report on activities in wp5 and wp6 from university of...
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Report on activities in WP5 and WP6 from University of Copenhagen
Mathias MiddelboeMarine Biological SectionUniversity of Copenhagen
Secondments from UCPH
Roi H. Cristiansen (visiting INTA, Chile, 2013)
Mathias Middelboe (visiting INTA, Chile 2013 and 2015 and EIMBV, Georgia 2014)
Nanna Rørbo (Visiting EIMBV, Gerogia 2014)
Secondments to UCPH
Gastón Higuera (Visting from University of Santiago de Chile, 2014)
Natela Toklikishvili (Visiting from Georgia, 2013)
Elene Didebulidze (Visting PhD from Georgia, 2013)
Tamara Qoqashvili (Visting from Georgia, 2013)
Daniel Castillo (Visiting from INTA, Chile 2011 and 2013)- Leading to a 2-year post doc employment at UCPH
Ekaterina Jaiani (Planned visit from Georgia, February 2015)
Nino Janelidze (Planned visit from Georgia, February 2015)
Publications (peer reviewed journals/theses):
1. Castillo, D, Higuera, G., Villa, M, Middelboe, M. Dalsgaard I, Madsen, L, Espejo, R. (2012). Diversity of Flavobacteriumpsychrophilum and the potential use of its phages for protection against bacterial cold water disease in salmonids. J. Fish Dis. 35:193-201
2. Madsen, L., Dalsgaard, I. and Middelboe, M. (2013) Dispersal and survival of Flavobacterium psychrophilum phages in vivo in rainbow trout and in vitro under laboratory conditions: Implications for their use in phage therapy. Appl. Environ. Microbiol. 79:4853-4861.
3. Castillo, D., Espejo, R. and Middelboe, M. (2014) Characterization and genomic structure of the temperate bacteriophage 6H, and its distribution as prophage in Flavobacterium psychrophilum strains. FEMS Microbiol. Lett. 351:51-58.
4. Martiny, JH, Riemann, L., Marston, M. and Middelboe, M.(2014) Antagonistic coevolution of planktonic viruses and their hosts. Ann. Rev. Mar. Biol. 6:393-414.
5. Castillo, D., Christiansen, RH., Espejo, R and Middelboe, M. (2014) Diversity and geographical distribution of Flavobacterium psychrophilum isolates and their phages: Patterns of susceptibility to phage infection and phage host range. Microb. Ecol. (DOI 10.1007/s00248-014-0375-8).
6. Christiansen, RH (2014) "Phage-host interactions in Flavobacterium psychrophilum and the potential for phage therapy in aquaculture". PhD thesis, University of Copenhagen, 185 pp
7. Castillo, D., Christiansen, RH., Dalsgaard, I., Madsen, L., and Middelboe, M. (2014) Bacteriophage resistance mechanisms in the fish pathogen Flavobacterium psychrophilum: Linking genomic mutations to changes in bacterial virulence factors. Appl. Environm. Microbiol. (in press).
8. Christiansen, RH., Dalsgaard, I., Middelboe, M. Lauritsen, AH. and Madsen, L. (2014). Detection and quantification of Flavobacterium psychrophilum specific phages In vivo in rainbow trout upon different administration methods: Implications for disease control in aquaculture. Appl. Environm. Microbiol. 80:7683-7693.
9. Christiansen, Madsen, L. Dalsgaard, I., Castillo, D, Kalatzis, PG, and Middelboe, M. (2015): Effect of bacteriophages on the growth of Flavobacterium psychrophilum and development of phage-resistant strains. Submitted to Microb. Ecol.
10. Tan, D., Dahl, A. and Middelboe, M. (2015) Phage-host interactions in two strains of Vibrio anguillarum: The role of biofilm formation as a protection mechanism against phage infection. Submitted to Appl. Environm. Microbiol.
11. Tan, D., Svenningsen, SL, Middelboe, M. (2015) Quorum sensing determines the choice of anti-phage defense strategy in Vibrio anguillarum. Submitted to mBIO.
Publications in preparation:
12. Laigaard, HK ( in prep): Dynamics and stability of phage resistance mutations in Flavobacterium psychrophilum.
13. Castillo et al (in prep): Whole genome sequencing of Flavobacterium psychrophilum phages isolated from aquaculture farms in Denmark and Chile.
14. Castillo et al (in prep): Genetic diversity of Flavobacterium psychrophilum based on whole genome sequencing of isolates from Chile and Denmark.
15. Johnston et al. (in prep). Isolation and characterization of prophages from Flavobacterium psychrophilum and their distributon among host genomes
Presentations
1. Christiansen et al: Poster: Danish Marine Research Meeting, 2011,
2. Christiansen et al: Poster: Aquatic Virus Workshop, Holland, 2011,
3. Christiansen et al: Poster: Danish Microbiological Society møde, 2011.
4. Christiansen et al: Orals presentation: Danish Microbiological Soviety 2011
5. Middelboe: Oral presentation, AQUAPHAGE workshop, Crete, 2011
6. Middelboe et al: Oral presentation: ISME Meeting; Copenhagen, 2012
7. Christiansen et al: Oral presentation: KU, Section seminar, 2012
8. Laigaard et al: Oral presentation: KU, Section seminar, 2012
9. Laigaard et al: Oral presentation: Danish Marine Research Meeting, 2013
10. Christiansen et al: Oral presentation: Dafinet workshop, 2013
11. Christiansen et al: oral pres.: EAFP conference, Tampere Finland 2013
12. Middelboe et al: Oral presentation: AQUAPHAGE workshop, Kreta, 2013
13. Castillo et al: Oral pres: Copenhagen Microbiology Center, meeting, 2013
14. Castillo et al: Oral presentation: KU, Section seminar, 2013
15. Middelboe: Oral presentation: University of Aarhus, 2014
16. Tan et al: Poster presention: Bacteriophages 2015 meeting, London
Other students involved in the project (outside AQUAPHAGE)
1. Camille Johnston (MSc august 2014): Characterization and genetic compositon of prophages in Flavobacterium psychrophilum.
2. Lau Lyck Nielsen (B.Sc Febr. 2014): Review of the potential and challenges of phage therapy in aquaculture.
3. Andreas Ruth (B.Sc. Jan 2014): Phage-host interactions in model sea water systems
4. Amalie Dahl (M.Sc. Jan 2014): Phage-host interactions in bacterial bioifilms
5. Helle Kjær Laigaard (M.Sc. Sept 2013): Mechanisms of bacteriophage resistance in marine bacteria.
6. Caroline Amalie Brunbjerg Hey (B.Sc. June 2013): Implications of phage resistance for metabolicproperties in Flavobacterium psychrophilum
7. Camille Johnston (B.Sc. June 2012): Occurence and composition of prophages in Flavobacteriumpsychrophilum.
WP5: Evolution and distribution of broad-host range phagesspecific to Flavobacterium psychrophilum – from local to global scales.
Aim:
- To obtain a collection of F. psychrophilum phages from Danish and Chilean fish farms
- To examine the diversity, distribution sensitivity/infectivity of Flavobacterium psychrophilum and their phages across large spatial scales
- To determine the mechanisms of phage susceptibility and resistance in F. psychrophilum (and Vibrio angillarum)
√√ √
√ √√ x
x x
x x
x x
x x
x x x
Phage sensitive
Phage resistant
Selection phage resistant
Phage resistant replace and predominate population
Background: Classical view on phage resistance to phage infection
Spontaneous resistance mechanism: Phage-receptor mutation
The spontaneous resistance may occur through the loss or alteration of phage receptors
Phage-receptor
Phage
x
x
Random mutation (From Labrie et al 2010)
Sensitive strain950106-1/1
Sensitive strain 950106-1/1
Phage-resistant V1-20
Phage-resistant V2-20
Phage-resistant V3-5
Phage-resistant V4-24
Phage-resistant V4-28
Phage-resistant V4-33
Deletion and/or insertion mutation
Point mutation
Methodology: Whole genome sequencing
FpV4FpV21Cocktail 11 bacteriophages
√
x
x
x
x
x
x
V1-20
V2-20
V3-5
V4-24
V4-28
V4-33
Phage-resistant clone
GenomeSequenced
Genomecomparison
Castillo et al. 2015
Host-pathogen interactionsMembrane anchoring, Attachment and invasion of tissues
The evolution of LRRs is not well understood
Observed mutations associated with resistance: cell surface proteins (Leucine rich repeat)
Observed mutations associated with resistance: Gliding motility proteins
Leucine rich repeats
Mutations present in phage –resistant strains:
Model in F. johnsoniae
GldJGldMGldK
Castillo et al 2015
Mechanism of resistance: Mutations in O-antigens and peptidoglycan biosynthesis
Mutations in Leucine-rich, gliding motility , surface structural biosynthesis proteinsmay cause structural changes in the cell surface of phage-resistant isolates
UDP-N- acetyl-D-galactosamine dehydrogenase
UDP-N- acetyl tripeptide-D-alanyl-D-alanine ligase
Sensitive strain 950106-1/1
Phage-resistant V1-20
Phage-resistant V2-20
Phage-resistant V3-5
Phage-resistant V4-24
Phage-resistant V4-28
Phage-resistant V4-33
CRISPR activity in F. psychrophilum
These results suggest that F. psychrophilum cells do not have the ability to incorporate new spacers during phage infection
spacers
Castillo et al 2015
Mechanisms of resistance: Reduced adsorption rate
Castillo et al 2015
All strains resistant to phages had lost the 6H prophagefrom the genome
o Bacteriophage 6Ho Isolated in Chile like a free phageo Present in ~80 % of 90 environmental isolates from Chile, DK and USA-
Mechanisms of resistance: The role of prophages in F. psychrophilum genomes
Castillo et al 2013
Bacteriophages drive diversification in bacterial communities
….. is there a cost of resistance?
Strains*V2-17
V2-20
V2-23
V3-21
V3-24
V4-24
V4-28 V3-5V4-33
Water
Glycogen
Pyruvic Acid Methyl
Ester
α-Ketobutyric Acid
α-Ketovaleric Acid
L-Alaninamide
L-Alanine
L-Alanyl-Glycine
L-Asparagine
L-Aspartic-Acid
L-Glutamic-Acid
Glycyl-L-Aspartic-
Acid
Glycyl-L-Glutamic-
Acid
L-Lucine
L-Ornithine
L-Proline
L-Serine
L-Threonine
Urocanic Acid
Inosine
Uridine
Thymidine
Influence of resistance on host nutrient uptake
Christiansen et al, unpublished
Strains
950106-1/1 V1-20 V2-20 V3-5 V4-24 V4-28 V4-33
Ab
s 5
95
nm
0,0
0,2
0,4
0,6
0,8
1,0
1,2
Strains
950106-1/1 V1-20 V2-20 V3-5 V4-24 V4-28 V4-33
He
mo
lyti
c a
cti
vit
y
0,0
0,1
0,2
0,3
0,4
0,5
0,6
Strains
950106-1/1 V1-20 V2-20 V3-5 V4-24 V4-28 V4-33
To
tal
pro
teas
e a
cti
vit
y (
mm
)
0
5
10
15
20
25
30
Strains
950106-1/1 V1-20 V2-20 V3-5 V4-24 V4-28 V4-33
Ge
lati
nas
e a
cti
vit
y
0
5
10
15
20
25
Figure4. The F. psychrophilum phage-resistant isolates displayed reduced biofilm formation
and secretion of extracellular enzymes. A) Biofilm formation of the ancestral strain 950106-1/1
and the six phage-resistant isolates. B) Hemolytic activity. C) Total protease activity on skim milk
plates. D) Collagenase activity on gelatin plates. Results were plotted with standard deviation error
bars from triplicate experiments for each isolate; the p value was calculated using a paired Student's
T test. Asterisk indicate values significantly lower than control.
Biofilm formation Hemolytic activity
Proteinase activity Collagenase activity
Resistance against phages reduced virulence in F. psychrophilum
Castillo et al 2015
Tekst starter uden
dato og ”Enhedens
Stability of acquired phage resistance
Laigaard et al unpublished
Marine Biological Section
Multiple mechanisms of resistance:
Point mutations Loss of prophage Biofilm/aggregate formation CRISPR (aquired resistance) Downregulation of receptor sites (Quorum sensing) Restriction/modification systems
Conclusions
Marine Biological Section
Multiple costs of resistance:
Reduced virulence Reduced growth rates Loss of ability to utilize specific substrates Enhanced infection by other phages
Phages are key drivers of genetic and functionaldiversity in environmental bacterial communities
- Implications for bacterial evolution and virulence- Implications for cell aggregation and biofilm formation - Implications for phage-based control of pathogens
Conclusions
Marine Biological Section
WP6. Development of phage storage and delivery systems
Aim:
To examine
- methods for stable long term storage of phages as the basis for an efficient treatment of the disease.
- phage infectivity and decay at different conditions in vivo and during storage
- the efficiency and stability of the transfer of active phages to the target fish organs (e.g. brain, spleen, blood, and kidney) using different delivery methods
• Fish pellet has been coated
with bacteriophage FpV-9 (1x
109 CFU/ml) (BioMar A/S) and
stored at 4°C
• 25 ml phage stock used to coate 1 kg fish feed (2mm)
Time (days)
0 50 100 150 200 250
Pla
que f
orm
ing u
nit
s (P
FU
) g f
eed
-11e+5
1e+6
1e+7
1e+8
1e+9
FpV-9 4 C
Time (days)
0 50 100 150 200 250 300
Pla
que
form
ing u
nit
s (P
FU
) g f
eed
-1
1e+5
1e+6
1e+7
1e+8
1e+9
FpV-9 5 C
FpV-9 rt
FpV-9 -80 C
Phages coated on fish feed pellets
Christiansen et al 2014
Time (hours)
0 0,5 5 11 21 27 50 70 83 123 171 315
Pla
que
form
ing
unit
s m
g or
gan-1
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
Intestine, fish 1, tank 1
Intestine, fish 2, tank 1
Intestine, fish 1, tank 2
Intestine, fish 2, tank 2
Spleen in each fish
Time (hours)
0 0,5 5 11 21 27 50 70 83 123 171 315
Pla
que
form
ing
unit
s m
g or
gan-1
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
Intestine, fish 1, tank 3
Intestine, fish 2, tank 3
Intestine, fish 1, tank 4
Intestine, fish 2, tank 4
Spleen in each fish
Time (hours)
0 1 7 24 25 31 48 49 55 72 73 79 144 145 151 192 193 199 384
Pla
que
form
ing
unit
s m
g or
gan-1
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8Intestine, fish 1, tank B
Intestine, fish 2, tank B
Spleen in each fish
A
B
C
Quantification of phages in intestine and spleen
Immersion bath
Oral intubation
Phage coated feed
Christiansen et al 2014
WP4
Phage decay/propagation
Time (hours)
0 50 100 150 200
Pla
que
form
ing
unit
s m
g ti
ssue
-1
1e-1
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
Intestine
Spleen
Time (hours)
0 50 100 150 200
Pla
que
form
ing
unit
s m
g ti
ssue
-1
1e-1
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
Time (hours)
0 50 100 150 200
Pla
que
form
ing
unit
s m
g ti
ssue
-1
1e-1
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
1e+8
A
B
C
Phage
administratio
n method
Tissue/
phage
coated feed
Phage decay ratea Phage propagation ratea
h-1 r2 h-1 r2
Immersion
bath
Intestine 0.045 0.81
Spleen 0.013 0.18
Oral intubationIntestine 0.083 0.91
Spleen 0.060 0.81
Phage coated
feed
Intestine 0.0013 0.14
Spleen 0.0004 0.001
Phage coated
feed0.0043 0.21
Immersion bath
Oral intubation
Phage coated feed
Christiansen et al 2014
Dispersal and survival of F. psychrophilum phages in vivo
• Phages were rapidly spread to target organs by all three methods
• The oral delivery route via phage coating was a success
• High rate of phage decay in vivo
• Phage coated feed might be a promising application method. Continuous delivery of phages, early infection, prophylactic prevention
Conclusion
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