This document discusses bacteriophage therapy using the g4p3φ bacteriophage to combat Burkholderia dolosa infections in Galleria mellonella larvae, a model organism. It outlines methodologies for testing phage efficacy, including phage isolation, lethal dose determination, and survival assessments post-infection. The results indicate promising survival rates with optimal multiplicity of infection, while also highlighting challenges such as endotoxin effects and the need for further research on host ranges and persistence.

1. Bacteriophage therapy in Galleria mellonella (Wax
moth larvae) challenged with Burkholderia dolosa.
Ashley David Otter
Supervisor: Prof. Eshwar Mahenthiralingam

2. • Burkholderia cepacia complex (Bcc)
– Serious infections in cystic fibrosis sufferers
– Resistant to many antimicrobials
– Burkholderia dolosa
• Bacteriophages: bacterial viruses
– Lytic and lysogenic
– Phage therapy
– G4P3Φ, prophage of B. vietnamiensis str. G4
• Galleria mellonella (Wax moth larvae)
– Model organism for pathogenicity testing
Background

3. Aims and objectives
Can G4P3 infect B. dolosa?1
Selection of B. dolosa isolate that
gives most consistent plaque/kill2
Determination of LD50 of chosen isolate4
Six B. dolosa isolates
Can B. dolosa isolate kill G. mellonella?3
Bacteriophage therapy in
G. mellonella infected
with B. dolosa
5

4. • Phage proliferation and enumeration
– G4P3Φ spontaneously released
– Double agar overlay method: Adams (1959)
• Determine what B. dolosa isolates are
susceptible to G4P3Φ
– Drop test: solid media
– Bioscreen C: growth kinetics in liquid
Methods

5. • G. mellonella injection and maintenance
– Seed and Dennis (2009), Wand et al. (2011) and Harding et al. (2013)
Methods

6. • LD₅₀
– Lethal dose required to kill 50% of those injected
• Phage therapy in G. mellonella challenged with B. dolosa
– Lethal dose
– Prophage G4P3Φ
Methods
Alive Systemic Dead

7. Aim 1 and 2: G4P3 spot test onto six B. dolosa isolates:
Results
Aim 3 and 4: LD₅₀ B. dolosa BCC 1359 – 1.47x102 CFU/ml

8. Aim 2: Growth kinetics using Bioscreen C – B. dolosa BCC
1359
Results

9. Results
Aim 5:
Phage therapy in
G. mellonella
challenged with a
lethal dose of B.
dolosa.
Best multiplicity of
infection (MOI):
1:1 > 10:1 > 0.1:1

10. Aim 5: Survival graph
Results
MOI

11. • Potential for phage therapy
– Best MOI – 1:1
– Combination with antibiotics (Lu and Collins 2009)
• Problems
– Effect of endotoxin (Merabishvili et al. 2009)
– Difficulty and lysogenic phage
• Future work
– Endotoxin removal kit
– Host ranges
– Persistence
– Cloned lysin (Fischetti 2008)
Conclusions

12. Fischetti, V. A. (2008). Bacteriophage lysins as effective antibacterials. Current Opinion in Microbiology 11:393-400.
Harding, C. R., Schroeder, G. N., Collins, J. W. and Frankel, G. (2013). Use of Galleria mellonella as a Model Organism
to Study Legionella pneumophila Infection. Journal of Visualized Experiments(JoVE).
Lu, T. K. and Collins, J. J. (2009). Engineered bacteriophage targeting gene networks as adjuvants for antibiotic
therapy. Proceedings of the National Academy of Sciences 106:4629-4634.
Merabishvili, M., Pirnay, J.-P., Verbeken, G., Chanishvili, N., Tediashvili, M., Lashkhi, N., Glonti, T. et al. (2009). Quality-
controlled small-scale production of a well-defined bacteriophage cocktail for use in human clinical trials. PloS One
4:e4944.
Seed, K. D. and Dennis, J. J. (2009). Experimental bacteriophage therapy increases survival of Galleria mellonella
larvae infected with clinically relevant strains of the Burkholderia cepacia complex. Antimicrobial Agents and
Chemotherapy 53:2205-2208.
Wand, M. E., Müller, C. M., Titball, R. W. and Michell, S. L. (2011). Macrophage and Galleria mellonella infection
models reflect the virulence of naturally occurring isolates of B. pseudomallei, B. thailandensis and B. oklahomensis.
BMC Microbiology 11:11.
References