ENGINEERED BACTERIOPHAGES FOR TARGETED ELIMINATION OF ANTIBIOTIC RESISTANCE GENES: AN EMPIRICAL ASSESSMENT OF PROGRAMMABLE PAYLOADS AND RE-SENSITIZATION DYNAMICS

Abstract

Antimicrobial resistance (AMR) is a growing worldwide clinical crisis, with extensive horizontal transfer of antibiotic resistance genes (ARGs) among bacterial species playing a key role. ARG-bearing strains are under strong positive selection, a trend that may be accelerated by traditional chemotherapeutic agents, which can also kill susceptible microbial competitors. Highly selective alternative: Ge bacteriophages with programmable CRISPR–Cas payloads. These therapeutic vehicles can selectively kill host cells, disrupt important plasmid-borne vectors, or physically cure resistance factors, allowing re-sensitisation to classical drugs. We are employing different CRISPR–Cas systems (Cas9, Cas3, and Cas13a) delivered via engineered phage vectors in a comprehensive, empirical investigation of the molecular and phenotypic performance of these platforms. Here, we show that DNA-targeting systems (Cas9 and Cas3; >99.9% targeting clearance) and non-replicative phagemid vectors (up to 94.2% plasmid curing) are highly effective when active lytic cycles of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were tested against clinical isolates harbouring blaCTX-M, blaIMP-1 and mecA determinants, respectively. In contrast, the RNA-targeting Cas13a vectors efficiently inhibit resistance transcripts, leading to growth inhibition but not to clearance of the physical DNA pool. The combination of these specific vehicles and antibiotic sub-inhibitory dosing, with effective antibiotic levels, strongly restricts the development of evolutionary escape mutants in vitro. These findings provide a rigorous mechanistic framework to guide the use of sequence-specific antimicrobials to neutralise critical resistance reservoirs in heterogeneous microbial communities.