Spray-dried anti-Campylobacter bacteriophage CP30A powder suitable for global distribution without cold chain infrastructure

Campylobacter jejuni is a leading cause of foodborne illness globally. In this study, a spray drying and packaging process was developed to produce a thermally-stable dry powder containing bacteriophages that retains biological activity against C. jejuni after long distance shipping at ambient temperature. Spray drying using a twin-fluid atomizer resulted in significantly less (p 0.05) in biological activity after storage in suitable packaging for at least 3 weeks at room temperature and after ambient temperature shipping a total distance of approximately 19,800 kilometers, including with a 38°C temperature excursion. The bacteriophage powder therefore appears suitable for global distribution without the need for cold chain infrastructure

Development of a lyophilization process for Campylobacter bacteriophage storage and transport

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Bacteriophages are a sustainable alternative to control pathogenic bacteria in the post-antibiotic era. Despite promising reports, there are still obstacles to phage use, notably titer stability and transport‐associated expenses for applications in food and agriculture. In this study, we have developed a lyophilization approach to maintain phage titers, ensure efficacy and reduce transport costs of Campylobacter bacteriophages. Lyophilization methods were adopted with various excipients to enhance stabilization in combination with packaging options for international transport. Lyophilization of Eucampyvirinae CP30A using tryptone formed a cake that limited processing titer reduction to 0.35 ± 0.09 log10 PFU mL‐1. Transmission electron microscopy revealed the initial titer reduction was associated with capsid collapse of a subpopulation. Freeze‐dried phages were generally stable under refrigerated vacuum conditions and showed no significant titer changes over 3 months incubation at 4 °C (p = 0.29). Reduced stability was observed for lyophilized phages that were incubated either at 30 °C under vacuum or at 4 °C at 70% or 90% relative humidity. Refrigerated international transport and rehydration of the cake resulted in a total phage titer reduction of 0.81 ± 0.44 log10 PFU mL‐1. A significantly higher titer loss was observed for phages that were not refrigerated during transport (2.03 ± 0.32 log10 PFU mL‐1). We propose that lyophilization offers a convenient method to preserve and transport Campylobacter phages, with minimal titer reduction after the drying process

Jet nebulization of bacteriophages with different tail morphologies – structural effects

It was previously demonstrated that the loss of infectivity of a myovirus PEV44 after jet nebulization was closely related to a change in bacteriophage (phage) structure. In this follow-up study, we further examined the impact of jet nebulization on tailed phages, which constitute 96% of all known phages, from three different families, Podoviridae (PEV2), Myoviridae (PEV40) and Siphoviridae (D29). Transmission electron microscopy (TEM) identified major changes in phage structures after jet nebulization, correlating with their loss of infectivity. For the podovirus PEV2, jet nebulization had a negligible impact on activity (0.04 log10¬ pfu/mL loss) and structural change. On the other hand, the proportion of intact phages in the nebulised samples dropped from 50% to ~27% for PEV40 and from 15% to ~2% for D29. Phage deactivation of PEV40 measured by the TEM structural damage (0.52 log10¬ pfu/mL) was lower than that obtained by plaque assay (1.02 log10 pfu/mL), but within the range of variation (± 0.5 log10 pfu/mL). However, TEM quantification considerably underestimated the titer reduction of D29 phage, ~ 2 log pfu/mL lower than that obtained in plaque assay (3.25 log10 pfu/mL). In conclusion, nebulisation-induced titre loss was correlated with morphological damage to phages and in particular, the tail length may be an important consideration for selection of phages in inhaled therapy using jet nebulization.This work was financially supported by the Australian Research Council (Discovery Project DP150103953). The authors acknowledge the facilities and technical assistance of the Australian Microscopy and Microanalysis Research Facility at the Australian Centre for Microscop

Effects of storage conditions on the stability of spray dried, inhalable bacteriophage powders

This study aimed to develop inhalable powders containing phages active against antibiotic-resistant Pseudomonas aeruginosa for pulmonary delivery. A Pseudomonas phage, PEV2, was spray dried into powder matrices comprising of trehalose (0–80%), mannitol (0–80%) and L-leucine (20%). The resulting powders were stored at various relative humidity (RH) conditions (0, 22 and 60% RH) at 4 ºC. The phage stability and in vitro aerosol performance of the phage powders were examined at the time of production and after 1, 3 and 12 months storage. After spray drying, a total of 1.3 log titer reduction in phage was observed in the formulations containing 40%, 60% and 80% trehalose, whereas 2.4 and 5.1 log reductions were noted in the formulations containing 20% and no trehalose, respectively. No further reduction in titer occurred for powders stored at 0 and 22% RH even after 12 months, except the formulation containing no trehalose. The 60% RH storage condition had a destructive effect such that no viable phages were detected after 3 and 12 months. When aerosolised, the total lung doses for formulations containing 40%, 60% and 80% trehalose were similar (in the order of 105 pfu). The results demonstrated that spray drying is a suitable method to produce stable phage powders for pulmonary delivery. A powder matrix containing ≥ 40% trehalose provided good phage preservation and aerosol performances after storage at 0 and 22 % RH at 4 ºC for 12 months.This work was financially supported by the Australian Research Council (Discovery Project DP150103953). Authors are grateful to Tony Smithyman of Special Phage Services for his valuable discussion and advice. SSY Leung is a research fellow supported by the University of Sydney. T Parumasivam is a recipient of the Malaysian Government Scholarship. H-K Chan is funded by the National Institutes of Health (NIH Project no.1R21AI121627-01) and WJ Britton by the National Health and Medical Research Council Centre of Research Excellence in Tuberculosis Control (APP1043225)

Production of Inhalable Bacteriophage Dry Powders

Phage therapy is a possible alternative to conventional antibiotics to treat pulmonary infections caused by multidrug resistance (MDR) bacterial strains [1-4], due to its high specificity, low toxicity, capability of auto-dosing and biofilm penetration. However, most research has been confined to liquid presentations using intranasal instillation and nebulization. Therefore, there is an aim to develop efficacious and stable phage powder formulations for easy storage, transport and administration. We have previously demonstrated the suitability of spray drying and spray freeze drying to incorporate phages into inhalable dry powders [5], and shown the effect of storage humidity on the long term stability of the spray dried phage powders [6]. In the present work, we extend this to investigate the effect of storage temperature and leucine on the long term stability of inhalable powders of two types of Pseudomonas phages, PEV2 and PEV40.This work was financially supported by the Australian Research Council (Discovery Project DP150103953). SSY Leung is a research fellow supported by the University of Sydney. WJ Britton is funded by the National Health and Medical Research Council Centre of Research Excellence in Tuberculosis Control (APP1043225)

Production of inhalation phage powders using spray freeze drying and spray drying techniques for treatment of respiratory infections

Purpose The potential of aerosol phage therapy for treating lung infections has been demonstrated in animal models and clinical studies. This work compared the performance of two dry powder formation techniques, spray freeze drying (SFD) and spray drying (SD), in producing inhalable phage powders. Method A Pseudomonas podoviridae phage, PEV2, was incorporated into multi-component formulation systems consisting of trehalose, mannitol and L-leucine (F1 = 60:20:20 and F2 = 40:40:20). The phage titer loss after the SFD and SD processes and in vitro aerosol performance of the produced powders were assessed. Results A significant titer loss (~ 2 log) was noted for droplet generation using an ultrasonic nozzle employed in the SFD method, but the conventional two-fluid nozzle used in the SD method was less destructive for the phage (~0.75 log loss). The phage were more vulnerable during the evaporative drying process (~0.75 log further loss) compared with the freeze drying step, which caused negligible phage loss. In vitro aerosol performance showed that the SFD powders (~80% phage recovery) provided better phage protection than the SD powders (~20% phage recovery) during the aerosolization process. Despite this, higher total lung doses were obtained for the SD formulations (SD-F1 = 13.1 ± 1.7 ×104 pfu and SD-F2 = 11.0 ± 1.4 ×104 pfu) than from their counterpart SFD formulations (SFD-F1 = 8.3 ± 1.8 ×104 pfu and SFD-F2 = 2.1 ± 0.3 ×104 pfu). Conclusion Overall, the SD method caused less phage reduction during the powder formation process and the resulted powders achieved better aerosol performance for PEV2.This work was financially supported by the Australian Research Council (Discovery Project DP150103953). Authors are grateful to Tony Smithyman of Special Phage Services for his valuable discussion and advice. Sharon Leung is a research fellow supported by the University of Sydney. Thaigarajan Parumasivam is a recipient of the Malaysian Government Scholarship

An apparatus to deliver mannitol powder for bronchial provocation in children under six years old

Background: Currently bronchial provocation testing (BPT) using mannitol powder cannot be performed in children under 6 years. A primary reason is it is challenging for children at this age to generate a consistent inspiratory effort to inhale mannitol efficiently from a dry powder inhaler. A prototype system, which does not require any inhalation training from the pediatric subject, is reported here. It uses an external source of compressed air to disperse mannitol powder into a commercial holding chamber. Then the subject uses tidal breathing to inhale the aerosol. Method: The setup consists of a commercially available powder disperser and Volumatic™ holding chamber. Taguchi experimental design was used to identify the effect of dispersion parameters (flow rate of compressed air, time compressed air is applied, mass of powder, and the time between dispersion and inhalation) on the fine particle dose (FPD). The prototype was tested in vitro using a USP throat connected to a next generation impactor. The aerosols from the holding chamber were drawn at 10 L/min. A scaling factor for estimating the provoking dose to induce a 15% reduction in forced expiratory volume in 1 second (FEV) (PD) was calculated using anatomical dimensions of the human respiratory tract at various ages combined with known dosing values from the adult BPT. Results: Consistent and doubling FPDs were successfully generated based on the Taguchi experimental design. The FPD was reliable over a range of 0.8 (±0.09) mg to 14 (±0.94) mg. The calculated PD for children aged 1-6 years ranged from 7.1-30 mg. The FPDs generated from the proposed set up are lower than the calculated PD and therefore are not expected to cause sudden bronchoconstriction. Conclusion: A prototype aerosol delivery system has been developed that is consistently able to deliver doubling doses suitable for bronchial provocation testing in young children