Targeted Reduction of Antibiotic Resistance Via Mobilizable Genetic Payloads in Klebsiella Pneumoniae

Antibiotic resistance is a growing cause of mortality worldwide due to the emergence and proliferation of resistance genes in bacterial pathogens. One such pathogen is Klebsiella pneumoniae, a notorious carrier of many resistance genes harbored on numerous extrachromosomal elements. Most concerning is the increase in carbapenem-resistant strains, which has drastically reduced effective treatment options. Current treatments escalate antibiotic use but fail to address resistance genes themselves. To address this problem, we developed a plasmid-based therapy to enter K. pneumoniae cells and eliminate carbapenemase resistance genes directly. This therapy combines TP114, a self-transmissible plasmid with CRISPR-Associated Transposases (CASTs) for targeted DNA insertion into custom genomic loci, and I-C/Cas3 for targeted DNA cutting and bidirectional digestion. These tools together constitute a system that can effectively target and degrade extrachromosomal elements that harbor antibiotic resistance genes. Further, targeted insertion of I-C/Cas3 by CAST eliminates genomic copies of antibiotic resistance genes and prevents re-entry of resistance elements, thus preventing future acquisition of resistance. While eliminating target genes, this approach seeks to avoid killing all recipient cells of the broad-host TP114 plasmid, aiming to reduce further dysbiosis caused by off-target cutting events. This novel therapy class offers the unprecedented chance to selectively reduce antibiotic resistance of constituents within a bacterial community by physical removal of putative resistance genes. Future work will test various combinations of these elements to achieve the optimal resistance clearance payload.