Standard Bacteriophage Purification Procedures Cause Loss in Numbers and Activity.

Amanda Carroll-Portillo,Henry C Lin,Cristina N Coffman,Matthew G Varga,Sudha B Singh,Joe Alcock

doi:10.3390/v13020328

Amanda Carroll-Portillo, Henry C Lin + Show 4 more

Open Access

https://doi.org/10.3390/v13020328

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Abstract

For decades, bacteriophage purification has followed structured protocols focused on generating high concentrations of phage in manageable volumes. As research moves toward understanding complex phage populations, purification needs have shifted to maximize the amount of phage while maintaining diversity and activity. The effects of standard phage purification procedures such as polyethylene glycol (PEG) precipitation and cesium chloride (CsCl) density gradients on both diversity and activity of a phage population are not known. We have examined the effects of PEG precipitation and CsCl density gradients on a number of known phage (M13, T4, and ΦX 174) of varying structure and size, individually and as mixed sample. Measurement of phage numbers and activity throughout the purification process was performed. We demonstrate that these methods, used routinely to generate “pure” phage samples, are in fact detrimental to retention of phage number and activity; even more so in mixed phage samples. As such, minimal amounts of processing are recommended to introduce less bias and maintain more of a phage population.

Highlights

To determine if concentration of phages occurred with polyethylene glycol (PEG) precipitation and cesium chloride (CsCl) gradient purification, and to assess overall phage structure post-processing, Transmission Electron Microscopy (TEM) was
PEGand precipitation and CsCl performed on all phage samples
Speaking, phages remained consistent throughout the purification procedures except for T4 phages, which visibly stained differently after CsCl

Summary

Introduction

Beyond recognition of the advantages particular phages present for therapeutic reasons, there is an emphatic need for research into the role of phage as a key component and regulator of the microbiome [3,4]. Of particular interest is the mixed phage community that is part of the gastrointestinal microbiome, for its role in both regulation of the bacterial populations and activation of the metazoan immune system [5,6,7]. Better understanding of the dynamics of the phage population as a part of the microbiome offers promise for novel therapeutic strategies for treating gastrointestinal dysbiosis expanding upon the traditional definition of phage therapy. Successful purification of the entire mixed community while maintaining activity and numbers is necessary in order for research on phage dynamics within the gastrointestinal system to progress

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