CRISPR-Cas9 nanotherapeutic targeting of antibiotic resistance determinants in multidrug-resistant plant-associated bacteria

Abstract

Antimicrobial resistance (AMR) in plant-associated bacteria represents an emerging agricultural and environmental concern, highlighting the need for precision-based approaches to target resistance determinants. This study investigated a CRISPR-Cas9 nanotherapeutic strategy for targeted reduction of an antibiotic resistance gene in a multidrug-resistant (MDR) bacterium associated with tomato. Diverse culturable bacterial isolates were recovered from tomato samples and screened for antimicrobial susceptibility, resulting in the selection of a single isolate exhibiting resistance to multiple antimicrobial classes. 16S rRNA gene sequencing identified the isolate as closely affiliated with Pseudomonas aeruginosa, showing 99.4% sequence identity with strain PAO1. Molecular screening detected a bla-type β-lactam resistance determinant, which was selected as the target for CRISPR-Cas9-mediated intervention. Candidate target sequences were evaluated for specificity, conservation, protospacer-adjacent motif (PAM) compatibility, and predicted off-target potential; Candidate 1 showed the most favorable overall profile, with scores of 98.5, 95.0, 100.0, and 96.0, respectively. The CRISPR-Cas9 cargo was incorporated into a nanocarrier with a mean particle size of approximately 130 nm and a low PDI (0.12), indicating relatively homogeneous particles. Targeted treatment reduced bla abundance to 18.5% of the untreated control, corresponding to an 81.5% reduction, whereas nanocarrier-only and non-targeting controls showed minimal effects. This molecular response was accompanied by increased inhibition zones (20.8–24.5 mm) against ampicillin, amoxicillin, and ceftazidime. The nanotherapeutic also maintained substantial physicochemical stability and approximately 90.1% cargo retention over seven days under plant-associated conditions. Overall, CRISPR-Cas9 nanodelivery selectively targeted the resistance determinant and restored antibiotic susceptibility, warranting further validation under plant and field conditions.