Delivering active phage requires a prudent choice of inhalation device. The jet nebulizer was not a good choice for aerosolizing phage D29 under the tested conditions, due to substantial titer reduction likely occurring during droplet production. The vibrating mesh nebulizer is recommended for animal inhalation studies requiring large amounts of D29 aerosol, whereas the soft mist inhaler may be useful for self-administration of D29 aerosol.

Globally, more people die annually from tuberculosis than from any other single infectious agent. Unfortunately, there is no commercially available vaccine that is sufficiently effective at preventing the acquisition of pulmonary tuberculosis in adults. In this study, preexposure prophylactic pulmonary delivery of active aerosolized antituberculosis bacteriophage D29 was evaluated as an option for protection against Mycobacterium tuberculosis infection. An average bacteriophage concentration of approximately 1 PFU/alveolus was achieved in the lungs of mice using a nose-only inhalation device optimized with a dose simulation technique and adapted for use with a vibrating mesh nebulizer. Within 30 min of bacteriophage delivery, the mice received either a low dose (∼50 to 100 CFU) or an ultralow dose (∼5 to 10 CFU) of M. tuberculosis H37Rv aerosol to the lungs. A prophylactic effect was observed, with bacteriophage aerosol pretreatment significantly decreasing M. tuberculosis burden in mouse lungs at 24 h and 3 weeks postchallenge (P < 0.05). These novel results indicate that a sufficient dose of nebulized mycobacteriophage aerosol to the lungs may be a valuable intervention to provide extra protection to health care professionals and other individuals at risk of exposure to M. tuberculosis.

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Therapeutic bacteriophages offer a potential alternative approach in the treatment of drug resistant bacteria. In the present study, we examine the ability of atmospheric spray freeze-drying (ASFD) to process bacteriophage D29 into a solid dry formulation. Bacteriophage D29 is of particular interest due to its ability to infect Mycobacterium tuberculosis . A sugar solution containing bacteriophage D29 was sprayed and instantly frozen in a cold chamber. Cold drying gas was then passed through the chamber at a high flow rate and atmospheric pressure. Convective transport combined with the low temperature of the drying gas results in sublimation of ice, yielding a free-flowing, porous powder. The bacteriophages were atmospheric spray freeze-dried in solutions with varying concentrations of trehalose and mannitol. A solution of trehalose and mannitol at a mass ratio of 7:3 and a total mass concentration of 100 mg/mL led to powder with 4.9 ± 0.1% moisture content and an acceptable titer reduction of ∼0.6 logs. In comparison, a pure trehalose solution and a 1:1 ratio of trehalose and mannitol both had titer reductions of >1.5 logs. Spectroscopic analysis showed that trehalose in the powder was amorphous while mannitol completely crystallized during the drying process, both of which are desirable for preserving phage viability and storage in powders. The results highlight the potential for using ASFD as an alternative process in preserving biopharmaceutical products.