suggesting that the strains release an antibiofilm molecule into the external
environment. The characterization of these supernatants indicated that the inhibitory
activity was conserved in the < 3kDa fraction of the supernatants. Furthermore, one of the
molecules responsible for the antibiofilm activity appears hydrophilic, heat-resistant and
sensitive to the action of the RNase A. Finally, we observed that culture supernatants of
these bacteria had the ability to reduce or even prevent colonization of the mammary gland
by a strain of methicillin-resistant S. aureus in a murine model of mastitis. These
supernatants appear to be a promising alternative alone or in combination with antibiotics
in the control of BM.
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Prospecção de atividades biológicas da apamina e melitina / Prospecting of biological activities of apamin and melittinPicoli, Tony 28 November 2016 (has links)
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Previous issue date: 2016-11-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A busca por novos fármacos têm levado as pesquisas ao encontro dos produtos
naturais que oferecem uma quantidade inimaginável de princípios ativos ainda
desconhecidos. A apicultura moderna é um segmento da agropecuária que fornece
alguns produtos naturais como a própolis, o mel, a cera, a geleia real e a apitoxina,
que vêm sendo alvo de pesquisas devido às diversas atividades biológicas já
apresentadas. A apitoxina (veneno de abelhas) é uma complexa mistura de
substâncias bioativas, entre elas os peptídeos apamina e melitina. O primeiro tratase
da menor neurotoxina conhecida e com apenas 18 aminoácidos em sua
composição representa 2% do peso seco do veneno. Já melitina, composta de 26
aminoácidos, corresponde a 50% do peso seco do veneno e é um conhecido
peptídeo tóxico capaz de causar lise em diversos tipos celulares. Neste sentido, este
estudo objetivou caracterizar estes dois peptídeos quanto à sua citotoxicidade e seu
poder antimicrobiano. Através do ensaio de redução do MTT (3-(4,5 dimetiltiazol-
2yl)-2-5-difenil-2H tetrazolato de bromo) foi possível determinar as concentrações
citotóxicas de melitina para 50% dos cultivos celulares (CC50), que variou de 2,3 a
4,1 μg/mL e a CC90, que variou de 2,7 a 4,7 μg/mL para diferentes linhagens
celulares e, ao expor estas células frente à melitina por diferentes períodos pôde-se
observar que com apenas 5 minutos de exposição, houve queda nas viabilidades
celulares. Já os ensaios de toxicidade realizados em citometria de fluxo demonstram
que, com concentrações de melitina abaixo das consideradas tóxicas pelo ensaio de
redução do MTT, as células já apresentavam sinais de toxicidade da melitina,
alterando os padrões de apoptose e necrose, aumentando a peroxidação das
membranas lipídicas (LPO), assim como a produção intracelular de espécies
reativas ao oxigênio (ROS) e da fragmentação de seu DNA. Por microscopia
confocal foi possível a observação de focos de apoptose e necrose e do aumento da
fluidez das membranas das células proporcional ao aumento da concentração de
melitina em que essas foram expostas. Houve correlação positiva entre LPO e ROS
(r=0,3158, p<0,05), taxa de apoptose (r= 0,4978, p<0,05) e com a funcionalidade
das mitocôndrias (r= 0,3149, p<0,05). LPO parece iniciar os demais eventos de
toxicidade celular avaliados e aumentar a funcionalidade mitocondrial em resposta
ao estresse sofrido pelas células. Sendo assim, a citometria de fluxo e a microscopia
confocal demonstraram eficiência em auxiliar a desvendar os mecanismos de
toxicidade de melitina e se portam como técnicas complementares ao ensaio MTT,
que avalia a viabilidade celular apenas através da funcionalidade mitocondrial.
Quanto à atividade antiviral da melitina, houve a inativação de herpesvírus bovino
tipo 1 (BoHV-1), especialmente na concentração 2μg/ml. Apamina atuando isoladamente não apresentou poder antiviral, porém quando aliada à melitina,
apresentou efeito antiviral contra o vírus da diarreia viaral bovina (BVDV).
Resultados com padrões semelhantes ocorreram ao analisar o poder virucida das
substâncias, quando melitina inativou BoHV-1 em 2 horas de incubação com este
vírus mas não apresentou ação contra o BVDV, assim como a apamina, mas a
associação potencializou o efeito virucida contra BVDV. Já quanto à atividade
antibacteriana, a concentração inibitória mínima (CIM) e bactericida mínima (CBM)
de melitina foram, respectivamente (em μg/ml), frente a Staphylococcus aureus (6-7
e 32-64), Escherichia coli (40-42,5 e 64-128) e Pseudomonas aeruginosa (65-70 e
64-128). Biofilmes de S. aureus foram mais sensíveis à ação da melitina quanto
comparados aos biofilmes produzidos pelas Gram negativas. Melitina foi capaz de
destruir biofilmes pré-formados pelas três espécies bacterianas estudadas além de
inibir a formação dos biofilmes por estas espécies quando foram previamente
incubadas com melitina em concentrações abaixo da CIM. Os resultados são
promissores quanto à capacidade antimicrobiana das substâncias apresentando
potencial para o desenvolvimento de novos fármacos e/ou sanitizantes e, os
mecanismos de toxicidade avaliados podem auxiliar nesse desenvolvimento. / The search for new drugs leads the researchs to meeting the natural products that
offers an unimaginable amount of still unknown active principles. The modern
apiculture is a segment of agriculture that provides some natural products such as
propolis, honey, wax, royal jelly and apitoxin, which have been the subject of
research due to the various biological activities already presented. Apitoxin (bee
venom) is a complex mixture of bioactive substances, and between them the
peptides apamin and melittin. The first is the smaller known neurotoxin and, with
only 18 amino acids in its composition, represent 2% of the dry weight of the venom.
Melitin, composed of 26 amino acids, corresponds to 50% of the dry weight of the
venom and is a known toxic peptide capable of causing lysis in several cell types. In
this sense, this study aims to characterize these two peptides as to their cytotoxicity
and their antimicrobial power. By the reduction of MTT (3- (4,5-dimethylthiazol-2-yl) -
2-5-diphenyl-2H-tetrazolate bromide) test, it was possible to determine the cytotoxic
concentrations of melittin to 50% of the cell cultures (CC50) which ranged from 2.3 to
4.1 μg / mL and CC90, ranging from 2.7 to 4.7 μg / mL for different cell lines, and by
expose these cells to melina for different periods it was observed that only 5 minutes
of exposure, there was decrease in cellular viability. The toxicity tests performed in
flow cytometry shows that, with melittin concentrations below the toxic concentration
considerated by the MTT reduction test, the cells showed signs of melittin toxicity,
altering the patterns of apoptosis and necrosis, increasing the peroxidation of
membranes (LPO), as well as an intracellular production of oxygen reactive species
(ROS) and the fragmentation of their DNA. By confocal microscopy it was possible to
observe foci of apoptosis, necrosis and the increase of cell membranes fluidity
proportional to the increase in the concentration of melittin in which they were
exposed. There was a positive correlation between LPO and ROS (r = 0.3158, p
<0.05), apoptosis rate (r = 0.4978, p <0.05) and mitochondrial functionality (r =
0.3149, P0.05). LPO seems to initiate the other evaluated events of cellular toxicity
and increase mitochondrial functionality in response to the stress undergone by the
cells. Therefore, the flow cytometry and confocal microscopy have demonstrated
efficiency in helping to unravel the mechanisms of melittin toxicity and behave as
complementary techniques to the MTT assay, which evaluates the cellular viability by
mitochondrial functionality. As for the antiviral activity of melittin, there was
inactivation of bovine herpesvirus type 1 (BoHV-1), especially at 2μg / ml
concentration. Apamin acting alone did not present antiviral power, but when allied to
melittin, presented antiviral effect against bovine viral diarrhea virus (BVDV). Results
with similar patterns ocurred when analyzing the virucidal power of the substances, when melittin inactivated BoHV-1 with 2 hours of incubation with this virus but
showed no action against BVDV, as well as apamine, but the association potentiated
their virucidal effect against BVDV. As for antibacterial activity, the minimum
inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of
melittin were, respectively (in μg / mL), against Staphylococcus aureus (6-7 and 32-
64), Escherichia coli (40- 42,5 and 64-128) and Pseudomonas aeruginosa (65-70
and 64-128). Biofilms of S. aureus were more sensitive to the action of melitin
compared to biofilms produced by Gram negative bacteria. Melittin was able to
destroy preformed biofilms by the three bacterial species studied and inhibited the
formation of biofilms by these species when they were previously incubated with
melittin at concentrations below MIC. The results are promising as to the
antimicrobial capacity of the substances presenting potential for the development of
new drugs and / or sanitizers and the mechanisms of toxicity can aid in the
development.
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Recherche d'outils thérapeutique innovants pour lutter contre la bactérie Acinetobacter baumannii. / Research of innovative therapeutic tools against Acinetobacter baumanniiNicol, Marion 20 December 2017 (has links)
Acinetobacter baumanii fait aujourd’hui partie des bactéries les plus problématiques dans le monde. Responsable de nombreux pics épidémiques d’infections nosocomiales auxquelles sont associés de forts taux de mortalité, cette bactérie puise sa pathogénie dans de multiples caractéristiques qui lui permettent ainsi d’échapper au système immunitaire de l’hôte et à la plupart des traitements actuels. Capable d’adhérer à de multiples surfaces, A. baumanii persiste dans l’environnement hospitalier à travers un mode de vie communautaire au sein duquel ses capacités de survie sont exacerbées. Chez les espèces du genre Acinetobacter, le mode de vie communautaire peut prendre deux formes distinctes : le biofilm et la pellicule. Dans la première partie de cette thèse, nous avons cherché à discriminer ces deux modes de vie, chez la souche ATCC 17978, par une analyse protéomique à large échelle. Nous avons confirmé la présence de nombreux marqueurs communs aux deux communautés (transporteurs, systèmes de sécrétion, d’acquisition d’ions, adhésines et pili) et mis en exergue des systèmes spécifiquement reliés à la formation du biofilm (pilus Fim, T2SS, T1SS/pompe A1S_0535-38, LPS/LOS, motif capsulaire) et à celle de la pellicule (Gac). Grâce à l’étude de la souche A. baumannii SDF en mode biofilm, qui présente un génome plus compact, nous montrons que très peu de mécanismes moléculaires sont partagés par les deux souches étudiées. Ce résultat témoigne de la difficulté quant au développement d’un traitement dirigé contre les biofilms A. baumannii. Dans une deuxième partie, nous avons testé deux approches pour prévenir et éradiquer les biofilms à A. baumannii. La première a ciblé le Quorum Sensing (QS), système de communication essentielle à la coordination des cellules. Nous avons pu montrer que les acides gras mono-insaturés (acide palmitoléique et acide myristoléique), au même titre que la virstatine, limitait la formation de communautés à A. baumannii en inhibant l’expression du régulateur abaR nécessaire au QS. Dans une seconde stratégie, nous avons finalement évalué l’action antibactérienne et antibiofilm d’un nouveau composé d’origine naturelle : la squalamine. Dans cette étude, nous montrons pour la première fois qu’A. baumannii est capable d’entrer dans un état de dormance (persistant/VBNC) pour survivre à de fortes doses de ciprofloxacine, mais que la squalamine est capable d’éradiquer ces cellules persistantes grâce à des concentrations inférieures à la concentration hémolytique. / Today, Acinetobacter baumannii is one of the most problematic pathogens in the world. This bacterium is responsible for worldwide epidemic outbreaks associated with dramatic mortality rates. It possesses high capacities to evade the immune host system and to resist to numerous available antibacterial agents. A. baumannii is also able to persist into hospital environment due to high adhesion abilities which induce community development. This process is also associated to an enhanced survival rate. In Acinetobacter genus, community modes of lif can take two forms : biofilm and pellicle. In this study on the strain ATCC 17978, we tried to discriminate these two lifestyles by a large scale proteomic analysis. We have confirmed the presence of many common community markers (transporters, ion acquisition secretion systems, adhesins and pili) and highlighted systems specifically related to biofilm (pilus Fim, T2SS, T1SS / pump A1S_0535-38, LPS / LOS, capsular pattern) and pellicle communities. Furthermore the proteomic analysis of an avirulent A. baumannii strain, SDF, in biofilm allowed to highlight peculiar metabolic pathways, specific adhesion determinants but very few markers shared by ATCC 17978. This demonstrated the difficulty in developing a treatment directed against A. baumannii biofilm. Then, we tested different approaches to prevent and eradicate biofilms. The first one targeted the Quorum Sensing system (QS), an essential communication system for cell coordination. We have showed that monounsaturated fatty acids (palmitoleic acid and myristoleic acid), like virstatin prevent the community formation of A. baumannii by inhibiting the expression of the abaR regulator required for QS. In a second strategy, we have evaluated the antibacterial and antibiofilm activity of a new natural compound : the squalamine. We showed for the first time that if ciprofloxacin treatment was able to induce a dormancy population (persistent/VBNCs) in A. baumannii, squalamine was able to eradicate this population of dormant cells.
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Micro-organismos marinhos como fonte de metabólitos bioativos: atividade anti-trichomonas vaginalis, antibiofilme e antibacerianaSenger, Franciane Rios January 2016 (has links)
Nos últimos anos, os metabólitos isolados de fungos marinhos vêm ganhando considerável atenção em razão de possuírem estruturas químicas únicas com diversas atividades biológicas já descritas, incentivando novas pesquisas na área. A tricomoníase é a doença sexualmente transmissível (DST) de origem não viral mais comum no mundo, estando associada a sérias consequências à saúde, sendo relatado um aumento no número de isolados clínicos resistentes ao tratamento de escolha. Infecções causadas por bactérias com diferentes mecanismos de resistência, representam um grande desafio para a saúde pública atual, acarretando em altas taxas de mortalidade e morbidade. As bactérias patogênicas dispõem de fatores de virulência, como a formação de biofilme, que agravam infecções tornando-as persistentes. Devido ao potencial biológico dos produtos de origem marinha e a importância dessas infecções, o objetivo deste estudo foi avaliar a atividade anti-T. vaginalis, antimicrobiana e antibiofilme de moléculas obtidas da fermentação de fungos associados a organismos marinhos. As 14 cepas fúngicas foram isoladas de esponjas e corais marinhos, obtidos da costa do estado de Alagoas, Brasil. Após produção do metabólito, o micélio foi separado do meio. O micélio foi extraído com metanol e o meio com acetato de etila. As frações foram submetidas aos ensaios de atividade anti-T.vaginalis, antimicrobiana e antibiofilme (inibição da formação e erradicação). As frações que demonstraram atividade foram submetidas ao ensaio de hemólise, avaliação da citotoxicidade (HMVII e Vero) e toxicidade em modelo de Galleria mellonella. A fração que demonstrou os melhores resultados nos ensaios de atividade foi submetida ao fracionamento bioguiado. As frações orgânicas dos fungos Aspergillus niger (FMPV 03) e complexo Trichoderma harzianum/Hypocrea lixii (FMPV 09) foram ativas frente ao T. vaginalis ATCC 30236, com valores de MIC de 2 mg/mL e 1 mg/mL, respectivamente. Quando investigadas, essas frações mantiveram a atividade frente ao isolado clínico resistente ao metronidazol (TV-LACM2R), apresentado os mesmo valores de MIC encontrados para o isolado ATCC. Para a atividade antimicrobiana, as frações orgânicas do Aspergillus niger (FMPV 03), Aspergillus tubingensis (FMPV 06), complexo Trichoderma harzianu/Hypocrea lixii (FMPV 09) e Aspergillus sydowii (FMPV 10) foram ativas contra S. epidermidis ATCC 35984. Ainda, frente a P. aeruginosa ATCC 27853 somente as frações orgânicas do Aspergillus niger (FMPV 03) e Aspergillus tubingensis (FMPV 06) demonstraram atividade. Neste ensaio, para ambas as bactérias, os valores de MIC não ultrapassaram 1,5 mg/mL. A atividade destas frações também foi observada frente às mesmas bactérias no ensaio de antiformação de biofilme, já que ocorreu a morte das células. A habilidade de erradicar biofilmes foi detectada somente para a fração orgânica do fungo Aspergillus flavus (FMPV 01), o qual foi capaz de remover 52% do biofilme já formado de S. epidermidis ATCC 35984. A ausência de hemólise dos eritrócitos foi observada em todas as frações ativas estudadas. Na avaliação da citotoxicidade in vitro frente às linhagens celulares HMVII e Vero, apenas a fração orgânica do Aspergillus niger (FMPV 03), não apresentou efeito citotóxico. No entanto, no ensaio de avaliação da toxicidade in vivo, nenhuma das amostras testadas causou redução na sobrevivência das larvas de Galleria mellonella. Então, a fração orgânica do fungo Aspergillus niger (FMPV 03) foi submetida ao fracionamento bioguiado utilizando coluna RP-18. Sete frações foram obtidas, sendo que a primeira (100% água), foi ativa contra T. vaginalis, S. epidermidis e P. aeruginosa. Quando submetida à Cromatografia em Camada Delgada (CCD), demonstrou quatro bandas que foram coradas com anisaldeído-ácido sulfúrico e ninhidrina. Além disso, a fração 100% água não demonstrou redução da sobrevivência das larvas de Galleria mellonella, nas três concentrações testadas. Portanto, a gama de atividades relatadas corrobora o potencial dos fungos marinhos na produção de moléculas bioativas. / In recent years, the isolated metabolites from marine fungi have been attracted considerable attention due to unique chemical structures with diverse biological activities, encouraging further research in the area. Trichomoniasis is the most prevalent non-viral sexually transmitted disease (STD) worldwide. This has been linked to serious health consequences and an increase in the number of clinical isolates resistant to the treatment of choice has been reported. Infections caused by bacteria with different resistance mechanisms represent a major challenge to the current public health, resulting in high rates of mortality and morbidity. Pathogenic bacteria present virulence factors, such as biofilm formation, which migth enhance the persistence of infections. Due to the biological potential of marine products and the importance of these infections, the aim of this study was to evaluate the anti-T. vaginalis, antimicrobial and antibiofilm activities of the obtained molecule from the fermentation of fungi associated with marine organism. The 14 fungal strains were isolated from sponges and corals marine obtained from the coast of Alagoas, Brazil. After production of the metabolite the mycelium was separated from the medium. The mycelium was extracted with methanol and the medium with ethyl acetate. The fractions were subjected to the assays anti-T. vaginalis, antimicrobial and antibiofilm (inhibition of the formation and eradication) activities. The fractions that showed activity were subjected to the assays of hemolysis, cytotoxicity evaluation (HMVII and Vero) and toxicity Galleria mellonella model. The fraction which showed the best results in activity assays was subjected to fractionation bioguided. The organic fraction of Aspergillus niger (FMPV 03) and Trichoderma harzianum/Hypocrea lixii complex (FMPV 09) were active against T. vaginalis ATCC 30236 with MIC values of 2 mg/mL and 1 mg/mL, respectively. When investigated, these fractions maintained activity against resistant clinical isolate to metronidazole (TV-LACM2R), presented the same MIC values found to isolate ATCC. For the antimicrobial activity, the organic fractions of Aspergillus niger (FMPV 03), Aspergillus tubingensis (FMPV 06), Trichoderma harzianum/Hypocrea lixii complex (FMPV 09) and Aspergillus sydowii (FMPV 10) were active against S. epidermidis ATCC 35984. Even, against P. aeruginosa ATCC 27853 only the organic fractions of Aspergillus niger (FMPV 03) and Aspergillus tubingensis (FMPV 06) demonstrated activity. In this test, for both bacteria, MIC values did not exceed 1.5 mg/mL. The activity of these fractions was also observed across the same bacteria in the antibiofilm formation assay, since cell death occurred. The ability to eradicate biofilms was detected only for the organic fraction of the fungus Aspergillus flavus (FMPV 01) which was able to remove 52% of the already formed biofilm of S. epidermidis ATCC 35984. The absence of hemolysis of red cells was observed in all active fractions studied. In the assessment of cytotoxicity in vitro against the cell lines HMVII and Vero, only the organic fraction of Aspergillus niger (FMPV 03), showed no cytotoxic effect. However, in the test evaluation of in vivo toxicity, none of the samples tested caused a reduction in the survival of the larvae of Galleria mellonella. Then, the organic fraction of the fungus Aspergillus niger (FMPV 03) was submitted to bioguided fractionation using RP-18 column. Seven fractions were obtained, of which the first (100% water), was active against T. vaginalis, S. epidermidis e P. aruginosa. When subjected to thin layer chromatography (TLC) showed four bands were stained with ninhydrin and anisaldehyde-sulfuric acid. In addition, 100% water fraction showed no reduction of the survival of larvae of Galleria mellonella at the three concentrations tested. Therefore, the range of activities reported corroborates the potential of marine fungi to produce bioactive molecules.
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Caracterização do potencial antifúngico e antibiofilme do sal imidazólico cloreto de 1-metil-3-hexadecilimidazol (C16MlmCl) e das fraçoes purificadas de mate (Ilex paraguariensis A. St. Hil.) frente a células de biofilme de Candida tropicalis / Description of potential and antifungal antibiofilm imidazolium Salt 1- Methyl-3 – octilimidazolium chloride(c16mimcl) and purified fraction of mate ( ilez paraguariensis to. St. Hil.) front of cells biofilme Candida TropicalisBergamo, Vanessa Zafaneli January 2014 (has links)
Em contraste com a vasta descrição na literatura científica dos biofilmes bacterianos, poucos trabalhos focam o estudo da formação e as estratégias de inibição da constituição do biofilme fúngico. Este trabalho objetiva a caracterização do potencial antifúngico e antibiofilme do sal imidazólico 1-metil-3-octilimidazol cloreto (C16MImCl), e das frações (F70 e F90) purificadas de saponinas mate (Ilex paraguariensis A. st. Hil.), frente a células de biofilme de seis isolados clínicos de Candida tropicalis. A Concentração Inibitória Mínima (CIM) do C16MImCl foi de 0,014μg/mL frente às células planctônicas, ao passo que as Frações de Saponinas da Erva Mate (FSEM) não apresentaram atividade antifúngica. Utilizando o cateter traqueal (CT) como corpo de prova, foi utilizado para avaliar a capacidade de inibição e remoção do biofilme. Avaliou-se também a Concentração Mínima de Erradicação do Biofilme (CMEB) para remoção do biofilme pré-formado pelo método da microplaca. Para a atividade antibiofilme foi observado que o C16MImCl, apresentou melhor resultado quando comparado ao fluconazol. As FSEM também apresentaram atividade antibiofilme quando comparados ao fluconazol, entretanto menores do que o tensoativo sintético Pela análise dos resultados de CMEB, o C16MImCl foi o composto com maior capacidade de erradicar o biofilme pré-formado, na concentração de 0,9 μg/mL (92% a 100% de remoção do biofilme). Os demais compostos testados (fluconazol, FEM e a água) não apresentaram atividade removedora, observando-se valores menores que 80% de remoção. Tanto as concentrações nas quais C16MImCl inibiu as células planctônicas (0,014 μg/mL) como as de biofilme (0,028 -0,225 μg/mL) foram mais baixas que as obtidas pelo fluconazol. Os resultados obtidos demonstram a potencialidade destes tensoativos, principalmente o C16MImCl, que demonstrou baixa toxicidade e provável mecanismo de ação sobre a síntese do ergosterol. Assim, é possível afirmarmos que estes tensoativos representam uma potencial alternativa para o controle químico de fungos leveduriformes, especialmente as ocasionadas por células de biofilme por C. tropicalis. / In contrast to the extensive description in the literature of bacterial biofilms, few works focus on the study of the formation and strategies for inhibiting the formation of fungal biofilms. This work aims to characterize the antifungal potential and antibiofilm the imidazole salt 1 - methyl - 3 - octilimidazol chloride (C16MImCl), and fractions (F70 and F90) purified saponins mate (Ilex paraguariensis A. st. Hil.), against to biofilm cells of six clinical isolates of Candida tropicalis. The Minimum Inhibitory Concentration (MIC) of C16MImCl was 0.014 mg / mL to planktonic cells, whereas the Saponin Fractions of Yerba Mate (SFYM) showed no antifungal activity. Using the tracheal catheter (CT) as a specimen was used to evaluate the ability of the biofilm inhibition and removal. We also assessed the Minimum Biofilm Eradication Concentration (MBEC) to remove the preformed biofilms by the microplate method. For antibiofilm activity was observed that C16MImCl, showed better results when compared to fluconazole. The SFYM also had antibiofilm activity when compared to fluconazole, however smaller than the synthetic surfactant. By analyzing the results MBEC, the compound C16MImCl was capacity to eradicate pre- formed biofilm in a concentration of 0.9 mg / mL (92% to 100% biofilm removal) The other tested compounds (fluconazole, SFYM and water) showed no activity remover, observing less than 80 % removal values. Both concentrations at which they inhibit planktonic cells C16MImCl (0.014 μg/mL) and the biofilm (0.028 -0.225 μg/ml) were lower than those obtained by fluconazole. The results obtained demonstrate the potential of these surfactants, especially C16MImCl, which demonstrated low toxicity and probable mechanism of action on the synthesis of ergosterol. Thus, it is possible to assert that these surfactants are a potential alternative to chemical control of yeasts, especially those caused by biofilm cells of C. tropicalis.
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Antivirulent and antibiofilm salicylidene acylhydrazide complexes in solution and at interfacesHakobyan, Shoghik January 2015 (has links)
The growing bacterial resistance against antibiotics creates a limitation for using traditional antibiotics and requests development of new approaches for treatment of bacterial infections. Among the bacterial infections that are most difficult to treat, biofilm-associated infections are one of the most hazardous. Consequently, the prevention of biofilm formation is a very important issue. One of the techniques that are widely investigated nowadays for this purpose is surface modification by polymer brushes that allows generating antifouling antibacterial surfaces. Previously, it was reported that salicylidene acylhydrazides (hydrazones) are good candidates as antivirulence drugs targeting the type three secretion system (T3SS). This secretion system is used by several Gramnegative pathogens, including Pseudomonas aeruginosa, to deliver toxins into a host cell. Furthermore, the chemical structure of these substances allows formation of complexes with metal ions, such as Fe3+ and Ga3+. The antibacterial activity of Ga3+ is well known and attributed to its similarity to the Fe3+ ion. It has also been shown that Ga3+ ions are able to suppress biofilm formation and growth in bacteria. In this thesis the chemistry of antibacterial and antivirulence Ga3+-Hydrazone complexes in solution was studied. First, to get insights in the solution chemistry, the protonation and the stability constants as well as the speciation of the Ga3+-Hydrazone complexes were determined. Additionally, a procedure for anchoring one of the hydrazone substances to antifouling polymer brushes was optimized, and the resulting surfaces were characterized. Results showed that the complexation with Ga3+ ions stabilizes the ligand and increases its solubility. Ga3+ ion binds to the hydrazone molecule forming a strong chelate that should be stable at physiological conditions. The different biological assays, such as Ga3+ uptake, antivirulence and antibiofilm effects, indicated very complex interaction of these complexes with the bacterial cell. Negatively charged and zwitterionic surfaces strongly reduced protein adsorption as well as biofilm formation. Therefore, the antifouling zwitterionic poly-[2-(methacryloyloxy)ethyl]dimethyl-3- sulfopropyl)-ammonium hydroxide (pMEDSAH) brushes were post-modified and successfully functionalized with bioactive substances via a block-copolymerization strategy. However, in order to maintain the availability of the bioactive substance after functionalization, the hydrophobic polyglycidylmethacrylate (pGMA) top block is probably better to functionalize with a lipophilic molecules to reduce diblock copolymer brush rearrangement.
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