Poster Presentation BACPATH 2022

Assessing Resistance Evolution Dynamics Between Cystic Fibrosis-Derived Pseudomonas aeruginosa and Targeted Bacteriophages (#109)

Andrew Vaitekenas 1 2 , Anna S. Tai 3 4 5 , Joshua P. Ramsay 6 , Patricia Agudelo-Romero 2 , Anthony Kicic 1 2 7 8 9 , WAERP programme 2 3 8 10 11 , AREST CF 2 8 12 13
  1. Occupation, Environment and Safety, School of Population Health, Curtin University, Perth, WA, Australia
  2. Wal-Yan Respiratory Research Centre, Telethon Kids Institute, The University of Western Australia, , Perth, WA, Australia
  3. Department of Respiratory Medicine, Sir Charles Gairdner Hospital, Perth, WA, Australia
  4. Institute for Respiratory Health, Perth, WA, Australia
  5. Faculty of Health and Medical Sciences, The University of Western Australia, Perth, WA, Australia
  6. Curtin Medical School and Curtin Health Innovation Research Institute , Curtin University, Perth, WA, Australia
  7. Division of Paediatrics, School of Medicine, The University of Western Australia, Perth, WA, Australia
  8. Department of Respiratory and Sleep Medicine, Perth Children’s Hospital, Perth, WA, Australia
  9. Centre for Cell Therapy and Regenerative Medicine, School of Medicine and Pharmacology, The University of Western Australia and Harry Perkins Institute of Medical Research, Perth, WA, Australia
  10. School of Biomedical Sciences, University of Western Australia, Perth, WA, Australia
  11. St John of God Hospital, Perth, WA, Australia
  12. Murdoch Children's Research Institute, Melbourne, Vic, Australia
  13. Department of Paediatrics, University of Melbourne, Melbourne, Vic, Australia

Introduction: Pseudomonas aeruginosa is a prominent pathogen in cystic fibrosis (CF) and since it colonises early in life, it develops resistance to most antibiotics by early adulthood. Bacteriophages (phages) have been identified as a new therapeutic in this setting, however little is known about how this bacterium develops phage resistance. Here, we describe preliminary phage-resistance experiments using CF P. aeruginosa isolates. Methods: Four isolated and characterised phages were used to treat two CF-clinical isolates of P. aeruginosa (1 child-derived, 1 adult-derived) at multiplicities of infection (MOI) 10, 1, 0.1 and 0.01 in a 96 well plate. Optical density (OD600nm) was measured hourly, with phage (PFU/mL) and bacteria (CFU/mL) enumerated initially and every six hours. After 24 hours, 10 randomly selected colonies were isolated from each phage treatment, serially purified and efficiency of plating (EOP) performed to confirm resistance to the treating phage. Results: Phage and MOI dependent P. aeruginosa growth suppression was observed for 10-12 hours, and viable bacteria was reduced by 3 to 4-log after six hours before resistant re-growth. Once resistant, the child isolate returned to control CFU/mL values indicating growth comparable to wild type, whereas the adult isolate colonies never reached its control’s CFU/mL values. However, 136/160 adult isolate derived colonies were phage resistant compared to 105/160 childhood-derived colonies. This reduction in childhood isolate derived phage-resistant colonies was due to one phage remaining active against 34/40 of the colonies isolated after its treatment and another phage having reduced activity against 17/40 colonies isolated after its treatment. Conclusions: Results indicate that the adult-derived isolate may form more robust phage resistance which may have greater trade-offs for bacterial fitness. In contrast, the childhood-derived isolate appears to develop weaker resistance to phages, but does not experience as much of a fitness trade-off. To investigate further, the mechanism of phage resistance and off-target effects needs to be determined. Once fully understood it can be used to strategically formulate resistance-supressing phage cocktails.