Functional export of NDM-7 to outer membrane vesicles in Klebsiella pneumoniae compromises imipenem and cefiderocol activity

Abstract

The carbapenemases KPC and NDM are the most widespread determinants of carbapenem resistance in Klebsiella pneumoniae. Whereas KPC is a soluble periplasmic serine-{beta}-lactamase, NDM is a membrane-anchored metallo-{beta}-lactamase (MBL), a feature that promotes its incorporation into outer membrane vesicles (OMVs). OMVs are naturally released nanoparticles that deliver diverse bioactive cargo, including enzymes, virulence factors, and signaling molecules, and may contribute to antibiotic resistance. Here, we investigated the export and activity of carbapenemases in OMVs produced by carbapenem-resistant Klebsiella pneumoniae clinical isolates expressing NDM-7, an emerging variant, or KPC-2, as well as in isogenic laboratory-derived K. pneumoniae strains producing NDM-1, NDM-7 or KPC-2. NDM enzymes were detected in vesicles released by NDM-producing strains, whereas KPC-2 remained confined to the cellular fraction and was not observed in OMVs. OMVs contained catalytically active NDM enzyme and conferred protection to susceptible K. pneumoniae against imipenem. Importantly, NDM-positive OMVs also partially restored bacterial growth in the presence of cefiderocol, a siderophore cephalosporin used to treat infections caused by MBL producers. This protective effect was more pronounced for NDM-7 than for NDM-1. Together, these findings show that the clinically emerging NDM-7 variant is efficiently packaged into OMVs in K. pneumoniae and remains enzymatically active, allowing extracellular antibiotic degradation and conferring protection to susceptible bacteria exposed to carbapenems and cefiderocol.