CRISPR-Cas9-Mediated Carbapenemase Gene and Plasmid Curing in Carbapenem-Resistant <i>Enterobacteriaceae</i>

Abstract

Combating plasmid-mediated carbapenem resistance is essential to control and prevent the dissemination of carbapenem-resistant <i>Enterobacteriaceae</i> (CRE). Here, we conducted a proof-of-concept study to demonstrate that CRISPR-Cas9-mediated resistance gene and plasmid curing can effectively resensitize CRE to carbapenems. A novel CRISPR-Cas9-mediated plasmid-curing system (pCasCure) was developed and electrotransferred into various clinical CRE isolates. The results showed that pCasCure can effectively cure <i>bla</i>_KPC, <i>bla</i>_NDM, and <i>bla</i>_OXA-48 in various <i>Enterobacteriaceae</i> species of <i>Klebsiella pneumoniae</i>, <i>Escherichia coli</i>, <i>Enterobacter hormaechei</i>, <i>Enterobacter xiangfangensis</i>, and <i>Serratia marcescens</i> clinical isolates, with a >94% curing efficiency. In addition, we also demonstrated that pCasCure can efficiently eliminate several epidemic carbapenem-resistant plasmids, including the <i>bla</i>_KPC-harboring IncFIIK-pKpQIL and IncN pKp58_N plasmids, the <i>bla</i>_OXA-48-harboring pOXA-48-like plasmid, and the <i>bla</i>_NDM-harboring IncX3 plasmid, by targeting their replication and partitioning (<i>parA</i> in pKpQIL) genes. However, curing the <i>bla</i>_OXA-48 gene failed to eliminate its corresponding pOXA-48-like plasmid in clinical <i>K. pneumoniae</i> isolate 49210, while further next-generation sequencing revealed that it was due to IS<i>1R</i>-mediated recombination outside the CRISPR-Cas9 cleavage site resulting in <i>bla</i>_OXA-48 truncation and, therefore, escaped plasmid curing. Nevertheless, the curing of carbapenemase genes or plasmids, including the truncation of <i>bla</i>_OXA-48 in 49210, successfully restore their susceptibility to carbapenems, with a >8-fold reduction of MIC values in all tested isolates. Taken together, our study confirmed the concept of using CRISPR-Cas9-mediated carbapenemase gene and plasmid curing to resensitize CRE to carbapenems. Further work is needed to integrate pCasCure in an optimal delivery system to make it applicable for clinical intervention.