Nebulisation effects on the structure stability of bacteriophage PEV44

Abstract

With the present concern of widespread appearance of multi-drug resistant bacteria, bacteriophage therapy is currently being considered as a suitable alternative for bacterial infections. In the area of respiratory infections, inhalation by nebulisation is a preferred method for the delivery of phage formulations into the lung. However, the process of nebulisation is known to reduce infectivity levels (expressed by the tire loss) of phage preparations. It is unknown if any structural or morphological changes occur to the phage during nebulisation, and if any of such changes contribute to the titre loss. In this study, three different types of nebulisers were used relying on air-jet, vibrating mesh and static mesh mechanisms (SideStream, Aeroneb Go and Omron NE22, respectively) for aerosol generation. PEV44 was used as a bacteriophage model and nebulisation effects on its structure were assessed by transmission electron microscopy (TEM). Samples were negatively stained with 1% Phosphotungstic acid (PTA). Aerosol characteristics were measured by laser diffraction and phage viability by the plaque assay method. The viability within the aerosol respirable fraction was determined by particle size deposition using a multi-stage liquid impactor (MSLI). Air-jet and vibrating mesh nebulisations (SideStream and Aeroneb Go) caused a larger change on the phage morphology than static mesh nebulisation, with 50% of phages having their tail lost. In contrast, static mesh nebulisation (Omron NE22) caused only 15% tail detachment. Among the phages that conserved the tail, further observations showed that air-jet and vibrating mesh nebulisations caused tail contraction on almost 100% of phages while nebulisation by the static mesh mechanism affected about 80% of the phages. Moreover, the air-jet nebuliser caused the production of significantly more empty capsids compared with the other two nebulisers. All three nebulisers produced a similar reduction in phage viability of up to 80% (< 1 log reduction) from the initial titer. In terms of a viable respirable fraction (VRF), the Omron showed a better delivery of viable phages and had a lesser effect on the phage structure than the SideStream air-jet and Aeroneb Go vibrating mesh nebulisers.