Jean-Louis Nicolas

Bacterial flora associated with a trophic chain consisting of microalgae, rotifers and turbot larvae: Influence of bacteria on larval survival

Abstract In order to increase knowledge about the bacteria in cultures and their influence on the culture performance, bacterial analyses and physical and chemical measures were carried out for different biotopes of two similar trophic chains (A and B). These trophic chains consisted of microalgae ( Pavlova lutheri or Platymonas suecica ), rotifers ( Brachionus plicatilis ) and turbot larvae ( Scophthalmus maximus ). About 10 7 bacteria/ml were regularly observed by direct counts in the culture water of the algae and rotifers, and from 10 4 to 10 5 bacteria/individual in rotifers and turbot larvae. In general, the composition of the bacterial flora presented some elements of stability, since some taxa were found in successive samples of the same biotope. On the other hand, there were very few similarities between the bacterial flora of different biotopes, except between the gut of the rotifers and their medium. Nevertheless, among the Vibrionaceae, which were the chief constituent of the bacterial flora in the gut of the turbot larvae, some were probably introduced by the rotifers. The numbers of Vibrionaceae in both rotifer cultures increased over the course of the experiments. The turbot larvae B refused to ingest prey which contained large quantities of bacteria and died on days 7 and 8, whereas 40–60% of larvae A remained alive at day 10. The conclusions drawn from this experiment can be applied to improving the survival and growth of larvae as well as to developing sanitary procedures for hatcheries.

Evidence for the Involvement of Pathogenic Bacteria in Summer Mortalities of the Pacific Oyster Crassostrea gigas

A study was conducted to investigate the involvement of bacteria in oyster mortalities during summer. Moribund and apparently healthy oysters were sampled during mortality events along the French coast and in rearing facilities, usually when temperature reached 19°C or higher, and oysters were in the gonadal maturation phase. Hemolymph samples were aseptically withdrawn and submitted to bacteriological analysis. In healthy oysters, bacteria colonized hemolymph at low concentrations depending on the location. In most moribund oysters, bacteria were present in hemolymph and other tissues. These bacterial populations were more often diverse in oysters originating from the open sea than from facilities where animals were generally infected by a single type of bacterium. Only the dominant colonies were identified by phenotypic and genotypic characters (RFLP of GyrB gene and partial sequence of 16S rRNA gene). They belonged to a limited number of species including Vibrio aestuarianus, members of the V. splendidus group, V. natriegens, V. parahaemolyticus, and Pseudoalteromonas sp. The most frequently encountered species was V. aestuarianus (56% of isolates), which was composed of several strains closely related by their 16S rRNA gene but diverse by their phenotypic characters. They appeared intimately linked to oysters. The species within the V. splendidus group were less prevalent (25% of isolates) and more taxonomically dispersed. A majority of the dominant strains of V. aestuarianus and V. splendidus group injected to oysters induced mortality, whereas others belonging to the same species, particularly those found in mixture, appeared innocuous.

Roseobacter gallaeciensis sp. nov., a new marine bacterium isolated from rearings and collectors of the scallop Pecten maximus

Four bacterial strains were isolated from larval cultures and collectors of the scallop Pecten maximus. They showed a high level of intragroup genomic relatedness (84-95%) as determined by DNA-DNA hybridization. The cells were Gram-negative, strictly aerobic, motile, ovoid rods. They grew at temperatures from 15 to 37 degrees C and from pH 7.0 to 10, but did not grow in the absence of NaCl and required growth factors. They had the ability to use a wide variety of compounds as sole carbon source: D-mannose, D-galactose, D-fructose, D-glucose, D-xylose, melibiose, trehalose, maltose, cellobiose, sucrose, mesoerythritol, D-mannitol, glycerol, D-sorbitol, meso-inositol, succinate, propionate, butyrate, gamma-aminobutyrate, DL-hydroxybutyrate, 2-ketoglutarate, pyruvate, fumarate, glycine, L-alpha-alanine, beta-alanine, L-glutamate, L-arginine, L-lysine, L-ornithine and L-proline. They exhibited oxidase and catalase activities but no denitrification activity. The isolates did not contain bacteriochlorophyll a. The G + C content ranged from 57.6 to 58 mol%. Phylogenetic analyses of the 16S rRNA sequence revealed that these isolates belong to the genus Roseobacter. On the basis of quantitative hybridization data, it is proposed that these isolates should be placed in a new species, Roseobacter gallaeciensis. The type strain is Roseobacter gallaeciensis BS107T (= CIP 105210T).

The Benefit of a Roseobacter Species on the Survival of Scallop Larvae

Abstract A marine strain (BS107), identified as a Roseobacter species, was antagonistic to Vibrio species on agar plates. Results suggested that the inhibitory effect was displayed only in the presence of another bacterium. Quantification of the antibacterial activity showed that 48-hour-coculture supernatants from BS107 and another bacterial strain (V. anguillarum 408) reached the highest titers of bacterial inhibition. The antibacterial substance was also liberated when supernatants from V. anguillarum 408 were added to pure cultures of the inhibition-productive bacterium. The presence of a proteinaceous molecule may induce BS107 to display the inhibitory effect. The antibacterial substance was sensitive to trypsin (8000 U/ml) and stable at 100°C. Cell extracts of the isolate BS107 (106 cells/ml) significantly enhanced scallop larval survival, thus being beneficial to the rearing process.

Comparative analysis of Vibrio splendidus-related strains isolated during Crassostrea gigas mortality events.

French mollusc production is based mainly on the Pacific cupped oyster, Crassostrea gigas. Since 1991, annual mass mortality of juveniles has been reported during summer months. These recurring episodes concern professionals who fear that like Portugese oyster, C. angulata, C. gigas could in turn disappear following one of these epizooties. Previously, bacteriological analysis of moribund oyster juveniles yielded an isolate of a Vibrio splendidus biovar II strain, named TNEMF6. This isolate was demonstrated to be pathogenic to Crassostrea gigas spat by experimental challenge. To study the association between summer oyster mortality and presence of TNEMF6 cluster strains, Vibrionaceae fauna were isolated from infected spat along the French Atlantic coast between 1997-1998. Strains related to V. splendidus biovar II were selected. Comparison with TNEMF6 was performed by classical biochemical tests and polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP) of SSU rDNA, rpoD, and gyrB genes. Genomic similarities were confirmed by DNA/DNA hybridization. Only one strain out of 14, TNNIII7, was found to be closely related to the pathogenic bacteria. Neither the phenotypic nor the genotypic markers used in this study were able to distinguish pathogenic from non-pathogenic strains of the widespread V. splendidus. However, future genetic comparisons of TNEMF6 and TNNIII7 is likely to reveal genes involved in pathogenicity.

Vibrio pectenicida sp. nov., a pathogen of scallop (Pecten maximus) larvae.

Five strains were isolated from moribund scallop (Pecten maximus) larvae over 5 years (1990-1995) during outbreaks of disease in a hatchery (Argenton, Brittany, France). Their pathogenic activity on scallop larvae was previously demonstrated by experimental exposure. The phenotypic and genotypic features of the strains were identical. The G + C content of the strains was in the range 39-41 mol%. DNA-DNA hybridization showed a minimum of 73% intragroup relatedness. Phylogenetic analysis of small-subunit rRNA sequences confirmed that these strains should be affiliated within the family Vibrionaceae and that they are closely related to Vibrio tapetis and Vibrio splendidus. Phenotypic and genotypic analyses revealed that the isolates were distinct from these two vibrios and so constitute a new species in the genus Vibrio. They utilized only a limited number of organic substrates as sole carbon sources, including betaine and rhamnose, but did not utilize glucose and fructose. In addition, their responses were negative for indole, acetoin, decarboxylase and dihydrolase production. The name Vibrio pectenicida is proposed for the new species; strain A365 is the type strain (= CIP 105190T).

Purification and Partial Identification of Novel Antimicrobial Protein from Marine Bacterium Pseudoalteromonas Species Strain X153

A marine bacterium, X153, was isolated from a pebble collected at St. Anne du Portzic (France). By 16S ribosomal DNA gene sequence analysis, X153 strain was identified as a Pseudoalteromonas sp. close to P. piscicida. The crude culture of X153 was highly active against human pathogenic strains involved in dermatologic diseases, and marine bacteria including various ichthyopathogenic Vibrio strains. The active substance occurred both in bacterial cells and in culture supernatant. An antimicrobial protein was purified to homogeneity by a 4-step procedure using size-exclusion and ion-exchange chromatography. The highly purified P-153 protein is anionic, and sodium dodecylsulfate polyacrylamide gel electrophoresis gives an apparent molecular mass of 87 kDa. The X153 bacterium protected bivalve larvae against mortality, following experimental challenges with ichthyopathogenic Vibrio. Pseudoalteromonas sp. X153 may be useful in aquaculture as a probiotic bacterium.

Metabolites from the Sponge-Associated Bacterium Pseudomonas Species

Abstract: Quinolones and a phosphatidyl glyceride were isolated from the sponge-associated bacterial strain Pseudomonas sp. Structures were elucidated by spectroscopic analysis and chemical transformations.

Caractérisation de bactéries pathogènes de naissain d'huître creuse Crassostrea gigas

The French mollusc production is mainly based on the Pacific cupped oyster, Crassostrea gigas. Since 1991, outbreaks of mass mortality of juveniles are reported during the summer period. These outbreaks are a major concern of oyster industry. Several studies have established given bacterial strains to be pathogenic for bivalve species, including oysters. Here we present a study of mortality outbreaks of C. gigas, as initiated in 1995. In a first step, bacterial strains were isolated during mass mortality outbreak and were biochemically characterised. Among the isolated strains, some strains of Vibrio splendidus biovar II were found to be pathogenic by means of experimental challenge of oyster juveniles. In the second step, a genotypical identification of the pathogenic strain was undertaken, based on 16S RNA sequences and phylogenetic analysis. It confirmed that the pathogenetic strain belonged to Vibrio splendidus biovar II.

Integrative Study of Physiological Changes Associated with Bacterial Infection in Pacific Oyster Larvae

Background Bacterial infections are common in bivalve larvae and can lead to significant mortality, notably in hatcheries. Numerous studies have identified the pathogenic bacteria involved in such mortalities, but physiological changes associated with pathogen exposure at larval stage are still poorly understood. In the present study, we used an integrative approach including physiological, enzymatic, biochemical, and molecular analyses to investigate changes in energy metabolism, lipid remodelling, cellular stress, and immune status of Crassostrea gigas larvae subjected to experimental infection with the pathogenic bacteria Vibrio coralliilyticus. Findings Our results showed that V. coralliilyticus exposure induced (1) limited but significant increase of larvae mortality compared with controls, (2) declined feeding activity, which resulted in energy status changes (i.e. reserve consumption, β-oxidation, decline of metabolic rate), (3) fatty acid remodeling of polar lipids (changes in phosphatidylinositol and lysophosphatidylcholine composition`, non-methylene–interrupted fatty acids accumulation, lower content of major C20 polyunsaturated fatty acids as well as activation of desaturases, phospholipase and lipoxygenase), (4) activation of antioxidant defenses (catalase, superoxide dismutase, peroxiredoxin) and cytoprotective processes (heat shock protein 70, pernin), and (5) activation of the immune response (non-self recognition, NF-κκ signaling pathway, haematopoiesis, eiconosoids and lysophosphatidyl acid synthesis, inhibitor of metalloproteinase and antimicrobial peptides). Conclusion Overall, our results allowed us to propose an integrative view of changes induced by a bacterial infection in Pacific oyster larvae, opening new perspectives on the response of marine bivalve larvae to infections.