Development of a Multi-Host Enrichment Approach for Phage Isolation

Dominic Garza, Michelle Zorawik, Krisanavane Reddi, Tejas Bouklas, Amanda C Freise

Bacteriophages (phages) are often isolated using enrichment protocols that employ only a single host strain of bacteria. While this method has contributed to the isolation of tens of thousands of phages, single-host enrichments are time-consuming as they require a separate experimental set up for every host of interest. We sought to optimize phage isolation for multiple hosts by developing a multi-host enrichment process for soil-dwelling Arthrobacter phages. Multi-host enrichment cultures were prepared by incubating soil samples with PYCa liquid broth and a 48-hour multi-host Arthrobacter culture composed of A. globiformis (B-2880), A. atrocyaneous (NRRL-B-2883), A. sulfureus (ATCC 19098) and A. sp (ATCC 21022). The multi-host Arthrobacter culture was prepared by combining each of the four hosts at identical CFU/ml. All multi-host enrichment cultures were incubated with shaking at 30 ℃ for 48 hours before screening for phage presence via spot tests on each of the same four strains of Arthrobacter. For all samples that had a positive spot test, phage purification and amplification were carried out following SEA-PHAGES’ protocols. Using this approach we successfully isolated phages on all four Arthrobacter host strains, including the first A. sulfureus and A. atrocyaneous phages isolated at the University of California, Los Angeles (UCLA). These preliminary findings suggest that a multi-host enrichment protocol may offer a simple solution for saving labor during the phage isolation process, as well as possibly increase the number of phages isolated on currently undersampled hosts.