Volume 20/3

Bull. Eur. Ass. Fish Pathol., 21(4) 2001, 136

Conventional versus miniaturized systems for the phenotypic characterization of Lactococcus garvieae strains. Carmen Ravelo†, Beatriz Magariños, Jesús L. Romalde* & Alicia E. Toranzo Departamento de Microbiología y Parasitología. Facultad de Biología and Instituto de Acuicultura. Universidad de Santiago de Compostela. 15706, Santiago de Compostela. Spain. † Permanent address: Fundación La Salle, Apdo. 51. San Felix, Edo. Bolívar 8024, Venezuela

Abstract Lactococcus garvieae is an emerging pathogen in intensive freshwater aquaculture. In this study, twenty three isolates from different fish species and geographic origin were phenotypically characterized using conventional and miniaturized systems employing two different culture media to obtain the bacterial inocula. Based on the comparison with conventional tests, repetitive and accurate identification of the L. garvieae isolates with the miniaturized system Rapid ID 32 Strep was only achieved with spectrophotometrically adjusted inocula prepared from blood agar. The results obtained, under these conditions, indicated a high level of biochemical homogeneity among the strains, regardless of their source of isolation. Slight variability occurred among the strains for some reactions such as hydrolysis of hippurate, ß-galactosidase, N-acetyl-ßglucosaminidase, ß-mannosidase, and acid from melezitose and pululan. Our findings suggest different biotypes should not be established, since they would lack epidemiological or intraspecies taxonomical value.

Introduction Gram-positive cocci as aetiological agents of

tococcus parauberis (Doménech et al., 1996), Lactococcus piscium (Williams et al., 1990),

fish diseases have assumed importance in recents years in different parts of the world,

Vagococcus salmoninarum (Wallbanks et al., 1990) and Lactococcus garvieae (Collins et al.,

including Japan (Kitao, 1993; Kusuda & Salati, 1993), Australia (Carson & Munday,

1983; Kusuda et al., 1991; Doménech et al., 1993).

1990), South Africa (Bragg & Broere, 1986), Israel (Eldar et al., 1994), Spain (Toranzo et al., 1994; Doménech et al., 1996; Romalde et al., 1999) and the United States (Perera et al., 1994; Hawke & Camus, 2000). Molecular studies indicate that at least six diferent defined species of Gram positive cocci are pathogenic to fish: Streptococcus iniae, (Eldar et al., 1995), Streptococcus difficile (Eldar et al., 1994), Strep-

Lactococcus garvieae (junior synonym Enterococcus seriolicida) has been recovered from fish species living in diverse conditions; yellowtail cultured in marine environment in Japan and trout raised in temperate freshwater in Europe and Australia (Eldar et al., 1999). The typical signs exhibited by L. garvieae affected fish are exophthalmia, periocular haemorhage, con-

Bull. Eur. Ass. Fish Pathol., 21(4) 2001, 137 Source

Origin

TW I

Rainbow trout, Spain

Year of isolation Donor 1991

F. Sanz

TW II

Rainbow trout, Spain

1991

F. Sanz

TW 446.B3

Rainbow trout, Spain

1997

J. Aizpurúa

PP 60.1

Rainbow trout, Spain

1997

the authors

PP 61.1

Rainbow trout, Spain

1997

the authors

EW 175-97

Rainbow trout, Spain

1997

E. Planas

AR 1

Rainbow trout, Spain

1999

J.L. Muzquiz

AR 2a

Rainbow trout, Spain

1999

J.L. Muzquiz

AR 2b

Rainbow trout, Spain

1999

J.L. Muzquiz

3712

Rainbow trout, Spain

2000

T. Peraferrer

3682

Rainbow trout, Spain

2000

T. Peraferrer

3756

Rainbow trout, Spain

2000

T. Peraferrer

279-00

Rainbow trout, Spain

2000

J. Aizpurúa

321-00

Rainbow trout, Spain

2000

J. Aizpurúa

301-00

Rainbow trout, Spain

2000

J. Aizpurúa

657-1

Rainbow trout, Italy

1999

A. Manfrin

657-2

Rainbow trout, Italy

1999

A. Manfrin

2913

Rainbow trout, Italy

1999

A. Manfrin

297-8

Cat fish, Italy

1999

A. Manfrin

NG8206 KG+

Yellowtail, Japan

1982

T. Yoshida

NG8206 KG-

Yellowtail, Japan

1982

T. Yoshida

YT-3

Yellowtail, Japan

1974

R. Kusuda

SS 91014

Yellowtail, Japan

1991

R. Kusuda

Cow, UK

1973

NCDO

1949

ATCC

Reference strains L. garvieae NCDO 2155

Enterococcus faecalis ATCC 19433

Human, France

Table 1. Origin of Lactococcus garvieae isolates used in this study: F. Sanz & J. Aizpurúa, Trouw SA, Burgos, Spain; E. Planas, Proaqua Nutrición, Palencia, Spain; J.L. Murquiz, Dpto. Patología Animal, Univ. Zaragoza, Spain; T. Peraferrer, HIPRA Lab, Girona, Spain; A. Manfrin, Istituto Zooprofilatticoa Sperimentale delle Venezie, Italy; T. Yoshida, Faculty of Agriculture, Miyazaki Univ., Japan; R. Kusuda, Marine Biotechnology Laboratory, Fukuyama Univ., Japan.

Bull. Eur. Ass. Fish Pathol., 21(4) 2001, 138 gestion of some internal organs and

Biochemical and physiological tests

haemorrhagic enteritis.

Pure cultures of the isolated colonies in both TSA and CSB plates, were subjeted to stand-

Studies on phenotypic characterization of L. garvieae strains collected from different species and countries have been conduced using conventional methods and miniaturized systems (API 20 Strep, Rapid ID 32 Strep, API 50 CH) with variables results (Elliot et al., 1991; Teixeira et al., 1996; Eldar et al., 1999; Vela et al., 1999, 2000). Distinct intraspecies classification schemes were proposed on the basis of the described biotypes (Eldar et al., 1999; Vela

ard biochemical plate and tube assays (MacFaddin, 1993; Facklam & Washington, 1991). Acid production from carbohydrates was tested under aerobic and anaerobic conditions with O/F basal medium (Difco) supplemented with 1% of the appropriate sugar, and results were determined after 7 days incubation. The Rapid ID 32 Strep and API ZYM systems

et al., 1999, 2000) with no agreement among them and, therefore, lacking epidemiological

(bioMérieux) were incubated at 25ºC. Standardized inocula were prepared by suspend-

value.

ing cells in tubes containing sterile distilled water and ajusted spectrophotometrically to

The aim of the present paper is to evaluate the phenotypic variability within this species using both conventional and miniaturized systems in order to establish a standardized method so results are comparable among laboratories.

Materials and Methods Bacterial strains and culture conditions The twenty-three Lactococcus garvieae strains examined in this study, their host and geographic origin, are shown in Table 1. The reference strains of L. garvieae NCDO 2155, and Enterococcus faecalis ATCC 19433 were included for comparative purposes. The strains were routinely cultured on triptone soy agar (TSA, Oxoid) supplemented with 1% (w/v) NaCl and Columbia sheep blood agar (CSB, Oxoid) at 25ºC for 24-48 h. Size, morphology, and motility were observed by microscopic examination of cultures in triptone soy broth (TSB, Oxoid) at 25ºC.

DO = 0.8 (A 580). In addition, inocula adjusted to the McFarland scale No. 4 were used in parallel for comparative purpose. In order to evaluate the influence of culture media in the results obtained in the Rapid ID 32 Strep, each isolate was tested in parallel from inocula obtained from TSA and blood agar. The biochemical reactions obtained with each inoculum, using the miniaturized system, were compared with those achieved using standard plate and tube tests. All isolates were tested at least two times under the different conditions. The ability of the strains to grow at 4, 15, 25, 37 and 44.5ºC was tested in TSA over a period of 10 days. Growth at 3, 5, and 6.5% (w/ v) NaCl, and at pH 9.6 was determined in TSB at 25ºC after 1 week incubation. The susceptibility to antimicrobial agents was evaluated by the disc-difusion assay on Mueller-Hinton agar using the following chemotherapeutic

agents

(µg/disc):

Bull. Eur. Ass. Fish Pathol., 21(4) 2001, 139 enrofloxacin (5), ampicillin (10), erythromy-

and acid from D-arabitol, melezitose and

cin (5), chloramphenicol (30), nitrofurantoin (300), trimethoprim-sulfamethoxazole (23.75/

pululan (Table 2).

1.25), oxolinic acid (2) and oxytetracycline (30). Staphylococcus aureus ATCC 25923 was

The results of carbohydrate utilization with the Rapid ID 32 Strep system were affected

inlcuded as standard strain.

by the culture media (blood agar or triptone soy agar) utilized to obtain the bacterial in-

Results

ocula (Table 3). Diferences between the two media were apparent for acid production

The taxonomic analysis indicated a high level of phenotypic similarity among strains, regardless of the geographic origin or source. All strains were non-motile Gram-positive ovoid cocci, occurring in pairs and short chains, and produced a-haemolytic colonies on blood agar. The strains grew between 15 and 44.5ºC and at pH 9.6. Scant growth occurred in 6.5% NaCl for most strains after 48

from: lactose, maltose, sucrose, tagatose and cyclodextrin. In fact, with inocula prepared from TSA plates, the Rapid ID 32 Strep rendered false reactions for the sugars cited above when compared with results from traditional tube tests. However, when bacterial inocula were prepared from blood agar plates, biochemical profiles almost matched with those

h, and for all after 96 h.

obtained by conventional methods (Table 3). The delayed positive reaction detected in acid

The results of the biochemical patterns of the

production from lactose in the tube test (after 3-5 days incubation at 25ºC) can explain the

L. garvieae strains determined by conventional tests, as well as by the Rapid ID 32 Strep system are listed in Table 2. All isolates were

low percentage of positive strains for this reaction in the Rapid ID 32 Strep, even when

positive for Voges Proskauer (VP), pyrrolidonyl arylamidase (PIRA), arginine

blood agar was used as the initial inoculum medium. In addition, repetitive results were

dihydrolase (ADH), leucine arylamidase, αand ß-glucosidase, alanine phenylalanine pro-

only observed in the API profiles when the bacterial inocula were adjusted spectropho-

line arylamidase (APPA), esterase, lipase, valine arylamidase, cystine arylamidase,

tometrically.

chymotrypsine and acid phosphatase, and negative for oxidase, catalase, α-galactosidase, ß-glucuronidase, alkaline phosphatase (PAL), glicil triptophane arylamidase (GTA), ornithine and lysine decarboxilase, and urease. None of the strains hydrolyzed starch or gelatin and none were capable of utilizing citrate as the sole carbon source. Variable re-

Only the conjunction of two factors: i) a precise adjustement of the absorbance of bacterial inocula and, ii) use of blood agar as culture media, allowed a correct identification of the isolates as L. garvieae, with confidence percentages higher than 96% according to the API data base. All Spanish, Italian and Japanese isolates were

sults were observed among strains for: hydrolysis of hippurate, ß-galactosidase, N-

sensitive to enrofloxacin, and nitrofurantoin and resistant to oxolinic acid and

acetyl-ß-glucosaminidase, ß-mannosidase,

trimethoprim-sulphamethoxazole. However,

Bull. Eur. Ass. Fish Pathol., 21(4) 2001, 140 Rainbow trout Reaction(a)

Catfish

Yellowtail

Type strains

Spain (n=15)

Italy (n=3)

Italy (n=1)

Japan (n=4)

L.garvieae E. faecalis NCDO 2155 ATCC 19433

Oxidase

-

-

-

-

-

-

Catalase

-

-

-

-

-

-

Indole

-

-

-

-

-

-

Voges-Proskauer

+

+

+

+

+

+

Citrate

-

-

-

-

-

-

O/F

F

F

F

F

F

F

Hydrolysis of:

Hippurate

+

-

+

-

-

-

Gelatin

-

-

-

-

-

-

Starch

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Decarboxylation Lysine of: Ornithine ADH

+

+

+

+

+

+

PYRA

+

+

+

+

+

+

ß -glucosidase

+

+

+

+

+

+

α-galactosidase

-

-

-

-

-

-

ß -galactosidase

v(27)

-

-

v(25)

-

-

ß -glucuronidase

-

-

-

-

-

-

PAL ß NAG

-

-

-

-

-

-

v(87)

v(67)

+

v(75)

+

+ +

APPA

+

+

+

+

+

GTA

-

-

-

-

-

-

LA

+

+

+

+

+

+

v(13)

v(33)

-

v(50)

-

-

-

-

-

-

-

-

Lactose

(+)

(+)

(+)

v(75)

-

+ +

ß -mannosidase Urease Acid from:

Maltose

+

+

+

+

+

Rafinose

-

-

-

-

-

-

Sucrose

+

+

+

+

+

+ -

Sorbitol

-

-

-

-

-

L-arabinose

-

-

-

-

-

-

D-arabitol

-

-

-

v(50)

-

-

Mannose

+

Ribose

+

+

+

+

+

+

+

+

+

+ +

Mannitol

+

+

+

+

+

Melibiose

-

-

-

-

-

-

Trehalose

+

+

+

+

+

+

Melezitose

v(33)

-

-

v(75)

-

-

Tagatose

+

+

+

+

+

+

Cyclodextrin

+

+

-

+

+

+

Glycogen

-

-

-

-

-

-

v(7)

-

-

v(75)

-

-

Pululan

Table 2. Phenotypic characteristics obtained for L. garvieae isolates in conventional and Rapid ID 32 Strep system tests. aADH, arginine dihydrolase; PYRA, pyrrolidonyl arylamidase; PAL, alkaline phosphatase; ß NAG N-acetyl-ß-glucosaminidase; APPA, alanine phenylanaline proline arylamidase; GTA, glycyl tryptophan arylamidase; LA, leucine arylamidase. Symbols: + positive, - negative, (+) weak reaction, v variable (parentheses indicating percent positive).

Bull. Eur. Ass. Fish Pathol., 21(4) 2001, 141 Positive isolates (%) Rapid ID 32 Strep

Conventional tests*

TSA

CSB

Lactose

11

39

96

Maltose

78

100

100

Sucrose

78

100

100

Tagatose

67

100

100

Cyclodextrin

28

100

100

Acid from:

Table 3. Comparison of results for acid production from carbohydrates with L. garvieae isolates, employing miniaturized system and classical methods. * Using either TSA or CSB.

variable results among the strains were found

from melezitose and pululan. Interestingly, all

in the sensitivies to erythromycin, chloranphenicol, oxytetracycline and ampicil-

the Spanish isolates and the catfish strain, hydrolyzed the hippurate which has been

lin.

described as an uncommon characteristic of L. garvieae. However, some strains positive for

Discussion

this character have been reported (Cheng & Chen, 1998).

Streptococcosis caused by Lactococcus garvieae is an emerging disease in intensive aquaculture. At present, the disease is considered a major problem in trout farming worldwide, because of the economical losses occurring during the summer months, when water temperatures rises above 16ºC. On the basis of the results of the phenotypic characteristics, the Lactococcus strains isolated from fish were able to grow at 10 and 44.5 ºC, pH 9.6, and at 6.5% NaCl (after 48-96 h), which supports their assignation as L. garvieae. In fact, none of the other five species of grampositive cocci pathogenic for fish can grow under these conditions (Plumb, 1994; Schmidtke & Carson, 1994; Eldar et al., 1999). Our results indicated a high level of biochemical homogeneity among the strains, regardless of their origin of isolation. Some variability occurred among the strains for ß-galactosidase, N-acetyl-ß-glucosaminidase, and acid

Biotypes had been established for L. garvieae strains isolated from fish based on the acidification of carbohydrates and the presence of the enzymes using Rapid ID 32 Strep and API 50 CH systems. On the basis of the acidification of tagatose, ribose, and sucrose, Eldar et al. (1999) and Vela et al. (1999) independently defined three biotypes for L. garvieae but, unfortunately, no agreement existed between either scheme. Another intraspecies classification of this bacterial species was further proposed by Vela et al. (2000). These authors reported 13 different biotypes for isolates from trout, human, cow and water buffalo based on the acidification of sucrose, tagatose, mannitol, and cyclodextrin, and the presence of the enzymes pyroglutamic acid arylamidase, and N-acetyl-ß-glucosaminidase. However, in our study, all 23 fish isolates gave positive results in the acid production for the above carbohydrates.

Bull. Eur. Ass. Fish Pathol., 21(4) 2001, 142 The variability observed among some au-

Tecnología (CICYT), Ministerio de Educación

thors, might be due to the type of miniaturized system employed, density of the bacte-

y Cultura, Spain. C. Ravelo thanks Fundación La Salle (Venezuela) for a research fellowship.

rial inoculum and incubation temperature. In fact, Vela et al. (2000) reported variability in

The authors gratefully acknowledge the donors for the kind supply of some isolates.

the results obtained for the acidification of ribose between Rapid ID 32 Strep and API 50

References

CH systems. In addition, the results of the present study demonstrate the influence of the culture medium and initial inoculum density in the biochemical profiles of L. garvieae using the miniaturized Rapid ID 32 Strep system. Repetitive and accurate identification is only achieved with spectrophotometrically adjusted inocula obtained from blood agar. Under other conditions, some L. garvieae isolates can be misidentified as L lactis subsp. lactis or Streptococcus uberis. In conclusion, we consider that the Rapid ID 32 Strep system can be useful for a rapid diagnosis of fish streptococcosis caused by L. garvieae, precautions must be taken when carbohydrate utilization is used to differentiate biotypes. We recommend that, in addition to standardize the culture medium, inoculum density and incubation temperature used in the miniaturized system, the results must be always corroborated by conventional tests. In this manner, the intraspecific classification of L. garvieae has epidemiological and taxonomic value.

Acknowledgements This work was supported in part by Grants MAR99-0478 and PETRI 95-0471-OP from the Comisión Interministerial de Ciencia y

Bragg, R. and Broere, J.S.E. (1986) Streptococcosis in rainbow trout in South Africa. Bull.Eur.Ass.Fish Pathol. 6, 89-91. Carson, J. and Munday, B. (1990) Streptococcosis-an emerging disease in Aquaculture. Australian Aquaculture 5, 32-33. Collins, M.D., Farrow, F.A.E., Phillips, B.A. and Kandler, O. (1983) Streptococcus garvieae sp. nov. and Streptococcus plantarum sp. nov. J. Gen. Microbiol. 129, 3427-3431. Cheng, W. and Chen, J-C. (1998) Isolation and characterization of an Enterococcus-like bacterium causing muscle necrosis and mortality in Macrobrachium rosenbergii in Taiwan. Dis Aquat Org 34, 93-101. Doménech, A., Prieta, J., FernándezGarayzábal, J., Collins, M.D., Jones,D. and Domínguez, L. (1993) Phenotypic and phylogenetic evidence for a close relationship between Lactococcus garvieae and Enterococcus seriolicida. Microbiología (SEM) 9, 63-68. Doménech , A., Fernández-Garayzábal, J.F., Pascual, C., García, J.A., Cutúli, M.T., Moreno, M.A., Collins,, M.D. and Domínguez, L. (1996) Streptococcosis in cultured turbot, Scophthalmus maximus (L.), associated with Streptococcus parauberis. J. Fish Dis, 19, 33-38. Eldar, A., Bejerano, Y. and Bercovier, H. (1994)Streptococcus shiloi and Streptococcus difficile: two new streptococcal species causing meningoencephalitis in fish. Curr. Microbiol. 28,139.143.

Bull. Eur. Ass. Fish Pathol., 21(4) 2001, 143 Eldar, A., Frelier, P.F., Assenta, L. Varner, P.W., Lawhon, S. and Bercovier, H. (1995) Streptococcus shiloi, the name for agent causing septicemic infection in fish, is a junior synonym of Streptococcus iniae. Int. J. Syst. Bacteriol. 45,840-842. Eldar, A., Goria, M. Ghittino, C., Zlotkin, A. and Bercovier, H. (1999) Biodiversity of Lactococcus garvieae isolated from fish in Europe, Asia, and Australia. Appl. Environ. Microbiol. 65,1005-1008. Elliot, J.A., Collins, M.D., Pigott, N.E. and Facklam, R.R. (1991) Differentiation of Lactococcus lactis and Lactococcus garvieae from humans by comparison of whole-cell protein patterns. J. Clin. Microbiol. 20, 2731-2734. Facklam, R.R. and Washintong II., J.A. (1991)Streptococcus and related catalase-negative Gram-positive cocci. In: Manual of Clinical Microbiology, 5th. Ed. (Balows, A. Hausler Jr. WJ. Hermann, K.L., Isemberg, H.D. and Shadomy, H.J. eds.). Am.Soc. Microbiol. Washington, D.C. pp.238-257. Hawke, J.P. and Camus, A.C. (2000). Disease in farm raised tilapia caused by a group C Streptococus. Proceedings of the Meeting of the Fish Health Section of the American Fisheries Society. Pensacola Beach, Fl. p. 37. Kitao, T. (1993). Streptococcal infections. In: Bacterial diseases of fish (Inglis, V., Roberts, R.J. and Bromage, N.R., Eds.) pp. 196-210. Blackweel Scientific Publications, Oxford, UK. Kusuda, R., Salati, F. (1993) Major bacterial diseases affecting mariculture in Japan. Ann. Rev. Fish Dis. 3, 9-85. Kusuda, R., Kawai, K., Salati, F., Banner, C.R. and Fryer, J.L. (1991) Enterococcus seriolicida sp. nov. , a fish pathogen. Int. J. Syst. Bacteriol. 41, 406-409. Mac Faddin, J. F. (1991) Pruebas bioquímicas para la identificación de bacterias de importancia clínica. Editorial Médica Panamericana. México.

Perera, R.P., Johnson, S.K., Collins, M.D. and Lewis, D.H. (1994) Streptococcus iniae associated with mortality of Tilapia nilotica x T. aurea hybrids. J. Aquat. Anim. Health. 6, 335-340. Plumb, J.A. (1994) Health Maintenance of cultured fishes. Principal microbial diseases. CRC Press, London. Romalde, J.L., Magariños, B. and Toranzo, A.E. (1999) Prevention of streptococcosis in turbot by intraperitoneal vaccination: a review. J. Appl. Ichthyol. 15, 153-158. Schmidtke, L.M. and Carson, J. (1994) Characteristics of Vagococcus salmoninarum isolated from diseased salmonid fish. J. Appl. Bacteriol. 77, 229-236. Teixeira, L.M., Merquior, V.L.C., Vianni, M.C.E., Carvalho, M.G.S. , Fracalanzza, S.E.L., Steigerwalt, A.G., Brenner, D.J. and Facklam, R.R. (1996). Phenotypic and genotypic characterization of atypical Lactococcus garvieae strains isolated from water buffalos with subclinical mastitis and confirmation of L. garvieae as a senior subjective synonym of Enterococcus seriolicida . Int. J. Syst. Bacteriol. 46, 664668. Toranzo, A.E., Devesa, S., Heinen, P., Riaza, A., Núñez, S. and Barja, J.L. (1994). Streptococcosis in cultured turbot caused by an Enterococcus-like bacterium. Bull. Eur. Ass. Fish Pathol. 14, 19-23. Vela, I., Liebana, P., Albendea, C., Gibello, A., Blanco, M., Moreno, M., Cutúli, M., García, J.A., Domenech, A., Domínguez, L., Vázquez, J. and Fernández-Garayzabal, J. (1999) Caracterización fenotípica y molecular de Lactococcus garvieae. Proceedings of the XIII Congreso Nacional de Microbiología, Sociedad Española de Microbiología, Sept. 1924, 1999, Granada, Spain. p. 47.

Bull. Eur. Ass. Fish Pathol., 21(4) 2001, 144 Vela, A.I., Vázquez, J., Gibello, A., Blanco, M.M., Moreno, M.A., Liebana, P., Albendea, C., Alcalá, B., Méndez, A., Domínguez, L. and Fernández-Garayzabal, J.F. (2000) Phenotypic and genetic characterization of Lactococcus garvieae isolated in Spain from lactococcosis outbreaks in comparison with isolates of other countries and sources. J. Clin. Microbiol. 38, 3791-3795.

Wallbanks, S., Martínez-Murcia, A.J., Fryer, J.L., Phillips, B.A. and Collins, M.D. (1990) 16S rRNA sequence determination for members of genus Carnobacterium and related lactic acid bacteria and description of Vagococcus salmoninarum sp. nov. Int. J. Syst. Bacteriol. 40, 224-230. Williams, A.M., Fryer, J.L. and Collins, M.D. (1990) Lactococcus piscium sp. nov., a new Lactococcus species from salmonid fishes. FEMS Microbiol. Lett. 68, 109-114.