Tigecycline, a last-resort antibiotic in the tetracycline class, has been effective in treating infections caused by multidrug-resistant bacteria. However, the emergence of the tigecycline resistance gene cluster tmexCD-toprJ, which encodes a resistance-nodulation-division efflux pump, has significantly limited its therapeutic effectiveness. In this study, we developed two CRISPR/Cas9-based plasmids, pCas9Kill and pCas9KillTS, to target and cleave tmexCD-toprJ gene cluster from bacterial plasmids and chromosomal integrative conjugative elements (ICEs), respectively. The pCas9Kill plasmid designed to eliminate tmexCD-toprJ from plasmids through electroporation, resulting in the resensitization of the bacteria to tigecycline. Nanopore long-read sequencing revealed that the plasmids were repaired by insertion sequences after tmexCD-toprJ removal. In contrast, the pCas9KillTS plasmid introduced via conjugation to target tmexCD-toprJ gene cluster on ICEs within the chromosome. This approach led to chromosomal cleavage and subsequent bacterial cell death. Our results demonstrate that both plasmids effectively inactivated tmexCD-toprJ, with pCas9Kill restoring tigecycline susceptibility in plasmid-bearing strains and pCas9KillTS causing targeted cell death in chromosomal ICE-harboring bacteria. This study highlights the potential of CRISPR/Cas9 systems in addressing antibiotic resistance, providing a promising strategy to combat tigecycline-resistant pathogens.
Keywords: CRISPR/Cas9; genetic engineering; plasmid elimination; tmexCD-toprJ.
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