Phage-host interaction analysis by flow cytometry allows for rapid and efficient screening of phages

Abstract

Recently, phages have become popular as an alternative to antibiotics. This increased demand for phage therapy needs rapid and efficient methods to screen phages infecting specific hosts. Existing methods are time-consuming, and for clinical purposes, novel, quick, and reliable screening methods are highly needed. Flow cytometry (FC) allows a quick differentiation and enumeration of bacterial cell populations and has been used to assess in vitro the activity of antimicrobial compounds. In this work, we propose FC as a rapid and reliable method to assess the susceptibility of a bacterial population to phage infection. For that, the interaction of phages vB_PaeM_CEB_DP1 and vB_PaeP_PE3 with Pseudomonas aeruginosa PAO1 was characterized by FC. Synchronous infection assays were performed, and samples were collected at different time points and stained with SYTO BC and PI before analysis. Part of the collected samples was used to characterize the expression of early, middle, and late genes by qPCR. Both FC and qPCR results were correlated with phage propagation assays. Results showed that SYTO BC median fluorescence intensity (MFI) values increased in the first 25 min of PE3 and DP1 infection. The increase of fluorescence is due to the expression of phage genes observed by qPCR. Since SYTO BC MFI values increase with gene expression, it allows the determination of host susceptibility to a phage in a short period of time, avoiding false positives caused by lysis from without. In conclusion, this method may allow for a quick and high-throughput real-time screening of different phages to a specific host, which can be crucial for a quick phage selection in clinical practice.