Exploiting a conjugative endogenous CRISPR-Cas3 system to tackle multidrug-resistant Klebsiella pneumoniae

Abstract

Mobile plasmids play a key role in spurring the global dissemination of multidrug-resistant (MDR) K.pneumoniae, while plasmid curing has been recognized as a promising strategy to combat antimicrobial resistance. Here we exploited a K.pneumoniae native CRISPR system to cure the high-risk IncFII plasmids. We examined matched protospacers in 725 completely sequenced IncFII plasmids from K.pneumoniae genomes. Then, we re-engineered a native CRISPR-Cas3 system and deliver the CRISPR-Cas3 system via conjugation. Plasmid killing efficiency and G.mellonella infection model were applied to evaluate the CRISPR-Cas3 immunity invitro and invivo. Genomic analysis revealed that most IncFII plasmids could be targeted by the native CRISPR-Cas3 system with multiple matched protospacers, and the targeting regions were highly conserved across different IncFII plasmids. This conjugative endogenous CRISPR-Cas3 system demonstrated high plasmid curing efficiency in vitro (8-log decrease) and invivo (∼100% curing) in a Galleria mellonella infection model, as well as provided immunization against the invasion of IncFII plasmids once the system entering a susceptible bacterial host. Overall, our work demonstrated the applicability of using native CRISPR-mediated plasmid curing to re-sensitize drug-resistant K.pneumoniae to multiple antibiotics. This work provided strong support for the idea of utilizing native CRISPR-Cas systems to tackle AMR in K.pneumoniae. This work was supported by research grants National Natural Science Foundation of China [grant numbers 81871692, 82172315, 82102439, and 82202564], the Shanghai Science and Technology Commission [grant number 19JC1413002], and Shanghai Sailing Program [grant number 22YF1437500].