A survey of antibiotic-producing bacteria from the microbial flora attached to seaweeds and the study of their antibiotic capacities were carried out. From 5 species of green and brown marine algae, 224 bacterial strains were isolated and tested for antibiotic production. A total of 38 strains displayed antibiotic activity, withEnteromorpha intestinalis being the source of the highest number of producer strains. All epiphytic bacteria with antibiotic activity were assigned to thePseudomonas-Alteromonas group. Antagonism assays among the isolates demonstrated that each producer strain inhibits the growth of the other producers, as well as of some nonproducer strains also isolated from seaweeds. Likewise, an autoinhibitory effect was observed in all antibiotic-producing strains. Antibacterial spectra of all the strains include activity againstStaphylococcus, Alcaligenes, Pseudomonas, Vibrio, Pasteurella, andAchromobacter. A preliminary characterization of the antibiotic substances produced by these epiphytic bacteria demonstrated that they are low molecular weight compounds, thermolabile, and anionic and are not affected by proteolytic enzymes. The role that these inhibitory substances can play in the natural environment is discussed.

Two episodes of mortality of cultured carpet shell clams (Ruditapes decussatus) associated with bacterial infections were recorded during 2001 and 2002 in a commercial hatchery located in Spain. Vibrio alginolyticus was isolated as the primary organism from moribund clam larvae that were obtained during the two separate events. Vibrio splendidus biovar II, in addition to V. alginolyticus, was isolated as a result of a mixed Vibrio infection from moribund clam larvae obtained from the second mortality event. The larval mortality rates for these events were 62 and 73%, respectively. Mortality was also detected in spat. To our knowledge, this is the fist time that these bacterial species have been associated with larval and juvenile carpet shell clam mortality. The bacterial strains were identified by morphological and biochemical techniques and also by PCR and sequencing of a conserved region of the 16S rRNA gene. In both cases bacteria isolated in pure culture were inoculated into spat of carpet shell clams by intravalvar injection and by immersion. The mortality was attributed to the inoculated strains, since the bacteria were obtained in pure culture from the soft tissues of experimentally infected clams. V. alginolyticus TA15 and V. splendidus biovar II strain TA2 caused similar histological lesions that affected mainly the mantle, the velum, and the connective tissue of infected organisms. The general enzymatic activity of both live cells and extracellular products (ECPs), as evaluated by the API ZYM system, revealed that whole bacterial cells showed greater enzymatic activity than ECPs and that the activity of most enzymes ceased after heat treatment (100°C for 10 min). Both strain TA15 and strain TA2 produced hydroxamate siderophores, although the activity was greater in strain TA15. ECPs from both bacterial species at high concentrations, as well as viable bacteria, caused significant reductions in hemocyte survival after 4 h of incubation, whereas no significant differences in viability were observed during incubation with heat-killed bacteria.Culture of carpet shell clams (Ruditapes decussatus) is a traditional activity that has great economical importance in Spain, particularly in Galicia (northwest region of Spain). Therefore, losses in production of this clam species would seriously affect the economy of this region.Globally, clam production is often affected by vibriosis, which leads to high mortality rates mainly in nursery cultures of juvenile bivalves (20,35). In Spain serious mass mortalities associated with Vibrio tapetis infections have been reported previously (15, 21). V. tapetis causes brown ring disease in Ruditapes species, which is characterized by the appearance of brown conchioline deposits that have variable distributions and variable thicknesses on the inner shell of diseased clams (41, 42).Susceptibility of other cultured bivalve species to infections caused by bacteria belonging to the genus Vibrio has been found in several scallop species, including Aequipecten irradians (52), Euvo...

We describe in this work a new iron uptake system encoded by chromosomal genes in pathogenic strains of Vibrio anguilarum. This iron uptake system differs from the plasmid-encoded anguibactin-mediated system present in certain strains of V. anguilarum in several properties. The siderophore anguibactin is not utilized as an external siderophore, and although characteristic outer membrane proteins are synthesized under iron-limiting conditions, these are not related to the plasinid-mediated outer membrane protein OM2 associated with ferric anguibactin transport. Furthermore, the siderophore produced by the plasmidless strains may be functionally related to enterobactin as demonstrated by bioassays with enterobactin-deficient mutants, although its behavior under various chemical treatments suggested major differences from that siderophore. Hybridization experiments suggested that the V. anguillarum chromosome-mediated iron uptake system is unrelated genetically to either the anguibactin or enterobactin-associated iron assimilation systems.One of the most important virulence factors in many pathogenic bacteria is the ability to utilize iron-from the host fluids by means of an efficient iron-sequestering system (5,8,18,22,34). Vibrio anguillarum is a bacterium highly pathogenic for different species of marine fish, causing the disease known as vibriosis (6,15,25). In strain 775, isolated from the Pacific Northwest coast of the United States, an iron uptake system is mediated by a 65-kilobase plasmid designated pJM1 (7,11,13). This system allows bacteria to grow at low concentrations of available iron imposed by the high-affinity iron-binding proteins present in the host fluids. Experimental infections demonstrated that possession of the pJM1 iron uptake genetic determinants is essential to cause disease (7,11,33,35).Genetic characterization of the pJM1-mediated iron uptake system allowed the location of the iron uptake genes to a 25-kilobase region of the pJM1 plasmid (12,29,33). These genes determine the synthesis of the two essential components involved in the iron uptake process: the diffusible siderophore anguibactin (1, 33) and a component of the receptor for the iron-siderophore complex identified as the 86-kilodalton outer membrane protein OM2 (2, 10). Other pathogenic strains of V. anguillarum isolated from cultured turbot on the northwestern Atlantic coast of Spain also harbor a plasmid which exhibits a high structural and functional homology with pJM1 (28). However, in recent studies with other virulent strains of V. anguillarum that were also iron uptake proficient, ho plasmids have been detected, suggesting that the iron uptake system of these strains must be chromosomally encoded (30).In the present work we report the characterization of this chromosome-mediated iron uptake system present in certain plasmidless strains of V. anguillarum. Our results indicate that this new system is different from the one encoded by the pJM1-like plasmids. * Corresponding author. MATERIALS AND METHODSBacterial strains. Th...

Photobacterium damselae subsp. damselae (formerly Vibrio damsela) is a pathogen of a variety of marine animals including fish, crustaceans, molluscs, and cetaceans. In humans, it can cause opportunistic infections that may evolve into necrotizing fasciitis with fatal outcome. Although the genetic basis of virulence in this bacterium is not completely elucidated, recent findings demonstrate that the phospholipase-D Dly (damselysin) and the pore-forming toxins HlyApl and HlyAch play a main role in virulence for homeotherms and poikilotherms. The acquisition of the virulence plasmid pPHDD1 that encodes Dly and HlyApl has likely constituted a main driving force in the evolution of a highly hemolytic lineage within the subspecies. Interestingly, strains that naturally lack pPHDD1 show a strong pathogenic potential for a variety of fish species, indicating the existence of yet uncharacterized virulence factors. Future and deep analysis of the complete genome sequence of Photobacterium damselae subsp. damselae will surely provide a clearer picture of the virulence factors employed by this bacterium to cause disease in such a varied range of hosts.

From the cultures of Photobacterium damselae subsp. piscicida, the aetiological agent of fish pasteurellosis, a new siderophore named piscibactin (1), was isolated as its gallium and iron(III) complexes along with a possible intermediate of its biosynthesis, prepiscibactin (2). Analysis of the gene cluster involved in the siderophore biosynthesis allowed the partial prediction of the structures. Thus, an NRPS‐mediated mechanism similar to that for yersiniabactin was suggested by protein sequence comparisons. The final structures were solved by NMR and MS methods and by DFT molecular modeling. The results obtained in the structural and functional characterization of piscibactin enabled the proposal of a biosynthetic pathway.

Photobacterium damselae subsp. damselae causes infections and fatal disease in marine animals and in humans. Highly hemolytic strains produce damselysin (Dly) and plasmid-encoded HlyA (HlyA pl ). These hemolysins are encoded by plasmid pPHDD1 and contribute to hemolysis and virulence for fish and mice. In this study, we report that all the hemolytic strains produce a hitherto uncharacterized chromosome-encoded HlyA (HlyA ch ). Hemolysis was completely abolished in a single hlyA ch mutant of a plasmidless strain and in a dly hlyA pl hlyA ch triple mutant. We found that Dly, HlyA pl , and HlyA ch are needed for full hemolytic values in strains harboring pPHDD1, and these values are the result of the additive effects between HlyA pl and HlyA ch , on the one hand, and of the synergistic effect of Dly with HlyA pl and HlyA ch , on the other hand. Interestingly, Dly-producing strains produced synergistic effects with strains lacking Dly production but secreting HlyA, constituting a case of the CAMP (Christie, Atkins, and Munch-Petersen) reaction. Environmental factors such as iron starvation and salt concentration were found to regulate the expression of the three hemolysins. We found that the contributions, in terms of the individual and combined effects, of the three hemolysins to hemolysis and virulence varied depending on the animal species tested. While Dly and HlyA pl were found to be main contributors in the virulence for mice, we observed that the contribution of hemolysins to virulence for fish was mainly based on the synergistic effects between Dly and either of the two HlyA hemolysins rather than on their individual effects.

The activity of antibiotic-producing marine bacteria was assayed against bacterial fish pathogens belonging to the genera Vibrio, Aeromonas, Pasteurella, Edwardsiella, Yersinia and Pseudomonas with the aim of evaluating the possible use of these marine strains for controlling epizootics in aquaculture. Inhibition tests on solid medium showed that, in general, the majority of fish bacteria were strongly sensitive to the marine bacteria. Only two strains (Edwardsiella tarda and Pseudomonas aeruginosa), were resistant to all the antibiotic-producing strains. The results of antagonism assays in sea water, however, varied according to the fish pathogens examined. Experiments conducted using cell-free supernatant fluids of marine bacteria demonstrated the involvement of antibiotic substances in the inhibition of fish pathogens.

Vibrio anguillarum causes vibriosis, a hemorrhagic septicaemia that affects many cultured marine fish species worldwide. Two catechol siderophores, vanchrobactin and anguibactin, were previously identified in this bacterium. While vanchrobactin is a chromosomally encoded system widespread in all pathogenic and environmental strains, anguibactin is a plasmid-encoded system restricted to serotype O1 strains. In this work, we have characterized, from a serotype O2 strain producing vanchrobactin, a novel genomic island containing a cluster of genes that would encode the synthesis of piscibactin, a siderophore firstly described in the fish pathogen Photobacterium damselae subsp. piscicida. The chemical characterization of this siderophore confirmed that some strains of V. anguillarum produce piscibactin. An in silico analysis of the available genomes showed that this genomic island is present in many of the highly pathogenic V. anguillarum strains lacking the anguibactin system. The construction of single and double biosynthetic mutants for vanchrobactin and piscibactin allowed us to study the contribution of each siderophore to iron uptake, cell fitness, and virulence. Although both siderophores are simultaneously produced, piscibactin constitute a key virulence factor to infect fish, while vanchrobactin seems to have a secondary role in virulence. In addition, a transcriptional analysis of the gene cluster encoding piscibactin in V. anguillarum showed that synthesis of this siderophore is favored at low temperatures, being the transcriptional activity of the biosynthetic genes three-times higher at 18°C than at 25°C. We also show that iron levels and temperature contribute to balance the synthesis of both siderophores.