Development of synthetic bacteriophages with extended host range to overcome resistant Klebsiella pneumoniae

Klebsiella pneumoniae (Kp) is a leading cause of bacterial nosocomial infections. The rapid emergence of multidrug-resistant (MDR) Kp strains poses a significant global health threat, challenging current antibiotic-based therapies. While bacteriophage therapy offers a promising alternative, its effectiveness is often limited by the narrow host ranges of natural phages and the rapid emergence of phage resistance. Synthetic phages, with their diverse and customizable genomes, have the potential to overcome these limitations. In this study, we engineered synthetic bacteriophage variants using two in-house phage isolates, ɸ115 and ɸ100, which exhibited limited efficacy against a range of clinical Kp strains. In contrast, the synthetic phage libraries derived from the recombination of tail fiber genes and genomes of ɸ115 and ɸ100 exhibited a significantly extended host range and the ability to lyse phage resistant Kp strains. Indirect measurement of bacterial respiration as a growth indicator in presence of phage, using the OmniLog system revealed that no phage resistance Kp emerged against the synthetic phage libraries within 24 h, while other Kp strains developed resistance to the parental phages. Host range analysis, burst size measurements, and genomic DNA comparisons of individual synthetic phage isolates indicated that a short central tail fiber of ɸ100 likely plays a pivotal role in extending the host range and lysing resistant Kp strains. Our findings highlight the potential of synthetic bacteriophages to overcome the dual challenges of narrow host specificity and phage resistance. This represents a significant advancement toward developing viable phage therapeutics against MDR Kp infections.