Eliminating the tigecycline resistance RND efflux pump gene cluster tmexCD-toprJ in bacteria using CRISPR/Cas9.

Objectives: 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. This study aims to explore the potential of CRISPR/Cas9-based plasmids to target and cleave tmexCD-toprJ gene cluster from bacterial plasmids and chromosomal integrative conjugative elements (ICEs), respectively.

Methods: We developed two CRISPR/Cas9-based plasmids, pCas9Kill and pCas9KillTS. The pCas9Kill plasmid designed to eliminate tmexCD-toprJ from plasmids through electroporation, while the pCas9KillTS plasmid, delivered through conjugation, targeted tmexCD-toprJ within ICEs on the bacterial chromosome. The plasmid modifications were assessed using nanopore long-read sequencing.

Results: Electroporation with the pCas9Kill plasmid resulted in the removal of tmexCD-toprJ from plasmids, restoring bacterial susceptibility to tigecycline. Nanopore 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.

Conclusion: 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-harbouring bacteria. This study highlights the potential of CRISPR/Cas9 systems in addressing antibiotic resistance, providing a promising strategy to combat tigecycline-resistant pathogens.