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

Abstract

Bacteriophages (phages) are currently available for use by the food indus- try to control the foodborne pathogen Listeria monocytogenes. Although phage biocon-trols are effective under specific conditions, their use can select for phage-resistant bac-teria 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 mu-tations 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. monocyto-genes and L. monocytogenes strains previously found to be broadly resistant to phage in-fection. 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 recom-binant phages with adapted host ranges. These laboratory-evolved phages may be use-ful for limiting the emergence of phage resistance and for targeting strains that show general resistance to wild-type (WT) phages. IMPORTANCE Listeria monocytogenes is a life-threatening bacterial foodborne patho-gen that can persist in food processing facilities for years. Phages can be used to control L. monocytogenes in food production, but phage-resistant bacterial subpopu-lations can regrow in phage-treated environments. Coevolution experiments were conducted on a Listeria phage-host system to provide insight into the genetic varia-tion that emerges in both the phage and bacterial host under reciprocal selective pressure. As expected, mutations were identified in both phage and host, but addi-tionally, 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 im-prove the long-term efficacy of phage-based control of L. monocytogenes. This ap-proach may also be applied to other phage-host systems for applications in biocon-trol, detection, and phage therapy.