Combinations of mutations in envZ, ftsI, mrdA, acrB and acrR can cause high-level carbapenem resistance in Escherichia coli

The worldwide spread of ESBL-producing Enterobacteriaceae has led to an increased use of carbapenems, the group of beta-lactams with the broadest spectrum of activity. Bacterial resistance to carbapenems is mainly due to acquired carbapenemases or a combination of ESBL production and reduced drug influx via loss of outer-membrane porins. Here, we have studied the development of carbapenem resistance in Escherichia coli in the absence of beta-lactamases. We selected mutants with high-level carbapenem resistance through repeated serial passage in the presence of increasing concentrations of meropenem or ertapenem for similar to 60 generations. Isolated clones were whole-genome sequenced, and the order in which the identified mutations arose was determined in the passaged populations. Key mutations were reconstructed, and bacterial growth rates of populations and isolated clones and resistance levels to 23 antibiotics were measured. High-level resistance to carbapenems resulted from a combination of downstream effects of envZ mutation and target mutations in AcrAB-TolC-mediated drug export, together with PBP genes [mrdA (PBP2) after meropenem exposure or ftsI (PBP3) after ertapenem exposure]. Our results show that antibiotic resistance evolution can occur via several parallel pathways and that new mechanisms may appear after the most common pathways (i.e. beta-lactamases and loss of porins) have been eliminated. These findings suggest that strategies to target the most commonly observed resistance mechanisms might be hampered by the appearance of previously unknown parallel pathways to resistance.