Nebulization effects on structural stability of bacteriophage PEV 44

Reduced infectivity of phage due to the nebulization process has been reported previously, but no visual evidence on structural change upon nebulization has been established, or whether these structural changes can be attributed to the titer reduction. In this study, transmission electron microscopy (TEM) was employed to assess the impact of three different types of nebulizers, air-jet, vibrating-mesh and static-mesh nebulizers, on the structural stability of a Myoviridae phage, PEV44, active against Pseudomonas aeruginosa. The morphology of the phage in the nebulized samples was categorized into two groups: "whole" (the capsid and tail held together) and "broken" (the capsid separated from the tail) phages. The "whole" phage group was further divided into three sub-groups: (1) intact, (2) contracted tail and (3) empty capsid. The starting stock suspension was found to contain considerable portions of "broken" phages (35.5 ± 6.7%), "whole" phages with contracted tails (9.9 ± 5.4%) and empty capsids (19.3 ± 8.9%). The fraction of "broken" phages was significantly increased after nebulization, with the air-jet nebulizer (83%) being more pronounced than the mesh type nebulizers (50-60%). While the amount of intact phages (2-10%) and whole phages with contracted tails (0-3%) were significantly decreased, the proportion of phages with empty capsids (15-36%) were not significantly different. Phages with broken features obtained by TEM quantification were 92.9 ± 3.2%, 74.8 ± 10.4% and 71.2 ± 11.0% for the jet, vibrating-mesh and static-mesh nebulizers, respectively. These results were found to be comparable with the titer loss obtained by the conventional plaque assay results. The in vitro aerosol performance and viable phage delivery of the three nebulizers was also assessed. The Omron nebulizer achieved a significantly higher viable respirable fraction (VRF) than the SideStream and Aeroneb Go (15.1 ± 5.8%, 2.4 ± 2.0%, 4.1 ± 2.7% respectively). In conclusion, this study identified various changes in the phage structure and viability of phage from different types of nebulizers. Understanding these effects and the phage tolerance to nebulization stresses can potentially improve our choice of the delivery method for inhaled phage therapy.