Local and global scale host range patterns and distribution of bacteriophages infecting the fish pathogen Flavobacterium psychrophilum

Transcription

1 Local and global scale host range patterns and distribution of bacteriophages infecting the fish pathogen Flavobacterium psychrophilum Mathias Middelboe 1, Daniel Castillo 2, Rói H Cristiansen 1,3, Lone Madsen 3, Inger Dalsgaard 3, Romilio Espejo 2 1 Biological Institute, University of Copenhagen, Denmark 2 Instituto de Nutrición y Technologia de los Alimentos, Universidad de Chile, Chile 3 National Veterinary Institute, Technical University of Denmark, Denmark

2 Flavobacterium psychrophilum The causative agent of rainbow trout fry syndrome world wide Mortalities of 50-90% in rainbow trout fry Increasing antibiotic resistance problems No commercially available vaccines F. psychrophilum colonies on TYES agar Strong need for alternative treatments of F. psychrophilum infections

3 Objectives Study the interactions, diversity and temporal dynamics of the Flavobacterium psychrophilum phage system across global and local scales Study the development and dynamics of phage resistance in the host and the costs associated with resistance. Study the dynamics and distribution of a specific prophage in F. psychrophilum communities.

4 DGREA analysis of F. psychrophilum Marine Biological Section Danish Strains Chilean strains

5 Clustering of F. psychrophilum phage isolates based on their host ranges of infectivity against the 89 bacterial isolates. Danish phages Chilean phages

6 Phage 6H genome organization Integrase Tail protein Hypothetical protein 1 Hypothetical protein 2

7 Presence of four ORFs from genome 6H in F. psychrophilum strains isolated in Chile and Denmark 21 strains isolated in Chile 28 strains isolated in Denmark

8 Phage influence on host abundance and isolation of phage-resistant hosts Marine Biological Section A Low phage concentration. MOI:~0.01 Bacteria ml Control bacteria Phage cocktail Infected bacteria Time (h) B Control bacteria Phage cocktail Infected bacteria Phages ml -1 Medium phage concentration: MOI:~ C Time (h) Control bacteria Phage cocktail Infected bacteria Time (h) High phage concentration: MOI:~5

9 Phage influence on host abundance and isolation of phage-resistant hosts Marine Biological Section A FpV-4 infection FpV-21 infection Phage cocktail infection Low phage concentration. MOI:~ % Bacteria B Time (h) post phage infection FpV-4 infection FpV-21 infection Phage cocktail infection Medium phage concentration: MOI:~ Time (h) post phage infection 80 C FpV-4 infection FpV-21 infection Phage cocktail infection High phage concentration: MOI:~ Time (h) post phage infection

10 Phage influence on host abundance and FpV-4 isolation of phage-resistant hosts Sample time post phage infection (h) Early incubation (21-26) Phage concentration Low Medium High Low Mid incubation (37-47) Mediu m Late incubation (48-91) High Low Medium High Marine Biological Section V2-5 V2-6 V3- V3- V4-1 V4-2 V4-3 V2-11 V2-12 V3-10 V3-11 V4-13 V4-14 V4-15 V2- V2-18 V2-19 V3-16 V3-17 V4-25 V4-26 V4-27 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

11 Phage influence on host abundance and isolation of phage-resistant hosts - Multiple types of resistant strains develop within a short time scale after phage exposure - The types and degree of resistance is influenced by the phage-host ratio

12 Presence of four ORFs from genome 6H in F. psychrophilum resistant strains V1-17 v1-20 v2-13 v2-17 v2-20 v2-23 v3-4 v3-5 v3-15 v3-16 v3-21 v3-24 v4-14 v4-24 v4-28 v4-33 All Danish phage-resistant strains had lost the 6H prophage

13 Complete host range analysis including all Chilean and Danish F. psychrophilum strains and phages A B C Chilean phages Danish phages

14 Complete host range analysis including all Chilean and Danish F. psychrophilum strains and phages Chilean strains A B Danish Strains C Danish and Chilean Strains

15 Resistance against some phages provides increased sensitivity to others A B C

16 Resistance against some phages provides increased sensitivity to others E.g.: FpV-15 log [Free phage] Strain /1 PFU ml E E E E E Time (min) 1.00E Time (h) Strain Burst size latency time (hr) Sensitivity /1: WT 9 ± 1 5 3x10 6

17 Resistance against some phages provides increased sensitivity to others E.g.: FpV E E+07 6 log [Free phage] Strain /1 Strain v3-5 Strain v3-16 Strain /1 cured PFU ml E E E E Time (min) Time (h) Strain Burst size latency time (hr) Sensitivity /1: WT 9 ± 1 5 3x /1c: Cured 195 ± 10 3 ND v3-5: Resistant 125 ± 20 3 >10 10 v3-16: Resistant 183 ± 9 3 5x10 9

18 Influence of resistance on host growth rate Strains Growth rate (h -1 ) (mean ± SD) Significance* / ± 0.08 V ± 0.07 ns V ± 0.03 ns V ± 0.01 ns V ± 0.08 ns V ± 0.03 ns; P= V ± 0.10 ns V ± 0.00 P=0.006 V ± 0.02 P=0.007 V ± 0.03 ns V ± 0.05 ns V ± 0.08 ns V ± 0.06 ns

19 Influence of resistance on host nutrient uptake Strains* 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 /1 V2-13 V2-17 V2-20 V2-23 V1-20 V3-15 V3-21 V3-24 V4-24 V1-17 V4-28 V3-4 V3-5 V4-33 Marine Biological Section

20 Multiple costs of resistance: Enhanced infection by other phages Reduced growth rates Loss of ability to utilize specific substrates Metabolic and enhanced infection costs of resistance may be key drivers of host strain diversity and the co-existence of phages and hosts on local and global scales (Avrani et al 2012)

21 Stability of acquired phage resistance Laigaard et al unpublished

22 Development of host range mutants Clear plaques Turbid plaques No lysis Traving et al unpublished

23 Conclusions Positive cross-reactions between Danish hosts and Chilean phages and vice versa. Presence of a specific (phage 6H) prophage in both Danish and Chilean host strains. Development of phage-resistance were associated with metabolic costs and/or increased susceptibility to other phages. Resistance patterns and phage life cycle characterization of several phages were linked to the presence of 6H prophage in the host. Phylogenetically, the Chilean and Danish strains are similar, but separated into two distinct clusters based on direct genome restriction fragment analysis (DGREA).

24 The study suggests. a global distribution of phenotypic traits related with phagesusceptibility, and a global distribution of a specific prophage. fast and highly dynamic changes in phage susceptibility in the global F. psychrophilum community in response to phage exposure. despite the identification of local communities of phages and hosts, some key properties determining phage infection patterns seem to be universally distributed - independent of environmental conditions, geographic separation and other selection pressures.

25 Acknowledgements: Sachia Jo Traving and Helle Kjær Laigaard Jeanett Hansen AQUAPHAGE (EU Marie Curie Actions, International Research Staff Exchange Scheme, IRSES-2010) Directorate for Food Fisheries and Agri Business Danish Council for Independent Research