Phage-Bacteria Evolution via Next-Generation Phage Bioreactors - Project Summary/Abstract Multi-drug resistance to conventional antibiotics is precipitating a medical crisis. An estimated 3 trillion in costs and excess loss of 10 million lives is expected by 2050. The chemical antibiotic pipeline has run dry, disincentivized by the rapid development of resistance and lack of financial gain after product development. Phage therapy has emerged as a viable treatment alternative, where bacteriophage (phage for short), which are viruses that only infect bacteria, are used as naturally adaptive drugs to treat infections that no longer respond to currently available antibiotics. However, bacteria also mutate to resist phage infections, which limits treatment. Unlike antibiotics, phage can counter-adapt to these bacterial mutations, thereby killing so- called “resistors.” This raises the possibility of developing phage cocktails populated with phage that “anticipate” the resistance trajectories of bacteria and thus limit or prevent resistance altogether. For this purpose, we have developed a co-culture system that maintains continuous logarithmic bacterial growth so as to maximize phage production on a permissive bacterial host, thereby facilitating the emergence of rare phage variants that “train” to kill target resistors in a secondary vessel. Hypothesizing that this system will allow us to chart all resistor mutations for a given bacterial species and counter-adaptations by phage, the aims of this work are to optimize the growth parameters to maximize novel evolutionary adaptations for both agents. In Aim 1, we study the effect of nutritional status, aeration, temperature, and the bacterial/viral propagation strains in driving lineages of adaptation, both in the presence and absence of chemical and genetic mutators. For Aim 2, we sequence all lineages that emerge every 6 hours for 7 weeks (~ one evolutionary cycle) to chart the mutations for bacteria-phage interactions to identify how resistance undermines phage therapy and phages that will contribute to anti- resistor cocktails. With this knowledge, we can develop phage cocktails that block common avenues for bacteria to escape phage treatment.
