As part of my undergraduate degree in Microbiology, I was required to perform a laboratory research project as part of my dissertation. Here, I was based in the laboratory of Prof. Eshwar Mahenthiralingam at Cardiff University School of Biosciences undertaking a project entitled "Bacteriophage therapy in Galleria mellonella challenged with Burkholderia dolosa"
Discovery of an Accretion Streamer and a Slow Wide-angle Outflow around FUOri...
Dissertation Presentation - Ashley Otter
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
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
Editor's Notes
The aims and objectives of this project were
can g4P3 infect burkholderia dolosa
Can B. dolosa kill G. Mellonella?
Choice of Burkholderia to use
Ld50 of BCC 1359
Then finally establish if bacteriophage therapy is possible using galleria mellonella as a model for testing whether phage G4P3 is able to prolong the life of those infected with a lethal dose of B. dolosa
Phage G4P3 found in supernatant of B. vietnamiensis g4
Proliferated as described by Adams 1959, using double agar overlay method and isolating individual plaques and repeating
Using B. dolosa isolates, find out which isolates are susceptible to phage G4P3 using a simple drop test onto an overlay of each isolate and using a bioscreen C plate reader to determine the effect if any, g4p3 has on the growth rate of each isolate
Injected into the haemocoel between the last set of pro legs
Injections of between 10 and 20ul were used – bacteria or phage resuspended in MgS04 with 10 mg Ampicillin to prevent contamination or injection of bacteria on the surface of larvae
3 groups of 10 larvae – replicates
Control injections – life control – MgSO4, death control – Psuedomonas aeruginosa – 10 ul of overnight culture
1:1 1x106 1x10^6 - same concentration offered best percentage of survival
100:1 1x108 1x106
10:1 1x107 1x106
0.1:1 1x105 1x106
0.01:1 1x104 1x106
0.001:1 1x103 1x106
Small sample size
Unable to perform multivariate tests
. Statistical comparison using a non-parametric data obtained from the phage therapy experiment (Kruskal-Wallis test, multivariate states) and post-hoc tests (pairwise comparison) yielded no results due to the small sample size. Further experiments will need to be conducted with more replicates to determine if results are stastically significant.