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 clinical use is challenged by the rapid rise of resistant bacterial clones. To overcome this problem, phages can be trained in vitro to improve their ability to cope with the possible resistance that may arise. Here, we co-evolved phages with their hosts under different conditions and assessed their ability to infect an adapted bacterial panel using qPCR. The co-evolution experiment yielded a panel of bacterial clones adapted either to a phage, a competing phage, or a cocktail of both. Phages were adapted either in the continuous presence of an evolutionarily naïve host, in a cocktail with a competing phage, under both conditions, or under neither condition. We assessed each resulting phage for its ability to infect evolved bacterial clones in the panel we created, using qPCR for preliminary high-throughput assessment. This allowed us to evaluate 500 phages-bacteria interactions. Overall, qPCR-detected phage production following infection of different bacterial clones improved to varying degrees when evolutionary naïve hosts were added during the training. However, the screening suggests that optimal training conditions are phage-specific. For Enterobacter cloacae phages EC151 and EC152, the broadest increase in qPCR-estimated phage production 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 ten passages consistently infected a broader range of bacterial clones than those sampled after five passages. Sequencing of eight EC152-derived phages identified recurring mutations in a transcriptional regulator, and in some cases, in baseplate and tail fiber genes.