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. 2011 Nov;77(21):7868–7872. doi: 10.1128/AEM.05574-11

Phage Specificity of the Freshwater Fish Pathogen Flavobacterium columnare▿

Elina Laanto 1, Lotta-Riina Sundberg 1, Jaana K H Bamford 1,*
PMCID: PMC3209183  PMID: 21890667

Abstract

Flavobacteria and their phages were isolated from Finnish freshwaters and fish farms. Emphasis was placed on finding phages infecting the fish pathogen Flavobacterium columnare for use as phage therapy agents. The host ranges of the flavobacterial phages varied, phages infecting F. columnare being more host specific than the other phages.

TEXT

Species of the genus Flavobacterium are widely distributed in nature, and they have been found in diverse habitats (3, 19, 30, 32, 36, 37, 38, 39). In general, flavobacteria are nonpathogenic, but some species are opportunistic pathogens (2). Columnaris, a disease caused by the fish pathogen Flavobacterium columnare, can cause up to 100% mortality among salmonid fingerlings (28). Antibiotic treatment must be applied to prevent mass mortalities at fish farms. Despite effective treatment, columnaris occurs repeatedly during the summer (18). Therefore, the number of antibiotic treatments and the amount of antibiotics used can be extremely high. Increased resistance of environmental bacteria to antibiotics in fish farms and their surroundings has been reported (10, 24, 26, 31), and antibiotic-resistant fish-pathogenic flavobacteria have also emerged (7). To avoid risks related to antibiotic use, enrichment of bacteriophages could be used as an ecological method of decreasing the number of F. columnare infections. To our knowledge, this is the first study on European F. columnare phages.

In this study, a total of 53 flavobacterial isolates were received during the warm-water period (May to August) in 2008 and 2009 from water samples that included both open freshwater environments (rivers and lakes not connected to fish farming) and three inland land-based fish farms rearing mainly salmonid fingerlings in Finland (Fig. 1; Table 1). One bacterium (B67) was isolated from diseased fish in 2007. Water samples were cultured on Shieh agar (25) and 1/5× Luria Bertani agar (22), and yellow- and orange-pigmented colonies were selected for analyses. The flavobacterial isolates were subjected to PCR with universal primers (UP-PCR) (for methods, see references 5, 6,, and 13), and the 16S rRNA genes of different groups were sequenced (Table 1). All F. columnare isolates fell into the same UP-PCR group, and thus they were further analyzed with ribosomal intergenic spacer analysis (RISA) (8, 29). According to RISA, the isolates were assigned to five groups (Table 1). Based on our data, the occurrence of F. columnare seems to be connected to the fish farming environment; this organism was not isolated from natural waters. However, it is likely that the initial source of F. columnare at the farms is nature, because there is evidence that it is also present outside fish farms (20, 21; H. Kunttu, L.-R. Sundberg, and E. T. Valtonen, unpublished data).

Fig. 1.

Fig. 1.

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(A) Sites in Finland where flavobacteria and their phages were isolated. Sampling sites are marked on the free map obtained from the National Land Survey of Finland (Maanmittauslaitos, 2005). The sites and their abbreviations and coordinates are listed on the right. (B) Electron micrographs of purified and negatively stained tailed Flavobacterium phages. (a) FCV-1; (b) FCL-2; (c) FJy-3; (d) FKo-2; (e) FKy-1. Bar, 50 nm.

Table 1.

Bacterial strains isolated and used in the study

Bacterial straina UP-PCR or RISA groupb EMBL accession no.c Sampling site Source or reference
B67 A Fishery L This study
B28 1 FR696328 Lake Konnevesi This study
B80 2 FR696329 Lake Jyväsjärvi This study
B105 3 FR696330 River Vantaanjoki This study
B110 4 FR696331 River Vantaanjoki This study
B114 5 FR696332 River Kevojoki This study
B121 6 FR696333 River Tsarsjoki This study
B127 7 FR696334 Lake Kevojärvi This study
B130 8 FR696335 Lake Kevojärvi This study
B167 9 FR696336 Lake Jyväsjärvi This study
B169 10 FR696337 Lake Jyväsjärvi This study
B171 11 FR696338 Lake Valtimojärvi This study
B174 12 FR696339 River Kymijoki This study
B176 13 FR696340 Lake Leppävesi This study
B178 14 FR696341 Lake Äkässaivo This study
B180 15 FR696342 River Äyskoski This study
B183 16 FR696343 Fishery L This study
B185 G FR696344 Fishery L This study
B187 17 FR696345 Fishery L This study
B207 18 FR696346 Lake Inari This study
B209 19 FR696347 Lake Inari This study
B214 20 FR696348 Fishery V This study
B218 21 Fishery V This study
B222 22 FR696349 River Kymijoki This study
B223 23 FR696350 River Kymijoki This study
B224 24 River Kymijoki This study
B225 24 FR696351 Fishery V This study
B226 25 FR696352 Fishery V This study
B230 I Fishery V This study
B234 C Fishery V This study
B235 C Fishery V This study
B236 ND Fishery V This study
B237 C Fishery V This study
B241 26 FR696353 Fishery V This study
B243 27 FR696354 Fishery V This study
B244 ND Fishery V This study
B245 C Fishery V This study
B247 C Fishery V This study
B257 26 FR696355 Fishery V This study
B259 C Fishery V This study
B260 ND FR696356 Fishery V This study
B261 C Fishery V This study
B262 ND FR696357 Fishery V This study
B263 ND FR696358 Fishery V This study
B267 C Fishery V This study
B268 C Fishery V This study
B269 ND Fishery V This study
B270 C Fishery V This study
B271 C Fishery I This study
B272 J Fishery I This study
B273 C Fishery I This study
B274 C Fishery I This study
B275 C Fishery I This study
Rz-A A 29
R-B B 29
Rz-C C 29
S-C C 29
R-D D 29
S-D D 29
Rz-E E 29
R-E E 29
S-E E 29
S-F F F. columnare type strain NCIMB 2248
Rz-G G 29
R-G G 29
S-G G 29
R-H H 29
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a

The previously studied F. columnare strains (genomic groups A to H and colony morphologies 1 to 4) are referred to in this study by placing the colony morphology after the genomic group: Rz, rhizoid (previously 1); R, rough (previously 2 and 3); and S, smooth (previously 4). For example Rz-C corresponds to the previous designation C1.

b

UP-PCR group (numbers) for Flavobacterium sp. or RISA group (letters) for Flavobacterium columnare. ND, not determined.

c

For the partial 16S rRNA sequence.

Previous reports describe phages infecting the genus Flavobacterium and their interaction with the bacterial host mostly in marine environments (4, 9, 12, 14), but the phage-host relationship of the fish pathogen Flavobacterium psychrophilum has also been studied (27). A total of 49 bacteriophages were isolated from water samples (Table 2). Phages were enriched using flavobacterial isolates from freshwaters, fish farms, and previously described F. columnare strains. Phage stocks were prepared, and selected phages were grown by infecting the host bacterium (multiplicity of infection, 5 to 10) at the proper cell density, concentrated, and purified (22). Many of the isolated phages produced low-titer lysates, and they were used only for infection tests (see below for host range studies). Phage genomic DNA was extracted (for methods, see references 1 and 23) and digested with BamHI, EcoRI, HindIII, and PstI. For the genomes that were cut, the genome size was calculated from the resulting restriction profile (Table 2). For the phages that were sequenced (FKj-2, FL-1, FCL-2, and FCV-1), approximately 3,000 bp of each genome (except FCV-1, for which 1,600 bp was used) was subjected to BLAST searching (http://blast.ncbi.nlm.nih.gov/Blast.cgi; May 2011), and no significant DNA sequence similarity was found in the database. Putative protein-coding genes were analyzed using Vector NTI 11.0.0 (Invitrogen). Best matches for FCL-2 were to a hypothetical protein of the Vibrio phage VP16T (score, 57.8) and to a hypothetical protein of the Vibrio phage VP16C (score, 55.1). For FCV-1, the best match was to a hypothetical protein, B40-8030, of the Bacteroides phage B40-8 (score, 53.9).

Table 2.

Bacteriophages isolated and characterized in this study

Phageb Sampling site Isolation strain Phage family Approximate genome size (kbp)c RISA groupd
FJy-1 Lake Jyväsjärvi B80 Myoviridae 30
FJy-2 Lake Jyväsjärvi B167 NDa >48
FJy-3 Lake Jyväsjärvi B169 Myoviridae L
FKo-1 Lake Konnevesi B28 Myoviridae 25–48
FKo-2 Lake Konnevesi B28 Myoviridae 20–30
FKe-1 River Kevojoki B114 Myoviridae ND
FTs-1 River Tsarsjoki B121 ND 25–48
FKj-1 Lake Kevojärvi B127 ND >48
FKj-2 Lake Kevojärvi B130 Myoviridae 25–48
FKy-1 River Kymijoki B222 Myoviridae 20–48
FKy-2 River Kymijoki B223 Podoviridae? 25–48
FKy-3 River Kymijoki B224 Myoviridae? L
FLe-1 Lake Leppävesi B176 Siphoviridae? 20–30
FL-1 Fishery L B183 Myoviridae 55
FV-1 Fishery V B214 Myoviridae 28
FV-2 Fishery V B218 ND 25–48
FV-3 Fishery V B225 Myoviridae ND
FV-4 Fishery V B226 Podoviridae 25–48
FV-5 Fishery V B241 ND L
FV-6 Fishery V B243 ND 30–40
FV-8 Fishery V B257 ND L
FV-9 Fishery V B260 Podoviridae? L
FV-10 Fishery V B262 ND L
FV-11 Fishery V B263 ND >48
FV-12 Fishery V B278 Podoviridae? 60
FCL-1 Fishery L B67 Myoviridae/Podoviridae 50 A
FCL-2 Fishery L B185 Myoviridae 30 G
FCL-3 Fishery L R-G ND 30 G
FCL-4 Fishery L R-G ND ND G
FCV-1 Fishery V Rz-C Myoviridae 50 C
FCV-2 Fishery V B235 ND ND C
FCV-3 Fishery V B236 ND ND C
FCV-4 Fishery V Rz-C ND ND C
FCV-5 Fishery V Rz-C ND ND C
FCV-6 Fishery V Rz-C ND ND C
FCV-7 Fishery V Rz-C ND ND C
FCV-8 Fishery V Rz-C ND ND C
FCV-9 Fishery V B245 ND ND C
FCV-10 Fishery V B247 ND ND C
FCV-11 Fishery V Rz-C ND ND C
FCV-12 Fishery V Rz-C ND ND C
FCV-13 Fishery V B261 ND ND C
FCV-14 Fishery V Rz-C ND ND C
FCV-15 Fishery V Rz-C ND ND C
FCV-16 Fishery V Rz-C ND ND C
FCV-17 Fishery V Rz-C ND ND C
FCV-18 Fishery V Rz-C ND ND C
FCV-19 Fishery V Rz-C ND ND C
FCV-20 Fishery V Rz-C ND ND C
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a

ND, not determined.

b

The first letter(s) of the phage name indicates the isolation host (F, Flavobacterium sp.; FC, F. columnare); subsequent letters refer to the sampling site.

c

L, much larger than the typical tailed phage genome (50 kb).

d

The F. columnare RISA group that the phage is specific to.

Transmission electron microscopy (TEM) was used to study phage morphology. All of the flavobacterial phages that were characterized were tailed phages of the families Myoviridae, Podoviridae, and Siphoviridae (Fig. 1 and Table 2). Most of these phages had an average head size of 50 to 70 nm, but some of the myovirus isolates had capsid sizes of about 100 nm or more (FJy-3, FKo-2, FKj-2, FKy-1, and FKy-3).

Phages infecting F. columnare were isolated only from fish farms during disease outbreaks. These phages might survive inside the host cell during the cold-water period and start a lytic cycle when nutrients become available for the host cell and enough energy is available.

Studies on aquatic phage-host interplay have been conducted extensively in marine environments (15), but less is known about this interplay in freshwaters (15, 34, 35). In our study, the host ranges differed greatly between the phage isolates (Fig. 2). In the initial screening for susceptibility of the bacteria for phages, each bacterium was infected with each phage isolate by spotting the phage lysate on top agar containing the host bacterium. Each bacterium was then infected with each phage using a plaque assay. Some of the isolated phages infecting Flavobacterium species were identified as having a broad host range. These phages infected bacteria isolated from both freshwater and fish farm samples, although it has been suggested that single-host enrichments select for phages with narrow host ranges from sewage and marine environments (11, 33). Some of the phages (especially all F. columnare phages) were more host specific, as determined with our collection of Flavobacterium strains. F. columnare strains isolated from the same location as the phage were the only ones susceptible to that specific phage.

Fig. 2.

Fig. 2.

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Host ranges of the phages infecting different Flavobacterium sp. strains. Dark gray squares indicate infection; white indicates no infection, and light gray squares mark the strain which was originally used for isolation of the phage. The numbers at right and bottom are the total number of different host ranges of the phage and susceptibility of bacteria to phages, respectively. The approximate titer of each phage on the bacteria is marked with plus signs (+, ≤105; ++, 106 to 108; and +++, ≥109 PFU/ml). The titer (PFU/ml) of the phage on the isolation strain is marked on the light gray squares. In parentheses after the name of the bacterial strain is the abbreviation of the place of isolation, which is also included in the phage nomenclature (Fig. 1). F. columnare phages infected only one specific F. columnare RISA group, and they are listed in Table 2.

No infection of F. columnare strains by Flavobacterium sp. phages was observed, and vice versa. However, 11 Flavobacterium sp. phage lysates (FTs-1, FKo-2, FL-1, FV-1, FKy-1, FKy-2, FKy-3, FV-3, FV-4, FV-5, FV-6, and FV-8) inhibited the growth or lysed the underlying bacterial culture on all F. columnare strains tested but produced no individual plaques (data not shown). It could be that the phages were able to bind to the bacteria and cause death but were not able to produce progeny, or the clear spots could be an indication of bacteriocin activity. The causative agent of this strong inhibition or lysis should be studied further for the possibility of developing antimicrobial agents. One of the phages (FCL-1) was isolated from a fish suffering from columnaris. The presence of the F. columnare phages in the fish indicates the possibility of controlling a fish disease by enrichment of these phages. A number of successful reports on phage therapy against fish diseases have been published; in these studies, the phages were applied directly to the water (16, 17). Another possible benefit related to phages is that they could be developed for use as diagnostic tools. In the present study, we show that bacteriophages and flavobacteria are widespread in northern freshwaters. We found phages of F. columnare to be host specific, making them good candidates for phage therapy.

Nucleotide sequence accession numbers.

All sequences have been submitted to the EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/) under the accession numbers given in Table 1 and FR714876 (FCL-2), FR714877 (FL-1), FR714878 (FKj-2), and FR865436 (FCV-1).

Acknowledgments

Petri Papponen, Katja Ryymin, and Irene Helkala are thanked for their excellent technical assistance. Some bacterial strains used in this study were kindly donated by Päivi Rintamäki and Finnish food safety authority EVIRA. Station manager Kari Saikkonen and field master Esa Karpoff from The Kevo Research Station (University of Turku) are acknowledged for providing water samples.

This work was supported by Finnish Centre of Excellence Program of the Academy of Finland (2006–2011) grant 1129648 (J.K.H.B.), Academy of Finland grant 127500 (L.-R.S.), and a grant from the Maj and Tor Nessling Foundation (J.K.H.B.).

Footnotes

▿

Published ahead of print on 2 September 2011.

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