Poster Presentation BACPATH 2022

The search for the next silver bullet – phage, antibiotics and EDTA (#102)

Renee N Ng 1 2 , Barbara J Chang 3 , Stephen M Stick 2 4 5 , Anthony Kicic 2 5 6 , WAERP Programme 1 2 4 7 , AREST CF 2 4 8 9
  1. School of Biomedical Sciences, The University of Western Australia, Perth, WA, Australia
  2. Wal-Yan Respiratory Research Centre, Wal-Yan Respiratory Research Centre, Telethon Kids Institute, Perth, Western Australia, Australia
  3. The Marshall Centre for Infectious Diseases Research and Training, School of Biomedical Sciences, The University of Western Australia, Perth, WA, Australia
  4. Department of Respiratory Medicine, Perth Children’s Hospital, Perth, WA, Australia
  5. 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
  6. Occupation and the Environment, School of Public Health, Curtin University, Perth, WA, Australia
  7. St John of God Hospital, Subiaco, Perth, WA, Australia
  8. Murdoch Children's Research Institute, Melbourne, VIC, Australia
  9. Department of Paediatrics, The University of Melbourne, Melbourne, VIC, Australia

Bacterial respiratory infections are extremely challenging to treat since bacteria typically develop resistance and occur in both planktonic and biofilm states. Due to the lack of development of new antimicrobials, alternative treatments including bacteriophage (phage) therapy are currently being explored. Since the use of singular therapy does not successfully reduce burden of infection in all cases, in this study we investigated the potential of combination treatment using phages, antibiotics and an adjuvant, CaEDTA, on overall treatment efficacy.

Overnight cultures of PAO1 were initially adjusted to 2x105 CFU/mL and 100 µL added to each well of a 96-well culture plate. PAO1 was then treated with 100 µL of tobramycin (0.0625-4 µg/mL), CaEDTA (50mM) or phage (E79, ϕRN1, ϕRN2, ϕRN3; MOI 0.1, 105 PFU/mL) singularly or in combination (tobramycin+CaEDTA, phage+CaEDTA and tobramycin+phage+CaEDTA) to assess treatment efficacy. Bacterial suspensions were incubated for 20 hours and overall bacterial growth (OD600nm) and enumeration of viable bacterial load (CFU/mL) were measured. When treated with agents singularly, only tobramycin was effective against PAO1 (minimum inhibitory concentration (MIC) 2 µg/mL). In contrast, when PAO1 was treated with tobramycin (1µg/ml)+CaEDTA, there was a 67.6% reduction in bacterial growth when compared to untreated controls. Combinations of tobramycin, phages and CaEDTA were also assessed using the fractional inhibitory concentration index (FICi).  Results showed that at 1 µg/mL of tobramycin and 105 PFU/mL of ϕRN1, ϕRN2, ϕRN3 or ϕE79, FICi were all 0.0 representing a synergistic interaction. Furthermore, combination treatment of tobramycin, phage and adjuvant successfully eradicated infection with no viable PAO1 recovered (Kruskal-Wallis test, p<0.05).  In conclusion, this study demonstrated synergistic effects of combination treatments and that the successful eradication of pseudomonas infections via this approach may delay onset of resistance development. Furthermore, the lower concentrations of antibiotics used that prove effectively will have a positive impact on the future stewardship of these compounds.