Photobacterium damselae subsp. damselae, an Emerging Fish Pathogen in the Black Sea: Evidence of a Multiclonal Origin

Abstract

Photobacterium damselae subsp. damselae is considered to be an emerging pathogen of marine fish of importance in aquaculture, with a notable increase in its geographical distribution during the last several years. In this study, we carried out for the first time to our knowledge a genetic and pathobiological characterization of 14 strains isolated from sea bass (Dicentrarchus labrax) reared in the Southeastern Black Sea, where high mortalities were observed at two aquaculture farms during the summer and autumn of 2011. Heterogeneity was evidenced among strains in phenotypical traits, such as sucrose fermentation, motility, and hemolysis. Although 11 of 14 isolates were hemolytic, we found that all of the isolates lacked the pPHDD1 virulence plasmid that encodes the phospholipase-D damselysin (Dly) and the pore-forming toxin PhlyP, two hemolysins previously reported to constitute major virulence factors for turbot. Subsequent PCR and sequencing analyses demonstrated that the 11 hemolytic isolates harbored a complete hlyA ch gene, a chromosome I-borne gene that encodes HlyA ch hemolysin, whereas the three nonhemolytic isolates contained hlyA ch pseudogenes caused by insertion sequence elements. Virulence challenges with two representative strains revealed that, albeit less virulent than the pPHDD1-harboring strain RM-71, the plasmidless hlyA ch -positive and hlyA chnegative Black Sea isolates were pathogenic for sea bass. A phylogenetic analysis based on the toxR gene sequence uncovered a greater diversity in the isolates, indicating that the presence of this pathogen in the Black Sea was not caused by the introduction and spread of a single virulent clone but by the proliferation of different clones. IMPORTANCEThe geographical distribution of marine bacterial pathogens is undergoing a worldwide increase. In particular, bacteria of the group vibrios are increasingly being isolated as the causative agents of disease in novel species of cultivated fish in areas where they had not been previously reported. Here we characterize for the first time to our knowledge a collection of isolates of the fish and human pathogen Photobacterium damselae subsp. damselae from diseased sea bass reared in the Black Sea. We uncovered great genetic diversity in the Black Sea isolates of this pathogen, suggesting a multiclonal origin. We also demonstrate for the first time that these isolates bear pathogenic potential for sea bass cultures by virulence challenges. P hotobacterium damselae subsp. damselae is a marine bacterium of the family Vibrionaceae that is recognized as a pathogen for a wide variety of aquatic animals, including fish, molluscs, and crustaceans. In addition, it is a pathogen of concern for humans, as it is capable of causing fatal infections (1). Most of the reported infections in humans originated from wounds inflicted during the handling of fish and fishing tools or from exposure to marine animals or seawater. Notably, it is a primary pathogen of fish species of economical importance in aquaculture. During ...

Chromosome-Encoded Hemolysin, Phospholipase, and Collagenase in Plasmidless Isolates of Photobacterium damselae subsp. damselae Contribute to Virulence for Fish

“…1). Although previous studies reported that plasmidless strains show virulence for a variety of fish species (7,17), our study constitutes evidence that hlyA ch (the gene that encodes PhlyC) is a major contributor to virulence of plasmidless strains.…”

“…P. damselae subsp. damselae is being isolated as a causative agent of disease in novel species of cultivated fish, as well as in fish from areas where it had not been previously reported, such as Egypt (19), Tunisia (20), and the Black Sea (7). The majority of P. damselae subsp.…”

“…The majority of P. damselae subsp. damselae strains isolated from outbreaks in aquaculture farms lack pPHDD1 (6,7,11), a virulence plasmid present in highly pathogenic P. damselae subsp. damselae isolates (9).…”

“…species include, among others, turbot (Scophthalmus maximus) (4), sea bream (Sparus aurata) (5), and sea bass (Dicentrarchus labrax) (6,7). The recent reports on the isolation of this pathogen from newly cultured species led to its consideration as an emerging pathogen in marine aquaculture (8).…”

“…However, the vast majority of the P. damselae subsp. damselae isolates from recent outbreaks in aquaculture farms do not produce Dly and PhlyP toxins and have been isolated mainly from sparid fish and from sea bass (6,7,11). Therefore, there is a need to investigate the genetic basis of virulence in strains that do not produce Dly and PhlyP.…”