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.