Container material dictates stability of bacteriophage suspensions: Light scattering and infectivity measurements reveal mechanisms of infectious titre decay

Abstract

Aims: To measure the infectious titre (IT) decay rate for various bacteriophages as a function of storage container material. Additionally, parallel light scattering and infectious titre measurements reveal distinct mechanisms for IT loss, depending on phage. Methods and Results: Suspensions of bacteriophages 44AHJD, P68 and gh-1 were stored in various labware. IT of each suspension was repeatedly measured over the course of 2 weeks. Large variability in IT decay was observed, with >4 log 10 loss in glass and low-binding polypropylene. Incubation of polymer containers with Bovine Serum Albumin (BSA) resulted in a consistent reduction in IT decay. Aggregation state of phage suspensions was studied by nanoparticle tracking analysis (NTA), revealing highest aggregation in glass-stored suspensions and lowest after storage in BSA-treated containers. Conclusions: Glass and 'low-binding' containers may aggravate IT decay while BSA treatment may present an easy mitigation strategy. IT versus NTA titre diagrams highlight the importance of phage inactivation in combination with aggregation.Significance and impact of the study: Container material is a significant determinant of bacteriophage IT decay. It is therefore essential to confirm IT following storage and tailor choice of phage storage containers accordingly. Aggregation of phages and adsorption onto labware surfaces are not only the mechanisms accounting for IT loss but also biological instability.

Heteroaggregation of virions and microplastics reduces the number of active bacteriophages in aqueous environments

“…Reports concerning other biomolecules assume the conformation in which biomolecular hydrophobic parts are in contact with the hydrophobic surface, whereas the more hydrophilic regions of the molecule are exposed to bulk (and water) (Rabe et al, 2011). O'Connell et al (2022) showed the effect of containers on phage titer, but they suggested that the topology of the surface governed the adsorption of phage virions on plastic surfaces. Based on these reports, we hypothesized that virions can be scavenged also by microplastic particles.…”

“…O'Connell et al. (2022) showed the effect of containers on phage titer, but they suggested that the topology of the surface governed the adsorption of phage virions on plastic surfaces. Based on these reports, we hypothesized that virions can be scavenged also by microplastic particles.…”

Approaching the Geometric Limit of Bacteriophage Conjugation to Gold: Synergy of Purification with Covalent and Physisorption Strategies

“…In conclusion, a phage-functionalized gold layer can easily approach the geometric limits of surface density when cross-linked to the surface with 11-MUA if special consideration is paid to upstream purification of the phage suspensions. Additionally, it is important to consider the impact of container material used for storage of phage suspensions as it has recently been shown that infectious titer can drop dramatically in the interval between the measurement of titer and immobilization depending on the container material …”

“…Additionally, it is important to consider the impact of container material used for storage of phage suspensions as it has recently been shown that infectious titer can drop dramatically in the interval between the measurement of titer and immobilization depending on the container material. 87 The immobilization of phages on appropriate substrates can be leveraged to detect a large variety of analytes, to reduce bioburden surrounding wounds, 88 and to prevent spoiling of food 2,3,89 and wine. 90 Phages can be engineered to express eukaryotic viral proteins on their surface to produce potent vaccines 91 or even attack these viruses directly.…”

Ultrafast and Multiplexed Bacteriophage Susceptibility Testing by Surface Plasmon Resonance and Phase Imaging of Immobilized Phage Microarrays

“…is controlled for since all phages are immobilized on the same surface. Phage titer can be unstable in aqueous suspensions, which brings the risk that otherwise identical substrates prepared on different days may be incubated with phage suspensions with different aggregation states and/or infectious titers [37,38]. Any differences in the phage response between the two chambers can reliably be considered to originate in the divergent response of each phage to each challenge strain, providing strong preliminary support for the feasibility of the proposed method of susceptibility testing.…”