The rapid emergence of phage-resistant bacterial mutants and the challenge of developing tailored phage cocktails have significantly hindered the broad application of phage therapy. This is particularly critical for infections caused by highly prevalent strains such as capsule locus 2 (KL2)-type carbapenem-resistant Acinetobacter baumannii (CRAB) in China.

We employed an iterative phage adaptive selection (iPAS) strategy to develop an optimised phage cocktail specifically targeting KL2-type CRAB strains. To facilitate efficient clinical application, we also established a rapid identification method for KL2-type isolates. The efficacy and safety of this rationally designed cocktail were subsequently evaluated in a clinical setting through its application in two compassionate use cases of CRAB infection as a proof-of-concept study. Whole genomic sequencing was conducted on the isolated phage-resistant mutants to reveal the related mutations.

KL2-type A. baumannii was the predominant lineage, accounting for 17.7% (159/896) of isolates reported across China over the past five years, and 33.3% (46/138) of clinical CRAB isolates obtained by our laboratory from five hospitals and one institute in Guangdong Province. The optimised phage cocktail effectively targeted 89.1% (41/46) of these KL2-type isolates. The phage-resistant A. baumannii mutants exhibited beneficial trade-offs, including increased antibiotic sensitivity, reduced virulence, susceptibility to immune clearance, and impaired biofilm formation. Genomic analysis revealed that these trade-offs were driven by concentrated and consistent mutations in genes involved in lipo-oligosaccharide and capsular polysaccharide biosynthesis. Crucially, the application of this cocktail in two clinical cases of CRAB infection demonstrated both clinical efficacy in resolving infections and a favourable safety profile.

This research underscores the potential of rational tailored phage cocktails developed through strategies like iPAS, to address the growing threat of CRAB infections. The successful clinical application highlights the translational impact of this study. Furthermore, the study provides valuable insights into the co-evolutionary dynamics between bacteria and phages, paving the way for broader and more effective clinical applications of phage therapy.

This work was supported by National Key R&D Programme of China; Shenzhen Medical Research Funds; Shenzhen Nanshan District Health Technology Major Project; Shenzhen Science and Technology Innovation Commission and Shenzhen Science and Technology Programme.

Phage therapy

KL-2 type carbapenem-resistant Acinetobacter baumannii

Trade-offs

Tailored phage cocktail

© 2025 The Author(s). Published by Elsevier B.V.