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. 2008 Jul;74(13):4070-8.
doi: 10.1128/AEM.00428-08. Epub 2008 May 9.

Isolation and characterization of bacteriophages infecting the fish pathogen Flavobacterium psychrophilum

Anne Rønnest Stenholm  1 , Inger Dalsgaard, Mathias Middelboe
Affiliations

Isolation and characterization of bacteriophages infecting the fish pathogen Flavobacterium psychrophilum

Anne Rønnest Stenholm et al. Appl Environ Microbiol. 2008 Jul.

Abstract

Flavobacterium psychrophilum is a serious pathogen in trout aquaculture, responsible for the diseases rainbow trout fry syndrome (RTFS) and cold water disease (CWD). Bacteriophage control of F. psychrophilum may constitute a realistic approach in the treatment of these diseases; however, a detailed understanding of the phage-host interactions is needed to evaluate the potential of F. psychrophilum bacteriophages for that purpose. Twenty-two F. psychrophilum phages from Danish rainbow trout farms were isolated and characterized. The phage genome sizes differed considerably and fell into three major size classes (8.5 to 12 kb, 48 kb, and 90 kb). The phage host ranges comprised from 5 to 23 of the 28 tested F. psychrophilum strains, and 18 of the phage isolates showed unique host ranges. Each bacterial strain had a unique pattern of susceptibility to the 22 phages, and individual strains also showed large variations (up to 10(7)-fold differences) in susceptibility to specific phages. Phage burst size (7 to 162 phages infected cell(-1)) and latency period (4 to 6 h) also showed pronounced differences both between phages and, for a specific phage, between host strains. In general, the characterization documented the presence of diverse F. psychrophilum phage communities in Danish trout farms, with highly variable patterns of infectivity. The discovery and characterization of broad-host-range phages with strong lytic potential against numerous pathogenic F. psychrophilum host strains thus provided the foundation for future exploration of the potential of phages in the treatment of RTFS and CWD.

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Figures

FIG. 1.
FIG. 1.
Host ranges of the isolated bacteriophages against selected F. psychrophilum strains.
FIG. 2.
FIG. 2.
Tree based on the unweighted-pair group method using average linkages for the F. psychrophilum phage isolates (“#” is followed by FpV number) based on their host ranges of infectivity against the 28 bacterial isolates. The data in Fig. 1 were scored and converted to pairwise distances by using the Dice similarity coefficient. Groupings of phages according to genome size and morphology are inserted to facilitate comparison.
FIG. 3.
FIG. 3.
Restriction digest analysis of selected bacteriophages with 48-kb genomes after treatment with enzymes ClaI and EcoRI. Lanes to the right are uncut phage DNA. The top band in the FpV-7 digest probably represents a fraction of undigested phage DNA.
FIG. 4.
FIG. 4.
TEM pictures of selected bacteriophages. (A) FpV-2; (B) FpV-4; (C) FpV-7; (D) FpV-9; (E) FpV-10; (F) FpV-14; (G) FpV-19. Scale bar, 50 nm.
FIG. 5.
FIG. 5.
Development in F. psychrophilum densities measured as changes in OD in the cultures (OD525) (A to C) and abundances of phages FpV-9 and FpV-10 measured as numbers of PFU (D to F) in enrichment cultures with various initial bacterial densities.
FIG. 6.
FIG. 6.
Temporal development in PFU during one-step growth experiments with F. psychrophilum phages as exemplified by FpV-2 and FpV-19.
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