Détection précoce de la sensibilité bactérienne aux antibiotiques

Following the discovery of antibiotics, the therapeutic successes foreshadowed a future where infectious diseases of bacterial origin would be eradicated. However, in less than a century, the massive use of broad-spectrum antibiotics led to the emergence of resistance thus reducing therapeutic options. My research project aims to understand early bacterial metabolic and morphological changes induced by antibiotics and to contribute to the development of rapid and reliable diagnostic tests to promote the implementation of more targeted antibiotic treatments. By monitoring changes in various metabolic and morphological parameters of bacteria after antibiotic treatment, we have shown the interest of viability markers such as DiBAC4(3), TOPRO®-3 or Alexa FluorTM Hydrazide for rapid detection (<3h) of bacterial susceptibility to antibiotics. In particular, we have shown for the first time that protein carbonylation, which is induced under conditions of oxidative stress and cellular aging, is a universal early marker of bactericidal antibiotic susceptibility. Following this first part of the study, we wanted to understand the mechanisms involved in bacterial response to lethal stress caused by antibiotics. Following this first part of the study, we wanted to understand the bacterial mechanisms involved in response to lethal stress caused by antibiotics. In our experiments, it was observed that when the conditions no longer allowed the organism survival, a fluorescence signal intrinsically linked to the bacterium allowed to predict the fatal outcome after only 2 hours of incubation. Indeed, following a treatment with a bactericidal antibiotic targeting the synthesis of bacterial peptidoglycan (ampicillin), we observed a maximum fluorescence of the cells at the dose of antibiotic corresponding to the Minimum Inhibitory Concentration (MIC). The fluorescence increase of bacterial cells was also observed during the lethal treatment with a biocidal agent (sodium hypochlorite). However, this phenomenon is no longer observable with bacteriostatic or bactericidal antibiotics that inhibit protein synthesis indicating active bacterial metabolism importance. The correlations of spectral properties allowed us to suspect the flavin molecules as responsible for the observed autofluorescence phenomenon. In addition, we showed an overactivation of the biosynthesis pathway of flavin-type cofactors and flavoproteins occurring during ampicillin treatment. Finally, we performed cell sorting and cell survival experiments of ampicillin-treated bacterial populations. Our results showed that highly fluorescent cells have an average survival 5 times higher than low fluorescent cells. This suggests that the fluorescence signal observed is a cellular response mediated by flavonoid compounds in an attempt to survive to antibiotic treatment. Exploratory work suggests that the phenomenon studied in bacteria is conserved among yeasts and human cells. These results open new perspectives in bacterial physiology understanding, the study of bacterial response to exogenous stress and the rapid monitoring of cell viability.Suite à la découverte des antibiotiques, les succès thérapeutiques ont laissé présager un avenir où les maladies infectieuses d’origine bactérienne seraient éradiquées. Cependant, en moins d’un siècle, l’utilisation massive des antibiotiques à large spectre a conduit à l’émergence de résistances réduisant ainsi les options thérapeutiques. Mon projet de recherche vise à comprendre les altérations métaboliques et morphologiques bactériennes induites précocement par les antibiotiques et à contribuer au développement de tests diagnostiques rapides et fiables pour favoriser la mise en place d’antibiothérapies plus ciblées. Grâce au suivi des modifications des divers paramètres métaboliques et morphologiques des bactéries après traitement aux antibiotiques, nous avons montré l’intérêt des marqueurs de viabilité tels que le DiBAC4(3), le TOPRO®-3 ou encore l’Alexa FluorTM Hydrazide pour la détection rapide (