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Escherichia coli – one of the most characterized bacteria and a major public health concern – remains invisible across the temporal landscape. Here, we present the meticulous reconstruction of the first ancient E. coli genome from a 16th century gallstone from an Italian mummy with chronic cholecystitis. We isolated ancient DNA and reconstructed the ancient E. coli genome. It consisted of one chromosome of 4446 genes and two putative plasmids with 52 genes. The E. coli strain belonged to thedoi:10.21203/rs.3.rs-1409634/v1 fatcat:kntgxvkuy5crtd2pgqvu3j6xti