In VitroEvolution to Increase the Titers of Difficult Bacteriophages: Rapid Appelmans Protocol

Abstract

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. Here, we present two distinct approaches to experimentally evolve novel New ZealandPaenibacillus larvaebacteriophages. The first approach uses the traditional agar-overlay method, whereas the other was a Rapid Appelmans Protocol (RAP) modelled after the established Appelmans Method. Both approaches resulted in an increase in plaque-forming units (PFU/mL). The RAP approach was significantly faster and simpler, and allowed us to evolve a set of bacteriophages in as little as four days, increasing titers 100-1000-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. We also propose a model that describes the parameters that allow the RAP approach to select improvement of bacteriophage titer. The RAP approach is an effective method for experimentally evolving previously intractable bacteriophages in a high-throughput and expeditious manner.

The potential of phage model systems as therapeutic agents

“…To answer the above questions, protocols could be adapted to specifically evolve ΦX174 (or, potentially, other phage) to infect pathogenic enterobacteria (Bono et al 2013;Burrowes et al 2019;Kok et al 2023;) (Fig. 1).…”

Directed evolution of bacteriophages: impacts of prolific prophage

“…Also referred to as phage training, one goal of directed evolution is to procure phages with altered or expanded host range targeting phageresistant or otherwise non-permissive hosts (2). The Appelmans protocol is one directed evolution method that is used to expand the host range of bacteriophages for phage therapy applications (2)(3)(4). This method employs a parallel presentation of bacterial hosts to a phage cocktail of two or more phages, where the phage mixture is serially diluted and allowed to propagate on individual hosts, followed by harvesting pooled lysate.…”

“…The end goal is to obtain a phage lysate or an individual phage isolate that has evolved to infect a desired number of target bacterial hosts. Subsequent infections increase the likelihood of coinfection and recombination, thus increasing the potential for diversity in the resulting pooled phage lysate, without the addition of new genetic information (4). In theory, as the input phage cocktail is propagated on the hosts, pooled lysate includes both input and evolved phage progeny.…”

“…The Appelmans protocol is one directed evolution method that is used to expand the host range of bacteriophages for phage therapy applications(2–4). This method employs a parallel presentation of bacterial hosts to a phage cocktail of two or more phages, where the phage mixture is serially diluted and allowed to propagate on individual hosts, followed by harvesting pooled lysate.…”