Choice of Ultrafilter Affects Recovery Rate of Bacteriophages

Abstract

Studies into the viral fraction of complex microbial communities, like in the mammalian gut, have recently garnered much interest. Yet there is still no standardized protocol for extracting viruses from such samples, and the protocols that exist employ procedures that skew the viral community of the sample one way or another. The first step of the extraction pipeline often consists of the basic filtering of macromolecules and bacteria, yet even this affects the viruses in a strain-specific manner. In this study, we investigate a protocol for viral extraction based on ultrafiltration and how the choice of ultrafilter might influence the extracted viral community. Clinical samples (feces, vaginal swabs, and tracheal suction samples) were spiked with a mock community of known phages (T4, c2, Φ6, Φ29, Φx174, and Φ2972), filtered, and quantified using spot and plaque assays to estimate the loss in recovery. The enveloped Φ6 phage is especially severely affected by the choice of filter, but also tailed phages such as T4 and c2 have a reduced infectivity after ultrafiltration. We conclude that the pore size of ultrafilters may affect the recovery of phages in a strain- and sample-dependent manner, suggesting the need for greater thought when selecting filters for virus extraction.

Fecal virome is sufficient to reduce necrotizing enterocolitis

“…1A). The phages were selected to represent a range of families commonly found in gut ecosystems including tailed, non-tailed, and enveloped viruses [32]. All phages showed good recovery from the spiked fecal solution to the 0.45 µm ltered FFT and further into the ultra ltered VIR solution.…”

“…We used an exogenous community of ve known phages (Φ29, T4, c2, Φ6, ΦX174) to estimate the active phage concentration in each step of the fractionating process prior to fecal separation of the intervention material. The community was made by propagating the phages on their respective hosts [32] to a concentration of more than 10 7 plaque-forming units (PFU) per mL, then diluting in SM buffer (200 mM NaCl, 10 mM MgSO4, 50 mM Tris-HCl, pH 7.5) with 20% glycerol and mixing to get one mock solution with a concentration between 10 4 and 10 7 PFU/mL for each phage.…”

“…This was based on previous protocols [32,33]. 2 g porcine feces were dissolved in 17 mL SM buffer along with 1 mL of mock community in a sterile 100 mL lter bag (BagPage, Interscience, Saint Nom la Brétèche, France) and homogenized for 2 minutes using a stomacher (Stomacher 80, Seward, Worthing, UK).…”

“…The mock phages in RES were enumerated using plaque assays without dilution. The laboratory methods are described in detail by Larsen et al [32].…”

“…Each phage was propagated on its respective host followed by 0.45 µm ltration (Filtropur S, Sarstedt, Germany). The phage solutions were mixed to reach a concentration of 5 × 10 9 plaque forming units (PFU) per mL total, as described by Larsen et al [32].…”