CRISPR-Cas9-Mediated Carbapenemase Gene and Plasmid Curing in Carbapenem-Resistant Enterobacteriaceae

Antimicrob Agents Chemother. 2020 Aug 20;64(9):e00843-20. doi: 10.1128/AAC.00843-20. Print 2020 Aug 20.

Abstract

Combating plasmid-mediated carbapenem resistance is essential to control and prevent the dissemination of carbapenem-resistant Enterobacteriaceae (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 blaKPC, blaNDM, and blaOXA-48 in various Enterobacteriaceae species of Klebsiella pneumoniae, Escherichia coli, Enterobacter hormaechei, Enterobacter xiangfangensis, and Serratia marcescens clinical isolates, with a >94% curing efficiency. In addition, we also demonstrated that pCasCure can efficiently eliminate several epidemic carbapenem-resistant plasmids, including the blaKPC-harboring IncFIIK-pKpQIL and IncN pKp58_N plasmids, the blaOXA-48-harboring pOXA-48-like plasmid, and the blaNDM-harboring IncX3 plasmid, by targeting their replication and partitioning (parA in pKpQIL) genes. However, curing the blaOXA-48 gene failed to eliminate its corresponding pOXA-48-like plasmid in clinical K. pneumoniae isolate 49210, while further next-generation sequencing revealed that it was due to IS1R-mediated recombination outside the CRISPR-Cas9 cleavage site resulting in blaOXA-48 truncation and, therefore, escaped plasmid curing. Nevertheless, the curing of carbapenemase genes or plasmids, including the truncation of blaOXA-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.

Keywords: CRISPR-Cas; antimicrobial resistance; carbapenem-resistant Enterobacteriaceae; plasmid.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / pharmacology
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • CRISPR-Cas Systems / genetics
  • Carbapenem-Resistant Enterobacteriaceae* / genetics
  • Carbapenem-Resistant Enterobacteriaceae* / metabolism
  • Enterobacter
  • Enterobacteriaceae Infections*
  • Microbial Sensitivity Tests
  • Plasmids / genetics
  • beta-Lactamases / genetics
  • beta-Lactamases / metabolism

Substances

  • Anti-Bacterial Agents
  • Bacterial Proteins
  • beta-Lactamases
  • carbapenemase

Supplementary concepts

  • Enterobacter hormaechei
  • Enterobacter hormaechei subsp. xiangfangensis