Resensitizing tigecycline- and colistin-resistant <i>Escherichia coli</i> using an engineered conjugative CRISPR/Cas9 system
Haijie Zhang, Bo Chen, Zeyu Wang, Kai Peng, Yuan Liu, Zhiqiang Wang
Abstract
ABSTRACT 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 tet (X4) and mcr-1 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 tet (X4) and mcr-1 genes and constitutively expressed Cas9. The conjugation efficiency was nearly 100% after conjugation condition optimization in vitro , and the resensitivity efficiency for clinical isolates was over 90%. In addition, when performing resensitization in vivo , 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 in vivo 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% in vivo . Our study demonstrates the efficacy and adaptability of CRISPR-Cas systems as powerful and programmable antimicrobials in resensitizing tet (X4)- and mcr-1- 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 tet (X4) and mcr-1 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 tet (X4)-mediated tigecycline-resistant and mcr-1 -mediated colistin-resistant bacteria. In this study, we developed a glutamate-based, chromosomal, plasmid-balanced lethal conjugative CRISPR/Cas9 system, which can simultaneously resensitize tet (X4)-mediated tigecycline-resistant and mcr-1 -mediated colistin-resistant Escherichia coli . The counts of tigecycline- and colistin-resistant bacteria decreased to 1% in vivo 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.