Background: Bacteriophages are becoming increasingly important in the race to find alternatives to antibiotics. Unfortunately, bacteriophages that might otherwise be useful are sometimes discarded due to low titers making them unsuitable for downstream applications.
Methods: Here, we present two distinct approaches used to experimentally evolve novel New Zealand Paenibacillus larvae bacteriophages. The first approach uses the traditional agar-overlay method, whereas the other was a 96-well plate liquid infection protocol that improved phage titers in as little as four days. We also used a mathematical model to probe the parameters and limits of the RAMP-UP approach to rapidly select mutants that improve bacteriophage titers.
Results: Both experimental approaches resulted in an increase in plaque-forming units (PFU/mL). The liquid infection approach developed here, which we call RAMP-UP for Rapid Adaptive Mutation of Phage - UP, was significantly faster and simpler, and allowed us to evolve high titer bacteriophages in as little as four days. Titers were increased from 100-100,000-fold relative to their ancestors. The resultant titers were sufficient to extract and sequence DNA from these bacteriophages. An analysis of these phage genomes is provided.
Conclusion: The RAMP-UP protocol is an effective method for experimentally evolving previously intractable bacteriophages in a high-throughput and expeditious manner.
Keywords: Appelmans protocol; Paenibacillus larvae; applied evolution; honeybee; phage; population biology.
Copyright 2023, Mary Ann Liebert, Inc., publishers.