A qPCR Based Screening Platform for Exploratory Assessment of Phage Training Outcomes in Enterobacter cloacae and Stenotrophomonas maltophilia

Bacteriophages (phages) represent promising therapeutic agents. Their use in treatments is challenged by the rapid rise of resistant bacterial clones. To overcome this problem, phages can be trained in vitro to adapt them to the possible resistance that may arise. Here, we co-evolved phages with their hosts under different conditions and assessed the outcomes using qPCR. The co-evolution experiment yielded a panel of bacterial clones that were either adapted to a phage, a competing phage, or to a cocktail of both. The adaptation of a phage was done either in the continuous presence of an evolutionarily naïve host, or in a cocktail with a competing phage, or both conditions, or neither conditions. We assessed each obtained phage ability to infect evolved bacterial clones in the panel we created, and we used qPCR to enable high-throughput assessment. This allowed us to evaluate 500 phage-bacteria interactions. While all phages benefitted from the presence of evolutionary naïve hosts, the screening suggests that optimal training conditions are phage-specific, based on the four phages tested. For Enterobacter cloacae phages EC151 and EC152, the most extensive infectivity in our experiments was observed when a competing phage and/or an evolutionarily naïve host was included during adaptation. For Stenotrophomonas maltophilia phages StM171 and StenM174, the presence of an evolutionarily naïve hosts appeared beneficial in both replicates; co-adaptation with a competing phage led to a complete loss of StM171 infectivity in both experiments, but benefited StenM174. Phages passaged for 10 passages consistently infected a broader range of bacterial clones than those sampled after 5 passages. Sequencing of 8 phages obtained after adapting EC152 identified recurring mutations in a transcriptional regulator, and in some cases, in the baseplate and tail fiber genes.