First isolation of Staphylococcus epidermidis from cultured gilthead sea bream ( Sparus aurata ) in Turkey

This study describes the first isolation of Staphylococcus epidermidis from gilthead sea bream (Sparus aurata), reared in the southwest of Turkey. During the spring of 2003 (April-May), the infection appeared in juvenile gilthead sea bream (3-5 g) in a net cage. The outbreak occurred after a sudden increase of water temperature in the middle of April. The fish losses were up to 12 % in one day. Affected fish showed haemorrhages on the fins and gills, the liver was anaemic and the abdomen was slightly distended as a result of ascitic fluid in the abdominal cavity. The gram-positive S. epidermidis was isolated from the anterior kidney, spleen and liver of sick fish. S. epidermidis was identified by morphological, physiological and biochemical features, using conventional methods and the ID 32 Staph system. Introduction Staphylococcus epidermidis has been reported previously as a fish pathogen in some marine and freshwater fish in Japan, Taiwan and Greece (Kusuda and Sugiyama 1981; Wang et al., 1996; Varvarigos, 2001). Several severe epizootics have been described in farmed fish i. e. in red sea bream (Chrysophrys major) and yellowtail (Seriola quinqueradiata) in Japan (Kusuda and Sugiyama, 1981); grass carp (Ctenopharyngedon idella) (Wang et al., 1996) and tilapia (Oreochromis spp.) in Taiwan (Huang et al., 1999) and sea bream (Sparus aurata) and sea bass (Dicenthrarchus labrax) at different fish farm locations in Greece (Varvarigos, 2001). Sugiyama and Kusuda (1981) thought that the bacteria originated from water or fish rather than from human beings, because of the pronounced antigenic differences when compared to human strains of S. epidermidis. This suggestion is supported by ecological studies, which have clearly demonstrated the presence of S. epidermidis in the aquatic environment (Gunn et al., 1982, Austin and Austin 1999). Staphylococci may be present in the fish throughout the year, but the disease is induced by a sudden rise in water temperatures or other stress factors in the aquatic environment. It usually appears in the spring and causes problems throughout the summer (Varvarigos, 2001). Staphylococcal infections in fish were also observed by Fryer and John, (1993) during specific and severe stress imposed on the fish by the enviBull. Eur. Ass. Fish Pathol. , 24(3) 2004, 138 ronment. In fish, typical signs of staphylococcal infections are exophthalmia, congestion and ulceration on the tail (Kusuda and Sugiyama, 1981). Staphylococcal infections involve systemic disease characterized by septicaemia (Varvarigos, 2001). This study describes isolation of S. epidermidis in juvenile gilthead sea bream (Sparus aurata) in a net cage farm, located in the coastal region of the Aegean Sea in Turkey. Materials and Methods Sampling The outbreak was restricted to a single net cage farm with a total of twenty-eight cages in the Aegean Sea. The disease was observed in six net cages of juvenile gilthead sea bream (S. aurata) during the spring, from April to May 2003. Moribund, diseased fish ranging in size from 3-5g were collected from the farm and transported to the Fish Disease Laboratory in Egirdir Fisheries Faculty at Süleyman Demirel University. Isolation and Identification of Bacteria For bacterial isolation, samples were taken from the kidney, spleen and liver of each fish and streaked on trypticase soy agar (TSA, Merck). The plates were incubated at 30oC for 48-72 hours. Based on the morphology, only one type of colony growth could be determined after 72 hours in all samples. Pure cultures of the isolates were obtained on TSA. Routine tests for determination of biochemical characteristics of the bacteria were carried out as described in Baird-Parker (1963,1965a, b), Cowan and Steel (1970), Collins and Lyne (1976), Kinkelin et al. (1985) and NCCLS (2001). A presumptive identification of the strain was performed by Gram staining, catalase, oxidase activity, motility, resistance to O/ 129 vibriostat and the glucose oxidation-fermentation test. The strain was identified by testing several biochemical reactions as described in Bergery’s Manual (Holt et al. 1994). The haemolytic ability of the bacteria was determined by growth on 5% sheep blood agar. For detection of the coagulase, rabbit plasma with lyophilized-EDTA (Merck) was used. Samples were also plated on Brain heart infusion agar (Difco), Baird Parker’s agar (Merck) and Mannitol salt phenol red agar (Merck) and incubated at 30oC. To determine the physiological characteristics of the bacteria, samples were inoculated into trypticase soy broth (TSB, Merck) and growth was determined at 4oC and at 45oC. The salt requirements/tolerance of the strain was determined in TSB containing 0 % and 15% NaCl. The ID 32 Staph system test strips (Biomerieux SA/ 69280 Marcy-l’ Etiole, France) were also used for bacteriological diagnosis. Antimicrobial Sensitivity Antibiogram tests for the isolate were performed using the disc diffusion techniques on Mueller Hinton agar (Oxoid). NCCLS standards were used for evaluation of the results. Also, ATB_VET (14 289 Biomerieux) strip system was used to obtain the antibiotic sensitivity of the isolated strain. Results and Discussion The external symptoms of the infected fish were congestion and haemorrhages on the pectoral and caudal fins, dark coloration of the skin, lethargy, and excessive secretion of mucus on the skin and gills. The mouth and lower jaw were haemorrhagic and the gills were anaemic. Internal symptoms included ascitic fluid in the abdominal cavity, with an Bull. Eur. Ass. Fish Pathol. , 24(3) 2004, 139 (+): positive reaction; (-): negative reaction. Table 1: Phenotypic characteristics of isolated Staphylococcus epidermidis. s c i t s i r e t c a r a h C e s n o p s e R s c i t s i r e t c a r a h C e s n o p s e R t n e m g i p y n o l o C e t i h W : m o r f n o i t c u d o r p d i c A ) m m ( A S T n o r e t e m a i d y n o l o C 1 e s o c u l G + n i a t S m a r G + l o t i n n a M y g o l o h p r o M i c c o C l a c i r e h p S l o t i s o n I y t i l i t o M l o t i b r o S ) F O ( n o i t a t n e m r e F n o i t a d i x O F e s o n m a h R 9 2 1 / O o t y t i v i t i s n e S e s o r c u S + : f o n o i t c u d o r P e s o i b i l e M e s a l a t a C + n i l a d g y m A e s a d i x O e s o n i b a r A ) a m s a l p t i b b a r ( e s a l u g a o C e s o r a h c c a S + e s a d i s o t c a l a g ß e s o t c a L +