Simple Two-step, High Yield Protocol for Isolation and Amplification of Bacteriophages Against Methicillin-resistant...

Bacteriophages are bacteria-targeting viruses that may prove useful as therapeutic agents against multidrug-resistant bacterial strains. Though phage therapy is a century-old concept, there is very limited progress on its therapeutic application due to the rapid expansion of antibiotics portfolios in the last few decades. However, the emergence of multidrug-resistant organisms has brought our attention back to bacteriophages. The first step towards developing effective phage therapy against multidrug-resistant bacteria is isolation, amplification, and purification of specific bacteriophages. There are many reported protocols for isolating host-specific bacteriophages from the environment. However, most of them are complex, multistep, low-yielding, resource-intensive protocols, requiring elaborate laboratory setup. We have demonstrated a simple two-step, high-yielding protocol for isolating and amplifying bacteriophages against methicillin-resistant Staphylococcus aureus (MRSA). We have shown that mixing various environmental samples (i.e., sample pooling) and phage amplification at two different temperatures significantly enhance the yield of MRSA phages. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of water sample filtrate for isolation of bacteriophages Basic Protocol 2: Bacterial strain and culture conditions Basic Protocol 3: Native bacteriophage count in water sample filtrate Basic Protocol 4: Isolation and enrichment of MRSA-specific bacteriophages Basic Protocol 5: Quantification of bacteriophages by drop cast method Basic Protocol 6: Effect of incubation temperature and heat shock on bacteriophage yield.