A Rapid Screening Strategy for Secondary Bacteriophages to Counter Phage Resistance in Carbapenem-Resistant...

Background: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major nosocomial pathogen; bacteriophage therapy is a promising alternative, but its principal challenge is the rapid emergence of phage resistance. Cocktails counter this yet they are usually assembled by simply pooling phages active against the bacterium, not designed against the resistant mutants that arise. Methods: Next evolutionary phage typing (NEPT) addresses this by inducing resistance to a primary phage and then selecting secondary phages that lyse the resulting resistant mutant. Because choosing effective secondary phages still relies on qualitative visual reading of plaques (size and clarity), this study aimed to identify a rapid quantitative criterion. Results: Using the clinical strain CRAB 43895 and primary phage ϕ8, we obtained 65 NEPT-derived secondary phages that lyse the ϕ8-resistant mutant (ϕ8R) and combined four quantitative indicators-relative bacterial growth, lytic capability, coverage rate, and plaque morphology-with binary logistic regression to predict effective inhibition by the ϕ8 + secondary-phage cocktail (96-h OD₆₀₀ < 0.1). Of the four, only lytic capability (the titer after 3 h of co-culture) independently predicted effective inhibition (ROC AUC = 0.76; at ≥10⁷ plaque-forming units (PFU)/mL, sensitivity 0.83, specificity 0.69). Conclusions: Lytic capability thus provides a rapid (~8-12 h), quantitative criterion for selecting effective secondary phages within the NEPT framework, improving on conventional qualitative typing.