Report on activities in WP5 and WP6 from University of...

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






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







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


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


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




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





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


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


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

Report on activities in WP5 and WP6 from University of...

Documents

Transcript of Report on activities in WP5 and WP6 from University of...