Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2016

Purification and safety assessment of bacteriophage preparations against Pseudomonas aeruginosa infection in children with cystic fibrosis (#259)

Stephanie Trend 1 2 , Barbara J Chang 3 , Hak-Kim Chan 4 , Stephen M Stick 1 2 5 6 , Anthony Kicic 1 2 5 6 , AREST CF 1 5 7 8
  1. Telethon Kids Institute, Perth
  2. School of Paediatrics and Child Health, The University of Western Australia, Perth
  3. School of Pathology and Laboratory Medicine, The University of Western Australia, Perth
  4. Advanced Drug Delivery Group, Faculty of Pharmacy, The University of Sydney, Sydney
  5. Department of Respiratory Medicine, Princess Margaret Hospital for Children, Perth
  6. Centre for Cell Therapy and Regenerative Medicine, School of Medicine and Pharmacology, The University of Western Australia, Perth
  7. Murdoch Children’s Research Institute, Melbourne
  8. Department of Paediatrics, The University of Melbourne, Melbourne

Background:

Chronic lung infections with Pseudomonas aeruginosa are very common in cystic fibrosis (CF), causing inflammation and lung destruction that ultimately contribute to early mortality. Current therapies include extensive intravenous or inhaled antibiotics, which are burdensome and do not always eradicate infection. As part of our current program exploring the potential of phage therapy to treat antibiotic resistant infections in CF, we aimed to develop a therapeutic preparation of bacteriophages (phages). Little is known about the impact of processing and purification of phages on phage infectivity and their safety for use in humans.  Here, we tested the hypothesis that the purified phage would not cause cytotoxicity or increased expression of inflammatory cytokines.

Methods:

Five bacteriophages, including two that were isolated from CF sputum were tested for host range against a panel of P. aeruginosa isolates from children in Perth with CF. A phage with broad activity was selected for further characterisation as an inhaled aerosol. Phage was propagated to high titre on a bacterial host (P. aeruginosa PA01). A commercial DNA extraction kit was used to collect phage DNA following DNase treatment. Phages were purified using a monolith anion-exchange column and applied using a nebuliser to primary airway epithelial cells, derived from children with CF, grown at the air-liquid interface. Phage will be optimally prepared as a dry powder and physicochemical properties and aerosol particle size assessed to estimate the effective dose in the lung. Retention of infectivity during drying and storage will also be tested to confirm suitability for inhaled therapy.

Results:

The phage selected for future work had antibacterial activity against 91% of P. aeruginosa isolates from children with CF (n=21), and showed specificity for P. aeruginosa. Phage retained infective activity for 10 weeks (to date) stored at 4°C in saline. DNA extraction yielded genomic material of sufficient quality to send for next-generation sequencing. This research is currently ongoing and latest results will be presented.