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Mise en évidence du système de communication "Quorum Sensing" impliquant les AHLs chez des bactéries marines isolées de la Méditerranée / Evidence of an AHL-based communication system quorum sensing in marine bacteria isolated from the Mediterranean SeaAye, Armande Mireille 20 March 2015 (has links)
Le contrôle du biofouling sur des surfaces inertes immergées ou en atmosphère humide est une nécessité dans le secteur marin, tant pour des raisons économiques qu’environnementales. La formation de biofilm microbien, étape préalable à la formation du biofouling, est souvent intrinsèquement liée chez les bactéries au système de communication “Quorum Sensing” (QS). Chez certaines bactéries Gram négatif, le QS est basé sur la perception de petites molécules diffusibles appelées N- Acyl Homosérine Lactones (AHLs). L’une des stratégies antifouling en voie de développement de nos jours repose sur l’inhibition du QS bactérien. L’objectif de cette thèse est d’utiliser certaines bactéries marines afin d’identifier des molécules anti-QS capables de perturber la formation de biofilm. Ce travail a donc porté sur la mise en évidence de molécules AHLs impliquées dans le QS chez certaines bactéries marines isolées de la rade de Toulon, l’étude de la modulation de certains phénotypes dont la formation du biofilm, par ces molécules et, la mise en place d’un test préliminaire d’inhibition du QS. Parmi les trois bactéries isolées de la rade de Toulon (TC8, TC14 et TC15) du genre Pseudoalteromonas, connues pour produire de nombreuses molécules actives, et testées pour leur capacité à sécréter des AHLs, seule Pseudoalteromonas sp. TC15 a produit la C12-HSL. P. ulvae TC14, capable de produire un biofilm conséquent et de la violacéine, ne produit aucune AHL. Afin d’évaluer la possibilité d’utiliser une bactérie marine comme outil de criblage anti-QS, interférant avec les AHLs et les conséquences sur son biofilm, des AHLs exogènes ont été testées sur la production de violacéine, la formation de biofilm et la mobilité de TC14. Certaines AHLs ont montré qu’elles pouvaient réguler la production de violacéine et la formation de biofilm chez TC14, suggérant l’existence d’un récepteur AHLs fonctionnel. Des tests préliminaires d’inhibition du QS ont été effectués avec des molécules commerciales et des analogues synthétiques. La 3-oxo-C6-HSL commerciale, ainsi que l’esculétine et la p- benzoquinone, connues pour interférer avec le QS bactérien, ont été capables d’inhiber la production de violacéine ainsi que la formation de biofilm de TC14 à des concentrations n’affectant pas sa croissance. Cette étude suggère donc que P. ulvae TC14 pourrait être utilisée comme un outil de recherche de molécules anti-QS en conditions proches de celles trouvées dans l’environnement marin, et ce dans le but d’être ultérieurement testées sur la formation de biofilm. L’objectif à plus long terme reste de trouver un moyen de limiter la formation du biofilm en utilisant des molécules non toxiques pour l’environnement. / The biofouling control on immersed inert surfaces or in moist atmosphere is a necessity in the marine sector for both economic and environmental reasons. Microbial biofilm formation, the initial step of biofouling development, is intrinsically linked to the communication system “Quorum sensing” (QS). In some Gram negative bacteria, QS is based on the perception of small diffusible signaling molecules called Acyl Homoserine Lactones (AHLs). The inhibition of bacterial QS is part of the different antifouling strategies currently developed. This present work focused on the detection of AHLs molecules involved in this communication system in bacteria isolated from Toulon harbor and the study of modulation of some phenotypes, including biofilm formation, by these molecules as well as the development of a preliminary anti-QS assay. Three marine bacteria isolated from Toulon harbor (TC8, TC14 and TC15), belonging to the Pseudoalteromonas genus, known to synthesize many active molecules, have been tested for their ability to produce AHLs. Only Pseudoalteromonas sp. TC15 produced the C12-HSL. P. ulvae TC14 a violacein-producing and biofilm-forming bacteria, did not secrete any AHLs. Few marine bacteria are used as an anti-QS screening tool, especially by interfering with AHLs with the goal of studying the consequences on biofilm formation. In order to evaluate the possibility to use TC14 with this purpose, exogenous AHLs were tested on the violacein production, the biofilm formation and the motility of TC14. Some AHLs were able to regulate violacein production and biofilm formation suggesting the presence of a functional AHLs receptor in TC14. Preliminary QS inhibition assays were performed with commercial molecules and synthetic analogues. The commercial 3-oxo-C6-HSL as well as esculetin and p-benzoquinone, known to interfere with bacterial QS, were able to inhibit QS and biofilm formation at a non-toxic concentration. Overall, this study suggests that the marine strain P. ulvae TC14 may be used as a tool for the detection of anti-QS molecules in conditions closed to the marine environment. These molecules may subsequently be tested on the biofilm formation of marine bacteria. The long term objective is to find a way to limit biofilm formation, using non-toxic molecules for the environment.
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Quorum Sensing Inhibitory Activities of Various Folk-Medicinal Plants and the Thyme-tetracycline Effect.Nagy, Maria M 14 December 2010 (has links)
Pseudomonas aeruginosa is an opportunistic, nosocomial pathogen for which antibiotic resistance and biofilm development is common. Quorum sensing communication is known to be a major controlling factor in virulence gene expression, biofilm development, antibiotic resistance factors, and specifically MexAB-OprM multi-drug efflux pump expression in P.aeruginosa. MexAB-OprM efflux pumps contribute to antibiotic resistance of tetracycline and other antibiotics in pseudomonads and other organisms. P.aeruginosa infections are problematic in cystic fibrosis and burn patients; it is also the number one causative agent of respiratory infections for intensive care unit patients. Present day antibiotics are losing the battle against these infections. In theory, quorum sensing inhibitors (QSI) reduce pathogencity of the organism; making it less virulent, thus allowing either the host immune system to clear the infection or use of a QSI in combination with an antibiotic to clear more persistent pathogens. For these reasons two alternative modes of treatment were explored in this study: quorum sensing inhibition by folk-medicinal plant extracts and an example of combination drug therapy, the “thyme-tetracycline effect”.
Fifty folk-medicinal plant extracts were screened for potential anti-quorum sensing activity using two quorum sensing inhibition (QSI) reporter strains, Pseudomonas aeruginosa QSIS2 and Chromobacterium violaceum 12725. These were used to test specifically for C4-C6 and C12 HSL quorum sensing inhibition. Of the fifty plants tested, thirty plant families were represented. Eleven plant extracts (basil, chaparral, clove, cranberry, oregano, pomegranate, rosemary, sage, sassafras, thyme and witch hazel) showed C4 HSL quorum sensing inhibition as determined by both assays. Interestingly, five of the plants were from the Lamiaceae family. Thymus vulgaris (thyme), also from the Lamiaceae family, was chosen for further assessment.
Previous research has shown that thyme extract can synergistically augment tetracycline activity against tetracycline-resistant Pseudomonas aeruginos, creating the “thyme-tetracycline effect.” Disc diffusion assay, thin layer chromatography (TLC), and TLC bioassay techniques were used to show that thymol is the active component in the thyme extract that augments tetracycline activity against resistant Pseudomonas. This study also showed that thymol is a potent C4 HSL quorum sensing inhibitor. The collective data suggests a potential mode of action for the thyme-tetracycline effect: thymol appears to prevent MexAB-OprM efflux pump gene expression. By blocking MexAB-OprM expression, tetracycline antibiotic accumulation can occur within the cell, thus allowing cellular damage.
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