Directed evolution of bacteriophages: impacts of prolific prophage.

Various directed evolution methods exist that seek to procure bacteriophages with expanded host ranges, typically targeting phage-resistant or non-permissive bacterial hosts. The general premise of these methods is to propagate phage on multiple bacterial hosts, pool the lysate, and repeat the propagation process until phage(s) can form plaques on the target host(s). In theory, this propagation process produces a phage lysate that contains input phages and their evolved phage progeny. However, in practice, this phage lysate can also include prophages originating from bacterial hosts. Here we describe our experience implementing one directed evolution method, the Appelmans protocol, to study phage evolution in the Pseudomonas aeruginosa phage-host system, in which we observed rapid host-range expansion of the phage cocktail. Further experimentation and sequencing analysis revealed that this observed host-range expansion was due to a Casadabanvirus prophage that originated from one of the Appelmans hosts. Host-range analysis of the prophage showed that it could infect five of eight bacterial hosts initially used, allowing it to proliferate and persist through the end of the experiment. This prophage was represented in half of the sequenced phage samples isolated from the Appelmans experiment. This work highlights the impact of prophages in directed evolution experiments and the importance of incorporating sequencing data in analyses to verify output phages, particularly for those attempting to procure phages intended for phage therapy applications. This study also notes the usefulness of intraspecies antagonism assays between bacterial host strains to establish a baseline for inhibitory activity and determine presence of prophage. IMPORTANCE: Directed evolution is a common strategy for evolving phages to expand host range, often targeting pathogenic strains of bacteria. In this study we investigated phage host-range expansion using directed evolution in the Pseudomonas aeruginosa system. We show that prophage are active players in directed evolution and can contribute to observation of host-range expansion. Since prophage are prevalent in bacterial hosts, particularly pathogenic strains of bacteria, and all directed evolution approaches involve iteratively propagating phage on one or more bacterial hosts, the presence of prophage in phage preparations is a factor that needs to be considered in experimental design and interpretation of results. These results highlight the importance of screening for prophages either genetically or through intraspecies antagonism assays during selection of bacterial strains and will contribute to improving experimental design of future directed evolution studies.

Complete Genome Sequence of a Serotype 7 Listeria monocytogenes Strain, FSL R9-0915.

Listeria monocytogenes serotype 7 lacks glycosidic constituents in wall teichoic acids. Here, we present the complete genome sequence of L. monocytogenes serotype 7 strain FSL R9-0915 and an analysis of genes known to affect L. monocytogenes antigenicity. This strain is used as a control strain in Listeria phage host range analyses.

Complete Genome Sequences of Three Listeria monocytogenes Bacteriophage Propagation Strains.

Bacteriophages can be used as a biocontrol for the foodborne pathogen Listeria monocytogenes Propagation of phages is a necessary step for their use in experimental studies and biocontrol applications. Here, we present the complete genomes of three Listeria monocytogenes strains commonly used as propagation hosts for Listeria phages.

Mutant and Recombinant Phages Selected from In Vitro Coevolution Conditions Overcome Phage-Resistant Listeria monocytogenes.

Bacteriophages (phages) are currently available for use by the food industry to control the foodborne pathogen Listeria monocytogenes Although phage biocontrols are effective under specific conditions, their use can select for phage-resistant bacteria that repopulate phage-treated environments. Here, we performed short-term coevolution experiments to investigate the impact of single phages and a two-phage cocktail on the regrowth of phage-resistant L. monocytogenes and the adaptation of the phages to overcome this resistance. We used whole-genome sequencing to identify mutations in the target host that confer phage resistance and in the phages that alter host range. We found that infections with Listeria phages LP-048, LP-125, or a combination of both select for different populations of phage-resistant L. monocytogenes bacteria with different regrowth times. Phages isolated from the end of the coevolution experiments were found to have gained the ability to infect phage-resistant mutants of L. monocytogenes and L. monocytogenes strains previously found to be broadly resistant to phage infection. Phages isolated from coinfected cultures were identified as recombinants of LP-048 and LP-125. Interestingly, recombination events occurred twice independently in a locus encoding two proteins putatively involved in DNA binding. We show that short-term coevolution of phages and their hosts can be utilized to obtain mutant and recombinant phages with adapted host ranges. These laboratory-evolved phages may be useful for limiting the emergence of phage resistance and for targeting strains that show general resistance to wild-type (WT) phages.IMPORTANCEListeria monocytogenes is a life-threatening bacterial foodborne pathogen that can persist in food processing facilities for years. Phages can be used to control L. monocytogenes in food production, but phage-resistant bacterial subpopulations can regrow in phage-treated environments. Coevolution experiments were conducted on a Listeria phage-host system to provide insight into the genetic variation that emerges in both the phage and bacterial host under reciprocal selective pressure. As expected, mutations were identified in both phage and host, but additionally, recombination events were shown to have repeatedly occurred between closely related phages that coinfected L. monocytogenes This study demonstrates that in vitro evolution of phages can be utilized to expand the host range and improve the long-term efficacy of phage-based control of L. monocytogenes This approach may also be applied to other phage-host systems for applications in biocontrol, detection, and phage therapy.