A method for evaluating the host range of bacteriophages using phages fluorescently labeled with 5-ethynyl-2′-deoxyuridine (EdU)

Abstract

The evaluation of bacteriophage (phage) host range is a significant issue in understanding phage and prokaryotic community interactions. However, in conventional methods, such as plaque assay, target host strains must be isolated, although almost all environmental prokaryotes are recalcitrant to cultivation. Here, we introduce a novel phage host range evaluation method using fluorescently labeled phages (the FLP method), which consists of the following four steps: (i) Fluorescently labeled phages are added to a microbial consortium, and host cells are infected and fluorescently labeled. (ii) Fluorescent cells are sorted by fluorescence-activated cell sorting. (iii) 16S rRNA gene sequences retrieved from sorted cells are analyzed, and specific oligonucleotide probes for fluorescence in situ hybridization (FISH) are designed. (iv) Cells labeled with both fluorescently labeled phage and FISH probe are identified as host cells. To verify the feasibility of this method, we used T4 phage and Escherichia coli as a model. We first used nucleic acid stain reagents for phage labeling; however, the reagents also stained non-host cells. Next, we employed the Click-iT EdU (5-ethynyl-2'-deoxyuridine) assay kit from Invitrogen for phage labeling. Using EdU-labeled T4 phage, we could specifically detect E. coli cells in a complex microbial consortium from municipal sewage. We also confirmed that FISH could be applied to the infected E. coli cells. These results suggest that this FLP method using the EdU assay kit is a useful method for evaluating phage host range and may have a potential application for various types of phages, even if their prokaryotic hosts are currently unculturable.