Resensitizing tigecycline- and colistin-resistant using an engineered conjugative CRISPR/Cas9 system.

UNLABELLED

Tigecycline and colistin were referred to as the "last resort" antibiotics in defending against carbapenem-resistant, Gram-negative bacterial infections, and are currently widely used in clinical treatment. However, the emergence and prevalence of plasmid-mediated (X4) and genes pose a serious threat to the therapeutic application of tigecycline and colistin, respectively. In this research, a tigecycline- and colistin-resistant bacteria resensitization system was developed based on efficient and specific DNA damage caused by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Associated Protein 9 (Cas9) nucleases. A conjugation method was used to deliver the resensitization system, which harbors two single-guide RNAs targeting (X4) and genes and constitutively expressed Cas9. The conjugation efficiency was nearly 100% after conjugation condition optimization , and the resensitivity efficiency for clinical isolates was over 90%. In addition, when performing resensitization , the resistance marker was replaced with a glutamate-based, chromosomal, plasmid-balanced lethal system to prevent the introduction of additional resistance genes in clinical settings, making this strategy a therapeutic approach to combat the spread of antibiotic resistance genes (ARGs) among bacterial pathogens. As a proof of concept, this resensitive system can significantly decrease the counts of tigecycline- and colistin-resistant bacteria to 1% . Our study demonstrates the efficacy and adaptability of CRISPR-Cas systems as powerful and programmable antimicrobials in resensitizing (X4)- and mediated, tigecycline- and colistin-resistant strains, and opens up new pathways for the development of CRISPR-based tools for selective bacterial pathogen elimination and precise microbiome composition change.

IMPORTANCE

The emergence of plasmid-encoded (X4) and isolated from human and animal sources has affected the treatment of tigecycline and colistin, and has posed a significant threat to public health. Tigecycline and colistin are considered as the "last line of defense" for the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections, so there is an urgent need to find a method that can resensitize (X4)-mediated tigecycline-resistant and -mediated colistin-resistant bacteria. In this study, we developed a glutamate-based, chromosomal, plasmid-balanced lethal conjugative CRISPR/Cas9 system, which can simultaneously resensitize (X4)-mediated tigecycline-resistant and -mediated colistin-resistant . The counts of tigecycline- and colistin-resistant bacteria decreased to 1% after the resensitization system was administered. This study opens up new pathways for the development of CRISPR-based tools for selective bacterial pathogen elimination and precise microbiome composition change.