In this study, an ertapenem-nonsusceptible Escherichia coli isolate was investigated to determine the genetic basis for its carbapenem resistance phenotype. This clinical strain was recovered from a patient that received, 1 year previously, ertapenem to treat a cholangitis due to a carbapenem-susceptible extendedspectrum--lactamase (ESBL)-producing E. coli isolate. Whole-genome sequencing of these strains was performed using Illumina and single-molecule real-time sequencing technologies. It revealed that they belonged to the ST131 clonal group, had the predicted O25b:H4 serotype, and produced the CTX-M-15 and TEM-1 -lactamases. One nucleotide substitution was identified between these strains. It affected the ompR gene, which codes for a regulatory protein involved in the control of OmpC/ OmpF porin expression, creating a Gly-63-Val substitution. The role of OmpR alteration was confirmed by a complementation experiment that fully restored the susceptibility to ertapenem of the clinical isolate. A modeling study showed that the Gly-63-Val change displaced the histidine-kinase phosphorylation site. SDS-PAGE analysis revealed that the ertapenem-nonsusceptible E. coli strain had a decreased expression of OmpC/OmpF porins. No significant defect in the growth rate or in the resistance to Dictyostelium discoideum amoeba phagocytosis was found in the ertapenem-nonsusceptible E. coli isolate compared to its susceptible parental strain. Our report demonstrates for the first time that ertapenem resistance may emerge clinically from ESBL-producing E. coli due to mutations that modulate the OmpR activity.KEYWORDS OmpR, ST131, avibactam, carbapenem, envZ, ESBL, temocillin C arbapenems are broad-spectrum antibiotics usually reserved for severe lifethreatening infections. Some enterobacterial isolates have developed carbapenem resistance, which resulted in limited options for the treatment of infections caused by these organisms. Except for the members of the Proteeae tribe, carbapenem resistance in Enterobacteriaceae is almost always attributable to the production of -lactamases, which can be distinguished according to their carbapenemase activity. True carbapenemases (e.g., KPC, OXA-48, and metallo--lactamases [MBLs]) confer per se resistance to carbapenems, whereas extended-spectrum -lactamases (ESBLs) and AmpC-type enzymes require an additional mechanism of resistance, such as decrease in the uptake