Abstract With the global rise of antimicrobial resistance, phage therapy is increasingly re-gaining traction as a strategy to treat bacterial infections. For phage therapy to be successful however, we first need to isolate appropriate candidate phages for both clinical and experimental research . Acinetobacter baumannii is an opportunistic pathogen known for its ability to rapidly evolve resistance to antibiotics, making it a prime target for phage therapy. Yet phage isolation is often hampered by A. baumannii ’s ability to rapidly switch between capsular states. Here, we report the discovery and structural characterisation of a novel lytic phage, Mystique. This phage was initially isolated against the wild-type AB5075: a commonly used clinical model strain against which no phage has previously been readily available for the capsulated form. When screening Mystique on 103 highly diverse isolates of A. baumannii , we found that it has a broad host range, being able to infect 85.4% of all tested strains when tested on bacterial lawns – a host range which expanded to 91.3% when tested in liquid culture. This variation between solid and liquid environments on phage infectivity was also observed for several other phages in our collection that were assumed unable to infect AB5075, and capsule negative mutants that initially seemed completely resistant to Mystique proved susceptible when assayed in liquid. Overall, through the discovery of a novel phage we demonstrate how environmental differences can drastically impact phage infectivity with important consequences for phage isolation and characterisation efforts. Author summary Bacterial infections caused by Acinetobacter baumannii are a major global health concern due to high antibiotic resistance, earning it a critical priority pathogen ranking by the WHO. Phage therapy is resurging as a treatment option, with some success against A. baumannii . However, the wild-type clinical model strain used to assess new therapies lacks an available phage, and isolating phages for A. baumannii is challenging due to its complex capsule. Here, we report the discovery of a novel lytic phage, Mystique, which exhibits a broad host range, infecting 94 out of 103 tested A. baumannii strains. We conducted genomic sequencing and structural analysis to fully characterise Mystique. Additionally, we found that the testing environment significantly impacts results; some phages that do not form plaques on bacterial lawns can still infect and amplify in liquid cultures of the same strain. Moreover, mutants resistant to Mystique based on plaque assays were susceptible in liquid culture assays. This work underscores the necessity of a multifaceted approach for phage isolation and characterisation, as traditional phage assays may not be sufficient for studying bacteria-phage dynamics in certain bacteria such as A. baumannii .

Environmental differences impact Acinetobacter baumannii phage isolation and infectivity