Fisheries Science 64(1), 62-64 (1998)

Phage Typing of garvieae (Formerly seriolicida) a of Cultured Yellowtail

Kyung Hyun Park, Hiroaki Kato, Toshihiro Nakai,t and Kiyokuni Muroga Faculty of Applied Biological Science, Hiroshima University, Higashihiroshima 739-8528, Japan (ReceivedMay 16, 1997)

Bacteriophages of Lactococcus garvieae, designated as PLgW and PLgS, were isolated from sea water and sediment samples by an enrichment method. Morphological and genomic features of these phages were in agreement with those of the L. garvieae phage, designated as PLgY, belonging to the fa mily Siphoviridae that was detected in an L. garvieae strain isolated from diseased yellowtail in the previ ous study. One hundred and eleven strains of L. garvieae examined were divided into 14 phage types (A•`N) by using the phage isolates which were differentiated from each other in the infectivity, with a major phage type (type A) containing 73 strains. One phage type (type N) consisting of 9 bacterial strains was insensitive to any of the phages used. However, there were no apparent correlations between the phage types and the geographical sources of the bacterial strains or between phage types and the an tigenic forms (KG- and KG+).

Key words: bacteriophage, phage typing, Siphoviridae, Lactococcus garvieae, Enterococcus seriolicida, yellowtail, Seriola quinqueradiata

The disease caused by Enterococcus seriolicida has been two reference strains, L. garvieae ATCC 43921T and E. responsible for significant losses in culture industry of yel seriolicida ATCC 49156T, were used in this study. They lowtail Seriola quinqueradiata in Japan.1,2) The causative were 93 strains from yellowtail, 6 strains from purplish am agent of this disease, which has long been called streptococ berjack Serbia dumerili, 3 strains from Japanese flounder cal , was first reported as Streptococcus sp.3) and Paralichthys olivaceus, 1 strain from goldstriped amber identified as Enterococcus seriolicida n. sp. in 1991.') jack S. lalandi, and 3 strains from striped jack Pseu However, it was recently demonstrated that E. seriolicida docaranx dentex cultured in 7 different prefectures, and 3 is a junior synonym of Lactococcus garvieae based on strains from sea water. Their geographical sources are phenotypic and genotypic homologies,4,5) thus we use the shown in Table 1. These strains were provided by some name L. garvieae instead of E. seriolicida in this paper. prefectural fish disease control centers or fisheries research In a previous study,6) we found a virulent bac stations and stored in (TSB; Nissui) with teriophages specific to L. garvieae isolated from diseased 10% glycerol at -80•Ž and subcultured on tryptic soy yellowtail and the phage was identified as a member of the agar (TSA; Nissui) at 25•Ž for 48 h prior to experiments. family Siphoviridae. The bacteriophage typing is based on Anti-L. garvieae rabbit sera raised against two strains be the susceptibility of the test strains to lysis by a panel of longing to different antigenic forms (KG and KG+),9) bacteriophages selected for differentiating strains within provided by Dr. T. Yoshida, were employed in typing L. species and its epidemiological utility in the medical field is garvieae strains. well documented for Staphylococcus aureus, Pseudomo nas aeruginosa, and Salmonella spp.7) However, our Phage knowledge on phage typing scheme for fish is The following 10 phages of L. garvieae were used in this limited except for a study in Aeromonas salmonicida,8) study (PLgY, W or S refers to phages of Lactococcus gar where 27 phage types of A. salmonicida were distinguished vieae from yellowtail, sea water or sediment, respectively). based on the sensitivity patterns to 18 phage isolates. In Three phages, PLgY16, PLgY30, and PLgY886 were ob the present study, we isolated L. garvieae phages from sea tained from cultures of L. garvieae isolated from diseased water and sediments in order to get phages with different yellowtail. The phage PLgY886 was provided by Dr. T. infectivities, and tried to establish a phage typing scheme Yoshida. As described later, six phages, PLgW 1 to of L. garvieae by using these new phage isolates from cul PLgW6, were isolated from sea water collected in fish ture environments and previously isolated phages from L. farms and a phage PLgSl was isolated from sediment sam garvieae. ples of fish farm. All of these 10 phages were investigated on their morphological and genomic features for their clas Meterials and Methods sification by electron microscopy and agarose gel elec trophoresis as in the previous study.6) One hundred and eleven strains of L. garvieae including

•ª Corresponding author. Phage Typing of Lactococcus garvieae 63

Table 1. Sources of the Lactococcus garvieae strains used in this study Results and Discussion

The enrichment method used in this study was highly ef fective for the recovery of the L. garvieae phage PLgY16 from sea water (Table 2), where one PFU or less phage in oculated in sea water was recovered at a concentration of 108 PFU/ml after the enrichment culture. Preliminary iso lation trials of L. garvieae phages from sea water using this enrichment method were not successful when sea water samples stored for several days after collection were used

(data not shown). It was demonstrated that the infectivity of the PLgY16 phage is not stable under an experimental condition using unsterilized sea water but stable in steri Duration of Phage Infectivity in Sea Water lized (autoclaved) sea water (Fig. 1). Based on these The PLgY16 phage was inoculated at 104 plaque form results, sea water samples for phage isolation were filtered ing unit (PFU)/ml in untreated sea water and autoclaved through bacterial filter immediately after collection. sea water, and kept at 25•Ž for two weeks. PFUs were Phages PLgW1•`W6 and PLgS1 were isolated from sea measured at 0, 3, 7, and 14 days after incubation by the water and sediment samples by the enrichment method. double agar layer method described previously6) using a PLgY16, PLgY30, PLgY886, PLgW1, PLgW2, PLgW3, strain of L. garvieae (no. 16) as host cell. and PLgW4 were differentiated each other based on their lysis patterns as shown in Table 3. Phages PLgW4, Isolation of Phage from Sea Water and Sediment PLgW5, and PLgW6 showed the same lysis pattern, thus An enrichment method described by Spencer10) was used they were represented by PLgW4. Phages PLgW2 and to isolate L. garvieae phages from fish culture environ PLgSI also exhibited the same lysis pattern, being ments. To examine the detection limits of phage by the represented by PLgW2. Consequently, 7 phages having method, 1 ml of serial 10-fold dilutions of the PLgY16 sus different infectivities were used for phage typing (Table 3). pension (initial concentration: 3.9 •~ 103 PFU/ml) was in Electron micrographs of negatively stained phages rev oculated in 150 ml of sea water mixed with 50 ml of quad ealed that these isolated phages had an isometric head ruple-strength TSB which contains L. garvieae no. 16 measuring 60 nm in diameter and a thin flexible tail of strain as indicator cells and incubated at 25•Ž overnight. After centrifugation at 3,000 •~ g for 30 min, the super natant was filtered through a 0.2ƒÊm-membrane filter and Table 2. Recovery of the phage (PLgY 16) from sea water by the PFUs were measured by the double agar layer method. enrichment method Phage isolation was made from 14 sea water samples and 5 sediment samples, which were collected in fish farms in Ehime Prefecture in 1996, by using the enrichment method. Sea water samples were filtered through a 0.45

µm-membrane filter within 2 h after collected. A 150 ml of sea water was mixed with 50 ml of quadruple-strength TSB, supplemented with 1 ml of an L. garvieae broth cul ture with a mixture of 5 to 20 strains as indicator cells, and incubated at 25•Ž for 24 h. After centrifugation at 3,000 •~ g for 30 min, the supernatant was filtered through a 0.2ƒÊm-membrane filter and this bacteria-free prepara tion was tested for lytic action against the appropriate strains of L. garvieae by the double agar overlay method. A small amount (0.2-0.5 g) of sediment was suspended in 100 ml of sterilized artificial sea water and centrifuged at 3,000 •~ g for 30 min. The supernatant was filtered and sub jected to the above mentioned enrichment method.

Sensitivity of L. garvieae Strains against Isolated Phages Prior to experiments, isolated phages were propagated by the double agar overlay method with each initial host strain or strains used for the enrichment method, and phage concentration was adjusted to 104PFU/ml. The sen sitivity of L. garvieae strains to isolated phages was exam ined by plaque formation using the double agar overlay method.

Fig. 1. Duration of infectivity of the phage (PLgY16) in sea water. -•œ: autoclaved sea water •œ , •› -•›: unsterilized sea water. 64 Park et al.

Table 3. Phage typing scheme of L. garvieae

7 •~ 140-180 nm. All phages had the nucleic acids larger sity for providing an phage PLgY886 and antisera. than 20 kbp, and the nucleic acids were broken down by EcoRI, but not by RNase. These results are in agreement References with those of PLgY16, that was classified into the family Siphoviridae in the previous paper.6) Thus, all of the isolat 1) R. Kusuda, K. Kawai, F. Salati, C. R. Banner, and J. L. Fryer: En ed phages of the present study also can be classified into terococcus seriolicida sp. nov., a fish pathogen. Int. J. Syst. Bac the family Siphoviridae in the current .") teriol., 41, 406-409 (1991). 2) T. Kitao: Streptococcal , in "Bacterial Diseases of Fish" The one hundred and eleven strains of L. garvieae exam (ed. by V. Inglis, R. J. Roberts, and N. R. Bromage), Blackwell ined were divided into 14 phage types (A •` N), with the Science Ltd, London, 1993, pp. 196-215. major phage type A including 73 strains examined (Table 3) R. Kusuda, K. Kawai, T. Toyoshima, and I. Komatsu: A new patho 3). The type strain (ATCC49156) of E. seriolicida and 3 genic bacterium belonging to the genus Streptococcus, isolated strains isolated from sea water belong to type A. All the from an epizootic of cultured yellowtail. Nippon Suisan Gakkaishi, Nagasaki strains (9 strains) belong to type A and the 8 Mie 42, 1345-1352 (1976). 4) L. M. Teixeira, V. L. C. Merquior, M. C. E. Vianni, M. G. S. strains belong to types A and D. However, there were no Carvalho, S. E. L. Fracalanzza, A. G. Steigerwalt, D. J. Brenner, apparent correlations between the phage types and the ge and R. R. Facklam: Phenotypic and genotypic characterization of ographical sources of the strains. As mentioned before, L. atypical Lactococcus garvieae strains isolated from water buffalos garvieae from diseased yellowtail were divided into two an with subclinical mastitis and confirmation of L. garvieae as a senior tigenic forms, KG and KG+,2) but no correlation could subjective synonym of Enterococcus seriolicida. Int. J. Syst. Bac be observed between phage types and the antigenic forms. teriol., 46, 664-668 (1996). The relationship between phage types and pathogenicity or 5) A. Eldar, C. Ghittino, L. Asanta, E. Bozzetta, M. Goria, M. Prearo, and H. Bercovier: Enterococcus seriolicida is a junior syno virulence should be investigated in the future. nym of Lactococcus garvieae, a causative agent of septicemia and The N type consisting of 8 strains isolated from yellow meningoencephalitis in fish. Curr. Microbiol., 32, 85-88 (1996). tail, 7 strains (nos. 1, 11, 39, 40, 41, 42, 43) in Ehime Pref. 6) K. H. Park, S. Matsuoka, T. Nakai, and K. Muroga: A virulent bac and 1 strain (no. 99) in Oita Pref., and the type strain teriophage of Lactococcus garvieae (formerly Enterococcusseriolici (ATCC43921) of L. garvieae was insensitive to any of the da) isolated from yellowtail Seriola quinqueradiata . Dis. Aquat. phages tested. These phage-insensitive strains could be sen Org., 29, 145-149 (1997). 7) M. A. Pfaller: Typing methods for epidemiologic investigation sitive to other types of the phage which could be isolated in "Manual of Clinical Microbiology , in the future. There is also a possibility that these insensitivi , 5th ed." (ed. by A. Balows, W. J. Hausler, K. L. Herrmann, H. D. Isenberg, and H. J. Shadowy), ties were due to lysogenic infection of the strains with the American Society for Microbiology, Washington DC, 1991, pp. phages. This was supported by the observation that a 171-182. strain (no. 1) of L. garvieae showed lytic reaction before 8) C. J. Rodgers, J. H. Pringle, D. H. McCarthy, and B. Austin: successive subcultures and a temperate phage (not includ Quantitative and qualitative studies of Aeromonas salmonicida bac ed in the present study) was detected in another strain (no. teriophage. J. Gen. Microbiol., 125, 335-345 (1981). 43) of L. garvieae when the strain was subcultured on TSA 9) T. Kitao: The methods for detection of Streptococcus sp., causative bacteria of streptococcal disease of cultured yellowtail (Seriola quin from stock at -80•Ž for the phage typing experiment. queradiata)-Especially, their cultural, biochemical and serological Therefore, further detailed investigations on the phage-in properties. Fish Pathol., 17, 17-26 (1982). sensitive strains are needed for the possible detection of 10) R. Spencer: Indigenous marine bacteriophages. J. Bacteriol., 79, temperate phages using various induction methods. 614 (1960). 11) F. A. Murphy, C. M. Fauquet, D. H. L. Bishop, S. A . Ghabrial, A. Acknowledgments The authors thank the staff of Prefectural Fish Dis W. Jarvis, G. P. Martelli, M. A. Mayo, and M. D. Summers: Virus ease Control Centers or Fisheries Research Stations of Ehime, Taxonomy 6th report of ICTV, Virology Division International Kagoshima, Nagasaki, Mie, Oita, Wakayama, and Miyazaki prefectures Union of Microbiological Societies, Springer-Verlag, New York, for providing bacterial strains and Dr. T. Yoshida of Miyazaki Univer 1995, p. 586.