Bacterial infections are a leading cause of morbidity and mortality. The increasing resistance to antibiotics among pathogenic bacteria is a major health concern. These infections are often chronic and due to the presence of self-structured multicellular communities called biofilms developing on medical devices or mucosa. In biofilms, bacteria undergo specific physiological changes and display characteristic but ill-understood high level of tolerance to both antimicrobial agents and host immune defences. As a consequence, there is currently no fully efficient method to prevent or eradicate biofilms. Whereas enhanced tolerance of biofilms towards antibiotics is a multifactorial process, relapse of infection is mainly explained by the presence within biofilms of high levels of so called persister bacteria that can sustain extremely high concentration of antibiotics but can regrow as biofilms when treatment is stopped. Persister bacteria are proposed to serve as a potential evolutionary reservoir from which resistance could emerge. The presence of these persister bacteria can be mainly explained by quiescence and stress-response caused by high physico-chemical heterogeneity and localized biofilm area in which bacteria have limited access to, for instance, nutrients or oxygen. The impact of bacterial persisters in clinical situations has been largely overlooked and evolution of tolerance due to persisters is not understood. However, recent studies have demonstrated that high-levels of antibiotic tolerance, but not resistance, could be rapidly achieved by exposure of batch planktonic cultures of E. coli and other pathogens to cyclic treatments of lethal concentration of antibiotics. Considering the importance of biofilms in chronic infections and the failure of their treatment there is an urgent need to characterize evolution of persistence and resistance within biofilms. We have developed an in vitro model of pathogenic E. coli biofilms on silicone disks and perform a first series of Adaptive Laboratory Evolution experiments (ALE), which mimic relevant clinical situations of biofilm-associated chronic infection that are treated by intermittent exposure of lethal antibiotic concentrations. We have shown that E. coli biofilms could rapidly evolved increased resistance towards 2 lethal concentration of the aminoglycoside, amikacin, through accumulation of mutations known to cause resistance. These promising preliminary experiments strongly suggest that the evolutionary path followed by biofilms and planktonic bacteria are different, with biofilms strongly favoring rapid emergence of genetic resistance, may be because of the high frequency of persister bacteria present in biofilms and increased mutability. These first results urge for a deepened analysis of emergence of antibiotic resistance within biofilms. Using ALE experiments, the developed in vitro biofilm model and a clinically relevant in vivo rat model of catheter-related infection, we will i/ pursue the analysis of the evolutionary trajectories that can lead to increased levels of tolerance and then resistance towards lethal concentration of different antibiotics in pathogenic E. coli within clinically relevant biofilm environments; ii/ determine whether the emergence of resistance within biofilms is directly link to the frequency of persister bacteria ; ii/ analyze, using DNA barcoding tagging, the dynamic of dissemination of emerged tolerant/resistant clones and repopulation of biofilms during intermittent antibiotic treatment. This project should contribute to improve our understanding of the dynamic relationships between biofilm tolerance and emergence of antibiotic resistance, as well as of the dissemination of this tolerance/resistance in highly structured environments. This could lead to the design of relevant strategies or clinical treatment protocols to mitigate the emergence of high tolerance and subsequent antibiotic resistance in clinically relevant situations.