Rapid quantification of bacteriophages and their bacterial hosts in vitro and in vivo using droplet digital PCR

Abstract

Viruses that infect bacteria, bacteriophages (phages), are well-studied across a wide range of environments and among diverse scientific fields. Nevertheless, current methods in phage research are lacking, in part due to limitations in culturability and the lack of a universal gene marker. Here, we demonstrate that droplet digital PCR (ddPCR) can be used as a repeatable and sensitive method to study bacteria-phage dynamics both in vitro and in vivo. Using fluorescent probes designed for the bacterial plant pathogen, Pseudomonas syringae, and two phages that prey upon it, we illustrate the use of ddPCR to enumerate phages, track bacteria and phage densities over time both in media co-culture and during infection of a tomato plant, compare phage time-to-lysis, and explore phage-phage competition. Overall, the ddPCR approach closely mirrors results from more traditional counts of plaque forming units (PFUs) but offers a much faster, lower waste, and more high-throughput way of studying these interactions. As such, we suggest that ddPCR will be a valuable new tool in bacteriophage research.