Paenibacillus larvae and their phages; a community science approach to discovery and initial testing of prophylactic phage cocktails against American Foulbrood in New Zealand.

Background: American foulbrood (AFB) is a devastating disease of the European honey bee (Apis mellifera) and is found throughout the world. AFB is caused by the bacterium Paenibacillus larvae (P. larvae). Treatment with antibiotics is strictly forbidden in many regions, including New Zealand. Safe and natural prophylactic solutions to protect honey bees from AFB are needed. Bacteriophages are a well-studied alternative to antibiotics and have been shown to be effective against P. larvae in other countries. Methods: We employed a community science approach to obtaining samples from around New Zealand to discover novel bacteriophages. Standard isolation approaches were employed for both bacteria and bacteriophages. Host range testing was performed by agar overlay spot tests, and cocktail formulation and in vitro testing were performed in 96-well plate assays, followed by sub-sampling and CFU visualization on agar plates. Results: Herein, we describe the discovery and isolation of eight P. larvae bacterial isolates and 26 P. larvae bacteriophages that are novel and native to New Zealand. The phage genomes were sequenced and annotated, and their genomes were compared to extant sequenced P. larvae phage genomes. We test the host ranges of the bacteriophages and formulate cocktails to undertake in vitro testing on a set of representative bacterial strains. These results form the basis of a promising solution for protecting honey bees in New Zealand from AFB.

In Vitro Evolution to Increase the Titers of Difficult Bacteriophages: RAMP-UP Protocol.

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.