Occurrence of Photobacterium damselae subsp. damselae in bivavlve molluscs from Northwest Spain

Photobacterium damselae subsp. damselae has been recognized as pathogen for poikilotherm and homeotherm organims. In this study we describe for the first time the isolation of this microorganism from Mytilus galloprovincialis in harvesting areas situated in the northwestern coast of Spain. The identity of the isolates as Ph. damselae subsp. damselae was confirmed by biochemical tests and multiplex PCR technique. Interestingly, the absence of gas production from glucose in the bivalve mollusc strains indicated that the importance of this biochemical trait for the presumptive identification of this microorganism need to be reevaluated. The family Vibrionaceae comprises bacteria inhabiting aquatic enviroments, especially marine and estuarine waters, where they are frecuently associated with organims ranging from plankton to fish. Currently, this family includes, among others, the marine genera Vibrio (Baumann et al., 1984) and Photobacterium (Baumann & Baumann, 1981). Within the genus Photobacterium, Ph. damselae subsp. damselae (formerly Vibrio damsela) is an halophilic bacterium that has been described as the causative agent of wound infections and fatal disease in a variety of marine animals and human (Grimes et al, 1984, Mc Garey et al, 1990). In fact, since the first isolation of Ph.damselae subsp. damselae as the causative organism of skin ulcers on the damselfish Chromis punctipinnis (Love et al., 1981), this bacterium has also been recognized as a pathogen for the others fish, such as turbot, seabream, seabass, yellowtail and sharks, reptiles, molluscs, and crustacean species ( Fouz et al. , 1992). However, to our knowledgment, no reports were published on the isolation of this microorganim from molluscs for direct consumption. The present work represents the first isolation and characterization of Ph. damselae subsp. damselae isolated from bivalve molluscs in harvesting areas situated in the northwest coast of Spain which could be regarded as a potential public health hazard. Sampling and Microbiological analysis: A total 153 samples of alive bivalve molluscs were taken from 30 points of harvesting areas situated in five “Rías” along the coast of Galicia (Northwest Spain). About 1 kg of shellBull. Eur. Ass. Fish Pathol., 23(1) 2003, 41 fish was placed in a sterile plastic bag for microbiological analysis. When the samples arrived to the laboratory, the molluscs were extracted from the bags and washed under sterile running water and subjected to bacteriological analysis. The presence of Ph. damselae subsp.damselae was determinated according to the standard Methods (DePaola & Kaisner, 2001). Fifty grams of the shell liqueur and meat of molluscs were collected into a sterile jar, added to 450 ml of phosphate buffered saline (PBS, pH 7.2) to make 1:10 dilution and blended for 90 s. Shellfish homogenates were added to peptone alkaline water plus 2% NaCl (APSW) in the three tube MPN enrichment technique. The tubes were incubated at 37oC for 24h, and then subcultured in thiosulphate citrate bile sucrose agar plates (TCBS, Oxoid). After incubation at 37oC for 24 hours, at least three of each type of sucrose negative colonies (if present), were picked and identified. The identification of colonies recovered from TCBS plates as presumptive Ph. damselae, was conducted following conventional plate and tube test procedures (Fouz, et al., 1992 ), as well as by API-20E systems. The taxonomical confirmation of the strains at subspecies level was carried out by molecular techniques using a multiplex PCR assay (Osorio et al., 2000). The primers Car1 and Car2 directed to internal region of the 16S rRNA and the primers Ure-5’ and Ure-3’ designed for ureC gene amplification, were used in conjunction. The amplification conditions were: 95oC for 4 min followed by 30 cycles consisted of denaturation at 95oC for 1min, annealing at 60oC for 1min, and elongation at 72oC for 40 s. Quantification of Ph. damselae subsp. damselae present in the positive samples was conducted by MPN tables (de Man, 1983). Although Ph. damselae subsp. damselae has been detected in some turbot farms in Spain (Fouz et al.,1991), it had not been detected until now in bivalve molluscs harvested for human comsumption. According the results of this study the prevalence of Ph. damselae subsp. damselae in molluscs can be considered low because from the 153 shellfish samples examined, only two of them were positive (Table 1). One sample was obtained from “Ria” de Arosa in February and the other from “Ria” de Vigo in March. The concentration of this bacterium in positive samples estimated by the MPN technique was 36 and 92 MPN/ 100g, respectively. Biochemical tests conducted in representative isolates from both positive samples indicated that they can be considered presumptively as Photobacterium damselae. The strains were gram negative motile rods, oxidase positive, fermentative, sensitive to the vibriostatic agent, produced green colonies on TCBS agar, and displayed urease activity (Table 2). Moreover, the patterns obtained when we used the API-20E system developed the typical profile number of Ph. damselae (6014004). a i R . o N d e z y l a n a e a l e s m a d . h P e a l e s m a d . p s b u s e v i t i s o P g 0 0 1 / N P M