Escherichia coli overexpressing a Baeyer-Villiger monooxygenase from Acinetobacter radioresistens become resistant to Imipenem

Antimicrobial resistance is a global issue currently resulting in fatalities of hundreds of thousands a year worldwide. Data present in literature illustrate the emergence of many bacterial species that display resistance to known antibiotics; Acinetobacter spp. is a good example of this. We report here that Acinetobacter radioresistens has a Baeyer-Villiger monooxygenase (Ar-BVMO) with 100% amino acid sequence identity to Ethionamide monooxygenase of the MultiDrug-Resistant (MDR) Acinetobacter baumannii. Both enzymes are phylogenetically only distantly related to other canonical bacterial BVMO proteins. Ar-BVMO is not only capable of oxidizing two anticancer drugs metabolized by human FMO3, Danusertib and Tozasertib, but can also oxidize other synthetic drugs such as Imipenem. The latter is a member of carbapenems, a clinically important antibiotic family used in the treatment of MDR bacterial infections. Susceptibility tests performed by the Kirby-Bauer disk diffusion method demonstrate that Imipenem sensitive E. coli BL21 cells overexpressing Ar-BVMO become resistant to this antibiotic. Agar disc diffusion assay proves that when Imipenem reacts with Ar-BVMO, it loses its antibiotic property. Moreover, NADPH consumption assay with the purified Ar-BVMO demonstrates that this antibiotic is indeed a substrate and its product is identified by LC-MS as a BV oxidation product of the carbonyl moiety of the β-lactam ring. This is the first report of an antibiotic-inactivating BVMO enzyme that whilst mediating its usual BV oxidation, is also operating by an unprecedented mechanism of carbapenem resistance.