A novel Acinetobacter phage reveals altered virulence traits in phage-resistant strains

Abstract

ABSTRACT Phage therapy represents a promising alternative for combating bacterial infections. This study employed an A. baumannii isolate harboring the I-F CRISPR-Cas system as a host to isolate phage and evaluate its biological characteristics. Phage-resistant mutants were screened using a double-layer agar plate assay, and the underlying molecular mechanisms were identified through whole-genome sequencing, followed by validation via gene knockout. Transcriptome sequencing was subsequently applied to alterations in the global regulatory networks of these mutants. Our results demonstrate the successful isolation of a novel myovirus, stable at 40–50°C, which was successfully isolated and found to utilize the capsule as its adsorption receptor. Whole-genomic analysis confirmed its distinction from currently published phages. Investigation into the primary resistance mechanism revealed that the capsule loss, due to an insertional mutation in the UDP-glucose 4-epimerase encoding gene galE. This conclusion was further validated through targeted gene knockout of galE. This defect concurrently attenuated bacterial virulence, as demonstrated by significantly reduced lethality in the Galleria mellonella infection model and enhanced susceptibility to serum killing, while concurrently enhancing the capacity for biofilm formation. Transcriptomic profiling indicated that the ΔgalE significantly upregulated multiple biofilm-associated genes and remodeled the transcriptomic-wide regulatory. Furthermore, the combination of carbenicillin or ceftazidime with the phage exhibited a synergistic effect in vitro, effectively inhibiting biofilm formation and suppressing the emergence of phage resistance. Overall, this work characterizes a novel phage and delineates the host’s biological network changes triggered by phage resistance, offering valuable insights for developing phage-based antimicrobial strategies.