Concept

Résistance aux antibiotiques

Résumé
La résistance aux antibiotiques ou antibiorésistance est la capacité d'une bactérie à résister aux effets des antibiotiques. C'est l'une des formes de la pharmacorésistance, différente du phénomène de tolérance aux antibiotiques. upright=1.2|vignette|Tests de résistance aux antibiotiques : une souche de bactérie cultivée en boîte de Petri est exposée à des disques blancs contenant chacun un antibiotique différent. À gauche, les bactéries sont sensibles à tous les antibiotiques testés (comme le montrent les anneaux sans bactéries autour des disques) alors que dans la boîte de droite, elles ne sont sensibles qu'à seulement trois des sept antibiotiques testés. vignette|redresse=1.2|Staphylococcus croissant dans la lumière d'un cathéter, cause possible de maladie nosocomiale. vignette|upright=1.2|Principe de la résistance aux antibiotiques. La sélection naturelle a doté les bactéries de mécanismes de résistance ou d'adaptation face à certains stress (rayonnement UV, chaleur, froid...) et
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Publications associées (100)

Design and Evaluation of New Quinazolin-4(3H)-one Derived PqsR Antagonists as Quorum Sensing Quenchers in Pseudomonas aeruginosa

Eduard Vico Oton

P. aeruginosa (PA) continues to pose a threat to global public health due to its high levels of antimicrobial resistance (AMR). The ongoing AMR crisis has led to an alarming shortage of effective treatments for resistant microbes, and hence there is a pressing demand for the development of novel antimicrobial interventions. The potential use of antivirulence therapeutics to tackle bacterial infections has attracted considerable attention over the past decades as they hamper the pathogenicity of target microbes with reduced selective pressure, minimizing the emergence of resistance. One such approach is to interfere with the PA pqs quorum sensing system which upon the interaction of PqsR, a Lys-R type transcriptional regulator, with its cognate signal molecules 4-hydroxy-2-heptylquinoline (HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS), governs multiple virulence traits and host-microbe interactions. In this study, we report the hit identification and optimization of PqsR antagonists using virtual screening coupled with whole cell assay validation. The optimized hit compound 61 ((R)-2-(4-(3-(6-chloro-4-oxoquinazolin-3(4H)-yl)-2-hydroxypropoxy)phenyl)acetonitrile) was found to inhibit the expression of the PA P-pqsA promoter controlled by PqsR with an IC50 of 1 mu M. Using isothermal titration calorimetry, a K-d of 10 nM for the P-qsR ligand binding domain (PqsR(LBD)) was determined for 61. Furthermore, the crystal structure of 61 with PqsR(LBD) was attained with a resolution of 2.65 angstrom. Compound 61 significantly reduced levels of pyocyanin, PQS, and HHQ in PAO1-L, PA14 lab strains and PAK6085 clinical isolate. Furthermore, this compound potentiated the effect of ciprofloxacin in early stages of biofilm treatment and in Galleria mellonella infected with PA. Altogether, this data shows 61 as a potent PqsR inhibitor with potential for hit to lead optimization toward the identification of a PA QS inhibitor which can be advanced into preclinical development.
AMER CHEMICAL SOC2021

Mécanismes d'action du TiO2 illuminé sur Escherichia Coli

Gaëtan Gogniat

The increase of bacterial resistance against various disinfection processes is a worrying phenomenon. As a consequence, the search for alternative techniques for fighting micro-organisms has become one of the major issues of these last few years. In this domain, water treatment is an important preoccupation for many states, either to guarantee the quality of drinking water supply or to detoxify industrial effluents, charged with chemically or biologically pollutants. The financing of this thesis project was provided by the European project AQUACAT. This project aims at evaluating an emerging technology, the titanium dioxide (TiO2) photocatalysis, as an alternative to the traditional water treatments. This technique is based on the illumination of the catalyst, which thus generates reactive oxygen species (ROS), particularly hydroxyls radicals. TiO2 photocatalysis can be used as a water treatment by detoxifying organic compounds and inactivate micro-organisms. Moreover, its bactericidal activity have a promising biomedical future, by killing cancerous cells for example. Although the bactericidal action of the TiO2 photocatalysis is well documented, its mode of action on bacteria was not yet well defined. The objective of this thesis is to contribute to a better comprehension of the mode of action of the TiO2 photocatalysis on the bacterium Escherichia coli. Few microbiologists ever worked on this subject. A review of published articles on the mode of action of TiO2 photocatalysis showed some experimental failures. Moreover, dogmas related to TiO2 photocatalysis showed contradictions, which will be discussed in this thesis. This thesis is divided in four chapters, including two major sections: i) interaction between TiO2 particles, bacteria, biomolecules and salts and ii) the defence mechanisms of the bacterial cells against TiO2 photocatalysis. The first chapter shows that bacterial cells are adsorbed onto TiO2 particles. So as to demonstrate this feature, bacterial cells and TiO2 particles were mix in the presence of two salts affecting at different degrees the effectiveness of TiO2 photocatalysis rate. In the presence of NaCl-KCl, photocatalysis was very effective and the cultivability of the cells decreased at the beginning of the illumination phase. On the contrary, in a phosphate solution, a latency phase of 20 minutes was observed. Using flow cytometry and microscopic observations, we observed very distinctly that the bactericidal effect begun when the cells started to be adsorbed on TiO2 particles. Bacterial adsorption on TiO2 particles could be correlated to the loss of cell cultivability and also to the loss of membrane integrity, measured by flow cytometry. We then studied the interaction between biomolecules (proteins and DNA), bacterial cells and TiO2 particles. Biomolecules strongly adsorbed onto TiO2 particles in a NaCl-KCl solution (up to 60 µg protein per ml of catalyst). A desorption buffer composed of phosphate (50mM, pH 7.0) and SDS (0.1%) is able to desorb 12% only of the proteins. When bacterial cells were in presence of a cell crude extract corresponding to the quantity of proteins belonging to their population (equal to 12µg protein/ml for 2×107 bacteria /ml), it was observed that the effectiveness of the treatment was decreased, suggesting that cell crude extract protected bacterial cells from the photocatalytical treatment. So as to improve our understanding of the mode of action of the TiO2 on the bacterial cells, we compared the survival of E. coli mutants with the wild type one. Knowing that TiO2 generated ROS, we assayed mutant affected in their defence mechanisms against these molecules. Thus, the sensitivity of mutants defected in the induction of DNA repair and of DNA protection systems suggested the induction of cytoplasmic damage by the photocatalytical process. We could also highlight the importance of the Fenton reaction and the formation of hydroxyls radicals in this inactivation process. The hypothesis is that these hydroxyl radicals were also produced in the cells, following an increase in reactive iron and hydrogen peroxide. Interestingly, the loss of E. coli cultivability after TiO2 illumination appeared not only during the illumination, but also when cells were incubate on Petri plates, whereas TiO2 was not illuminated. Thus bacteria underwent a secondary stress, leading also to their loss of cultivability. Finally, we identified genes expressed in the response to the treatment (primary stress) and after a short incubation in a rich culture medium to 37 °C (secondary stress). Thus after an illumination time (100% of bacteria were cultivable) and an other one which show first signs of letality (80% of the cultivable bacteria), no difference in expression of the mRNA could be observed with the transcriptome analysis (DNA chips) of the E. coli genome. On the other hand, as soon as the cell were resuspended in a rich medium after the treatment during only 5 minutes, we could observe differences, by a factor higher than two, in the induction of certain genes. The totality of genes induced during the TiO2 photocatalysis were repressed by Fur and were implied in the iron transport.
EPFL2006

Natural competence of the pathogen Acinetobacter baumannii: an elusive phenomenon

Nina Vesel

Natural competence for transformation is an important driver of horizontal DNA exchange between different organisms. This can result in accumulation of dangerous genetic features, such as antibiotic resistance genes, in a single organism. One example of an organism that frequently acquires antibiotic resistance genes is Acinetobacter baumannii, a Gram-negative opportunistic pathogen that has recently become problematic in hospital settings. Even though natural competence for transformation was demonstrated for some A. baumannii isolates, its competence regulon, the exact mechanism of DNA uptake, and the contribution of transformation to the acquisition of antibiotic resistance genes were largely unexplored at the beginning of this thesis. The aim of this thesis was therefore to better characterize optimal conditions of competence, the underlying competence regulon, and the necessary set of machinery proteins. Additionally, we aimed to identify the limiting factors that oppose transformation-mediated exchange of DNA in a small selection of strains. Here, we first addressed the general aspects of natural transformation, such as the competence window and the DNA uptake machinery. Our results showed that transformation is growth phase-dependent in A. baumannii and correlated with type IV pili (T4P) production. We demonstrated that T4P are essential for transformation and surface-associated motility but are only produced in a subfraction of the bacterial population. Furthermore, T4P production and assembly were under control of the PilSR two component system and the chemotaxis-like Pil-Chp system, respectively. These two regulatory systems were essential for competence development in A. baumannii, which is in contrast to what was observed for its close relative A. baylyi. We also investigated the reasons for non-transformability of certain A. baumannii isolates. We showed that comM interruption as well as the diversity of PilA protein sequences did not explain the strains' non-transformability. Instead, decreased expression of certain pilus genes was associated to the absence of transformability, which was also reflected in the protein level of the major component of T4P - PilA. Since the previously identified regulators could not explain the reduced transcript levels of the respective pilus genes, a transposon sequencing screen was performed to identify novel transformation-relevant genes. By comparison of transcriptional profiles of such genes between the transformable and non-transformable strains, we identified possible candidates that might explain non-transformability. Lastly, we explored whether the epigenome can influence A. baumannii's transformability. Our results showed that the source of transforming DNA has a significant effect on transformation of certain, yet not all tested strains. Specifically, for strain A118 the transformation levels were significantly decreased when non-self DNA was used as the donor DNA. Consequently, we identified a A118-specific restriction modification (RM) system that methylated specific DNA sequences in this strain and fostered the discrimination of self versus foreign DNA. Collectively, the findings of this thesis deciphered several important aspects of natural transformation in A. baumannii, which might ultimately help to better understand the spread of antibiotic resistance genes in this organism.
EPFL2022
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vignette|Gélules d'amoxicilline, l'un des antibiotiques les plus prescrits au monde, appartenant à la famille des bêta-lactamines. Un antibiotique (du grec anti : « contre », et bios : « la vie ») es
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