The retrieval of ancient DNA sequences from long-dead organisms offers a unique perspective on evolutionary biology. In this proposal, we aim to take advantage of cutting-edge approaches in next-generation sequencing, together with the contributions of other fields, including medicine, experimental biology and microbiology, in order to access the complete genome sequences of ancient E. coli organisms that lived several hundreds to thousands of years ago. DNA enrichment coupled with high-throughput DNA sequencing will be implemented and several lines of evidence will be used to demonstrate the authenticity of the ancient E. coli sequences recovered.The wide knowledge of modern E. coli genomes will be compared to ancient strains for the first time and the plasticity of E. coli genomes will be analyzed over millions of generations, at evolutionary scales that encompass by several orders of magnitude the scope of what is accessible with standard approaches in experimental evolution, promising to reveal important features about the evolutionary mechanisms of adaptation and selection as well as the origin of the variety of modern E. coli populations. In parallel, we aim at characterizing the prevalence of virulence factors (i.e. Shiga toxin) that still cause the onset of serious outbreak of foodborne illness in Europe and in the United States. This information will reveal the importance of E. coli digestive outbreaks in past human populations and could contribute in monitoring outbreaks in current populations.We are confident that the present project, which represents a perfect synthesis of the applicant education and will be carried out under the supervision of an internationally recognised researcher within the fields of paleogenomics and hosted in one of the world’s leading centres for ancient DNA research, will push the limits of archaeogenetics and receive a wide international and general public attention.