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Potentials and challenges of using phage therapy tocontrol bacterial infections in aquaculture
Mathias MiddelboeMarine Biological SectionUniversity of CopenhagenDenmark
Marine phages on a global scale
Abundance: ~1031 viruses
Biomass: ~200 million ton C
Total weight = 75 million blue whalesTotal lenght = 25 million light years
Sted og dato (Indsæt --> Diasnummer)Dias 5
Viruses influence global scale marine processes
Mortality of bacteriaand phytoplankton
Microbial population dynamicsand diversity
Turnover of carbon and nutrients Horisontal gene transfer
…and local scale pathogen mortality, evolution and ecology
Total aquacultureproduction in 2008
>80 million tonnes(value of USD 100 billion)
- China: 70 % of quantity- 50 % of value
Annual growth ratesince 1970:
- Aquaculture 8.8 %- Capture fisheries 1.2 %- Farmed meat prod. 2.8 %
Bacteriophage therapy in aquaculture
• The problemAquaculture and fish diseases- Rainbow Trout Fry Syndrome- Flavobacterium psychrophilum
• The solution?Phage therapy in aquaculture- potentials and limitations
• Conclusions and outlook
Disease ecology in aquaculture
Fish
pathogen Environment
Vibrio anguillarumAeromonas salmonicidaeYersinia ruckeriiFlavobacterium psychrophilumVirusesParasites
pH, temperature, oxygenNutrientsFish densityPhages
Flavobacterium psychrophilum
•The causative agent of rainbow troutfry syndrome
•Can be isolated from spleen, kidney,brain, eyes, ulcers, mucus and gills ofinfected fish
Photo: Lone Madsen
F. psychrophilum colonies on TYES agarIsolates from the kidney, spleen and brainof infected rainbow trout fry.
Rainbow trout fry syndrome
• Mortalities of 50-90% in rainbow trout fry• Mortalities up to 20-50% in larger fish
Rainbow trout fry syndrome
•Responsible for the loss of >30% ofall Danish rainbow trout fry
•Increasing antibiotic resistanceproblems
•No commercially available vaccines
Strong need for alternative treatments of F. psychrophilum infections
Phage-therapy in aquaculture
•Potential advantages:
• Self-replicating ”drug”• Highly specific• Easy to modify in response to changes inpatogens (e.g. resistance)
• Self-regulating• Naturally present in the environment
Phage-therapy in aquaculture•Challenges:
• Isolation of broad host range lytic phages (a phage cocktail)• Delivery of phages to target organs• Overcoming host diversity• Survival and reproduction of phages in the fish• Overcome development of resistance to phages in host bacteria• Understanding disease ecology of the pathogen and phage• Understanding fate of phages in environment
Challenge #1: Isolation of phages for target pathogens
•Enrichment cultures
•Lytic bacteriophages are visible as plaques
•Bacteriophages are extracted and purified
• Phages found in 48 % of all samplesand 53 % of all fish farms
•Phages found both in infected and non-infected farms
Plaques of various phageson a lawn of F. psychrophilum
Phage
Strain
FpV
-1
FpV
-2
FpV
-3
FpV
-4
FpV
-5
FpV
-6
FpV
-7
FpV
-8
FpV
-9
FpV
-10
FpV
-11
FpV
-12
FpV
-13
FpV
-14
FpV
-15
FpV
-16
FpV
-17
FpV
-18
FpV
-19
FpV
-20
FpV
-21
FpV
-22
NCMB 1947 T900406-1/3950106-1/1040615-1/2D040615-1/3A030522-1/1030522-1/2030522-1/3020612-2/1020612-2/2020612-4/1020612-4/2020529-2/1020529-2/2010418-2/1010418-2/2010418-2/3990512-1/1B990512-1/2A960625-3/1951004-1/1A951004-1/8A951004-1/11A951004-1/14C001026-1/35C001026-1/38B000720-1/59B000720-1/60C
Clear zone or plaques at spot: Turbid zone or plaques at spot: No growth inhibition at spot
Broad host ranges
Large diversity inhost ranges
Unique infection patternsamong phages
Unique susceptibilitypattern among hosts
Together isolated phageskill 23 of 27 examinedhost strains
Challenge #2: broad host range phages
T im e (h )
0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0
CF
U a
nd
PF
U m
l-1
1 0 1
1 0 2
1 0 3
1 0 4
1 0 5
1 0 6
1 0 7
1 0 8
1 0 9
1 0 1 0
T o ta l C F US T
S M
T im e (h )
0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0
CF
U a
nd
PF
U m
l-1
1 0 1
1 0 2
1 0 3
1 0 4
1 0 5
1 0 6
1 0 7
1 0 8
1 0 9
1 0 1 0
S T - re s is ta n t
S M - re s is ta n t
S T + S M - re s is ta n t
A
B
1 2 3 4 5 6
Challenge #3: host resistance against phages
Time of incubation with phages
Challenge #3: host resistance against phages
Strain MM#3 10dom 10:1 10:2 16dom 17dom 17:1 18dom 23dom 34dom 34:1Time of isolation 0 h 73 h 73 h 73 h 150 h 168 h 168 h 175 h 240 h 504 h 504 h
WaterDextrinGlycogenD-CellobioseD-FructoseL-FucoseD-GalactoseGentiobioseα-D-Glucosea-D-LactoseLactuloseMaltoseD-ManitolD-MannoseD-Melibioseß-Methyl-D-GluccosideD-RaffinoseSucroseD-TrehaloseTuranosePyruvic Acid Methyl EsterAcetic acidD-Gluconic AcidD-Glucoronic Acidα-Ketobutyric Acidα-Ketovaleric acidD,L-Lactic AcidPropionic AcidL-AlaninamideL-AlanineL-Alanyl-GlycineL-AsparagineL-Aspartic AcidL-Glutamic AcidGlycyl-L-Aspartic AcidGlycyl-L-Glutamic AcidL-HistidineL-LeucineL-OrnithinineL-ProlineL-SerineL-ThreoninUridine
Challenge #3: host resistance against phages
0,0
0,5
1,0
1,5
2,0
2,5
0 2 4 6 8 10Time (Days)
Optic
al de
nsity
control (-phages) + 1 phage+ 5 phages + 10 phages
Challenge #3: host resistance against phages
Challenge #3: host resistance against phages
Challenge #4: development of host range mutants
Clear plaques
Turbid plaques
No lysis
Time (hours)0 50 100 150 200 250
Plaq
ue fo
rmin
g un
its m
g ki
dney
-1
10-1
100
101
102
103
104
105
FpV-9 controlFpV-9 + F. psychrophilumFpV-9 + F. psychrophilum (dead fish)FpV-9 + F. psychrophilum (dead fish)
Challenge #5: Phage delivery and survival in fish organs
Time (h)0 20 40 60 80 100
Pla
que
form
ing
units
ml-1
100
101
102
103
104
105
106
107
108
Phage additionBloodAbdomenSpleenKidneyBrain
Challenge #5: Phage delivery and survival in fish organs
0 20 40 60 80 100
Pla
que
form
ing
units
ml-1
100
101
102
103
104
105
106
107
108
pH=4.5pH=7.5light exposure
Time (days)0 20 40 60 80 100
Pla
que
form
ing
units
ml-1
100
101
102
103
104
105
106
107
108
pH=3pH=4.5pH=6pH=7.5light exposure
FpV-9
FpV-4
Challenge #5: Phage delivery and survival in fish organs
Time (days)0 10 20 30 40 50 60
Pla
que
form
ing
units
ml-1
104
105
106
107
Challenge #5: Phage delivery and survival in fish organs
Time (days)0 10 20 30 40 50 60
Pla
que
form
ing
units
ml-1
104
105
106
107
-80 oC in glycerol20 oC in SM buffer
Challenge #5: Phage delivery and survival in fish organs
Time (days)0 10 20 30 40 50 60
Pla
que
form
ing
units
ml-1
104
105
106
107
Untreated dam waterAutoclaved dam waterFiltered dam water
Challenge #6: Fate of phages in the natural environment
Phage-therapy in aquaculture•Against Lactococcus gravieaeinfections of yellowtail (Nakai et al,1999)
•Against Pseudomonasplecoglossicida infections of Ayu (Parket al, 2000)
•Against Vibrio harveyi infections ofshrimp larvae (Vinod et al, 2007)
Phage-therapy of Lactococcus garviaeinfected yellowtail (Nakai et al, 1999)
+ phage
+ phage,+ pathogen
+ pathogen
Phage-therapy of Flavobacterium columnare infectingCatfish (Prasad et al 2011)
Phage-therapy in aquacultureConclusions
• Detailed characterization of F. psychrophilum phages• Strong lytic potential against F. psychrophilum• Large phage diversity• Broad host range phages (cover a wide spectrum of pathogenic hosts)• Long term storage possible• Can be applied via feeding pellets• Phages reach target organs• Fast decay in the absence of hosts in their natural environment• Phage-resistant strains can be overcome by applying a cocktail of phages• Lytic phage mutants can be selected for in lab experiments
•But…
Still has to be proven successful in controlling the pathogen in infected fish
The future
• Large scale infection experiments• Efficiency of phage infections on surfaces vs liquid cultures• Optimize delivery of phages to target organs• Optimize survival and reproduction of phages in infected fish• Examination of the importance of phage-resistance in infected fish• Disease ecology studies
Acknowledgements:
Partners:Dr. Inger Dalsgaard and Dr. Lone Madsen
Students:Rói Christiansen, Sif Bertelsen, Anne Stenholm, Sachia Jo Traving,Panos Kalatzis
Private partners:BioMar A/SAquasearch FarmChr. Hansen A/S
Funding:The Directorate for Food, Fisheries and AgricultureThe National Science FoundationThe Carlsberg Foundation
Emiliania huxleyi
E. Hux bloom in the North Atlantic
Acknowledgements:
The Directorate for Food, Fisheries and AgricultureThe National Science Foundation
Clustering of phages according to host range and genome size
Time (h)0 1 2 3 4 5 6 7 8
PFU
ml-1
103
104
105
106
107
FpV-19FpV-2
One-step growth experiments
Burst size: 7-160 phages infection-1
Latent period: 4-4.5 h
Sted og dato (Indsæt --> Diasnummer)Dias 45
Bacteriophage-host interactions and the potential or phage therapy tocontrol bacterial infections in aquaculture
Mathias Middelboe and Sif K. BertelsenMarine Biological LaboratoryUniversity of Copenhagen
Inger Dalsgaard, Lone Madsen & AnneStenholmFish Pathology LaboratoryTechnical University of Denmark
MM#3 10dom 10:1 10:2 16dom 17dom 17:1 18dom 23dom 34dom 34:1
f48:2f48:3f39:2f40:2f47:1f38:1f38:2f40:1f46:4f48:1f50:1f73:1f3:2
f3ST:1f3ST:2f12:2f14:1f18:4f19:4fSMf3:1fST
f13:1f19:2
1 1 2 2 2 2 2 2 2 3 4
Phage susceptibility group #
Bacterial strain
Ph
ag
e
The isolated bacteriophages• 18 different bacteriophage
stocks isolated from 27water samples
• Together they can control23 of 27 examined F.psychrophilum strains
• 107 fold variation in lyticefficiency against differenthosts
Annual growth rate since 1970:
Aquaculture 8.8 %Capture fisheries 1.2 %Farmed meat production 2.8 %
FpV
-5
FpV
-7
FpV
-9
FpV
-10
ClaI
FpV
-5
FpV
-9
FpV
-10
EcoRI
FpV
-7
FpV
-5
FpV
-9
FpV
-10
Uncut
FpV
-7
1.0 kb
0.5 kb
48.5 kb
RFLP of phage genomes with identical genome size
Podoviridae
Siphoviridae
Myoviridae
Flavobacteriumpsychrophilum•Fish pathogen•Gram-negative rods
•Growth temperature (5 to 20 °C,optimal at 15 °C)
•Growth at max. 0.5-1.0% NaCl
Foto: Lone Madsen
Phase-contrast picture of F. psychrophilumcells in TYES media after 72 h of growthbar: 20 μm
Phage genome sizes•Determined for 25 bacteriophage stocks
•Ranging from 10 to 150 kilobases
•Fall into 3 groups 8-12kb, 50 kb and 100 kb
E-1
/7L-
4/4
K-2
/4K
-1/4
C-3
/3,Ø
1.0
C-3
/3,Ø
0.5
C-3
/3,t
urbi
d
48.5 kb
97 kb
145 kb
Pulsed field gel electrophoresis ofbacteriophage stocks
B
D
A
D EC
Riemann & Middelboe 2002
Viruses and biogeochemical cycling
Host strainPhage
950106-1/1
010418-2/3
900406-1/3
960625-3/1 951004-1/11a 951004
-1/14c
FpV-4 7.8 x 108 ±1.2 x 108
2.7 x 109 ±4.2 x 108
3.5 x 102 ±1.2 x 102
1.5 x 109 ±3.3 x 108
1.5 x 108±5.3 x 107 nd
FpV-7 3.2 x 104 ±7.8 x 103
4.1 x 104 ±0.5 x 103 nd nd nd nd
FpV-9 >1012 6.9 x 109±3.5 x 108
1.5 x 105 ±3.3 x 104 nd 2.8 x 1010±
4.3 x 1092.8 x 1010 ±
2.3 x 109
FpV-10 3.7 x 109 ±2.5 x 108
5.4 x 109 ±5.6 x 108 nd nd nd nd
FpV-14 6.4 x 103 ±1.5 x 103
6.4 x 103 ±0.4 x 103 nd 6.5 x 103 ±
0.7 x 1035.7 x 103 ±2.9 x 103 nd
Efficiency of phage infection: Number of infecting units in a givenphage plate lysate exposed to selected host strains.