Sabela Balboa

Diversity and pathogenecity of Vibrio species in cultured bivalve molluscs.

Shellfish production is seriously affected by bacterial pathogens that cause high losses in hatcheries and in the aquaculture sector. A number of Vibrio species are considered important pathogens and have provoked severe mortality outbreaks. The pathologies caused by vibrios in bivalves have been described since the 1960s; however, over recent years, successive episodes of high mortality have been recorded due to these microorganisms. The present work provides an updated overview of the different studies performed in relation with the diversity of Vibrio spp. associated to bivalves. Special attention is given to the main Vibrio diseases and implicated species affecting the different life stages of cultured bivalves.

Diversity of Vibrios associated with reared clams in Galicia (NW Spain)

The aim of the present study was to characterize and identify vibrios isolated from cultured clams in Galicia (NW Spain). A total of 759 isolates were obtained, phenotypically characterized, grouped and assigned to the genus Vibrio. Subsequently, the genomic diversity of 145 representative strains was analyzed by means of amplified fragment length polymorphism (AFLP), which revealed a high genetic diversity amongst these isolates. Only 57 out of 145 strains could be identified to the species level, and they were distributed in 13 AFLP clusters. V. cyclitrophicus, V. splendidus and V. alginolyticus were the most abundantly represented species. Eighty-eight isolates remained unidentified, 59 were distributed over 16 clusters, while 29 were unclustered. Sequencing of the 16S rRNA and two house-keeping genes (rpoA and recA) from representative strains belonging to eight unidentified clusters with the highest number of isolates confirmed their assignation to the Vibrionaceae family, and some of these probably represent new species within the genus. The present study confirmed that the phenotypic characterization of vibrios is not sufficient to identify them at the species level. A wide diversity of vibrios was found in cultured clams from all four geographic locations analyzed. In total, more than 12 Vibrio species and at least three potential new species in this genus were identified.

New Vibrio species associated to molluscan microbiota: a review

The genus Vibrio consists of more than 100 species grouped in 14 clades that are widely distributed in aquatic environments such as estuarine, coastal waters, and sediments. A large number of species of this genus are associated with marine organisms like fish, molluscs and crustaceans, in commensal or pathogenic relations. In the last decade, more than 50 new species have been described in the genus Vibrio, due to the introduction of new molecular techniques in bacterial taxonomy, such as multilocus sequence analysis or fluorescent amplified fragment length polymorphism. On the other hand, the increasing number of environmental studies has contributed to improve the knowledge about the family Vibrionaceae and its phylogeny. Vibrio crassostreae, V. breoganii, V. celticus are some of the new Vibrio species described as forming part of the molluscan microbiota. Some of them have been associated with mortalities of different molluscan species, seriously affecting their culture and causing high losses in hatcheries as well as in natural beds. For other species, ecological importance has been demonstrated being highly abundant in different marine habitats and geographical regions. The present work provides an updated overview of the recently characterized Vibrio species isolated from molluscs. In addition, their pathogenic potential and/or environmental importance is discussed.

Vibrio celticus sp. nov., a new Vibrio species belonging to the Splendidus clade with pathogenic potential for clams.

A group of four motile facultative anaerobic marine isolates (Rd 8.15(T) [=CECT 7224(T), =LMG 23850(T)], Rd 16.13, Rd 6.8 [=LMG 25696] and Rd2L5) were obtained from cultured clams (Ruditapes philippinarum and Venerupis pullastra) in Galicia, north-western Spain. They formed a tight phylogenetic group based on sequences of the 16S rRNA gene and the four housekeeping genes rpoA (encoding the α-chain of RNA polymerase), rpoD (encoding the sigma factor of RNA polymerase), recA (encoding RecA protein), and atpA (encoding the α-subunit of bacterial ATP synthase). The phylogenies based on these sequences indicated that the four isolates represented a novel species in the genus Vibrio, and more precisely in the Splendidus clade. DNA-DNA hybridizations with the type strains of species showing more than 98.6% 16S rRNA gene sequence similarity, revealed a DNA-DNA relatedness below 70%. The isolates could be differentiated from the phylogenetically related Vibrio species on the basis of several phenotypic features. In addition, strain Rd 8.15(T) showed potential pathogenic activity for adult clams in virulence assays. The name Vibrio celticus sp. nov. is proposed for this new taxon, with the type strain being Rd 8.15(T) (=CECT 7224(T), =LMG 23850(T)).

Vibrio atlanticus sp. nov. and Vibrio artabrorum sp. nov., isolated from the clams Ruditapes philippinarum and Ruditapes decussatus.

Facultatively anaerobic marine bacteria isolated from cultured clams, Ruditapes decussatus and Ruditapes philippinarum, were previously investigated using amplified fragment length polymorphism (AFLP) and 16S rRNA gene sequence analyses. The isolates formed two AFLP clusters and belonged to the genus Vibrio, more precisely to the Splendidus clade. In this study, phylogenetic analyses based on sequences of the housekeeping genes rpoA, rpoD, pyrH, atpA and recA supported their inclusion in that clade forming two well differentiated groups with respect to the rest of the species within the clade, and confirmed that they formed two groups, separated from the rest of the species of the clade. DNA-DNA hybridization demonstrated that the isolates constitute two novel species of the genus Vibrio, which can be phenotypically differentiated from their closest relatives. The names Vibrio atlanticus sp. nov. and Vibrio artabrorum sp. nov. are proposed, with Vb 11.11(T) ( = CECT 7223(T)  = LMG 24300(T)) and Vb 11.8(T) ( = CECT 7226(T)  = LMG 23865(T)) as the type strains, respectively.

Arcobacter lekithochrous sp. nov., a new species isolated from a molluscan hatchery in Norway.

Four bacterial strains, LFT 1.7T, LT2C 2.5, LT4C 2.8 and TM 4.6, were isolated from great scallop (Pecten maximus) larvae and tank seawater in a Norwegian hatchery and characterized by a polyphasic approach including determination of phenotypic, chemotaxonomic and genomic traits. All were Gram-stain-negative, motile rods, oxidase- and catalase-positive and required sea salts for growth. Major fatty acids present were summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), summed feature 8 (C18 : 1ω7c or C18 : 1ω6c), C16 : 0, C14 : 0, summed feature 2 (C14 : 0 3-OH/iso-C16 : 1 I), C12 : 0 3-OH and C12 : 0. Strain LFT 1.7T contained menaquinone MK-6 as the sole respiratory quinone. Phylogenetic analysis based on 16S rRNA gene sequences indicated that all strains formed a distinct lineage within the genus Arcobacter with a low similarity to known species (94.77-95.32 %). The DNA G+C content was 28.7 mol%. Results of in silico DNA-DNA hybridization and average nucleotide identity confirmed that the isolates constitute a novel species of Arcobacter, for which the name Arcobacter lekithochrous sp. nov. is proposed. The type strain is LFT 1.7T (=CECT 8942T=DSM 100870T).

Multilocus sequence analysis of Vibrio tapetis, the causative agent of Brown Ring Disease: description of Vibrio tapetis subsp. britannicus subsp. nov.

Vibrio tapetis is the causative agent of an epizootic infection described in adult clams called Brown Ring Disease (BRD). The isolation of the pathogen from different hosts showed strain variability both at serological and genetic level, allowing the description of three major groups related to the host origin of the isolates. In this work we performed for the first time a phylogenetic study for this pathogen. When including the closest related Vibrio species, all strains of V. tapetis appeared as a robust monophyletic cluster in the trees generated from all genes studied, namely 16S rRNA, atpA, fstZ, gapA, pyrH, recA, rpoA and rpoD and topA, as well as from their concatenated sequences. On the other hand, V. tapetis strains appeared clearly separated in two main clusters, sharing a similarity percentage for the concatenated sequences from 95 to 95.2% and values of DDH between 65.5 and 79.8%. Both clusters are themselves variable, with isolates within each cluster grouped according their host origin. The two clusters are easily distinguishable for their capacity to produce acid from mannitol and arabinose and for the use of citrate. Therefore, the results obtained supported the existence of two subspecies within this pathogen for which the names V. tapetis subsp. tapetis and V. tapetis subsp. britannicus subsp. nov. are proposed.

Characterization of Vibrio tapetis strains isolated from diseased cultured Wedge sole (Dicologoglossa cuneata Moreau)

The first isolation of Vibrio tapetis from Wedge sole (Dicologoglossa cuneata) is reported. The bacterium was recovered from ulcers of ailing cultured fish, from two different outbreaks occurred in spring 2005. The four isolates found (a200, a201, a204 and a255) were biochemically, genetically and serologically characterized and diagnosis was confirmed by PCR V. tapetis specific primers and multilocus sequencing analysis (MLSA). The isolates constituted a homogeneous phenotypic and genotypic group, being distinct to the already serological and genetic groups defined within the species. A virulence evaluation of the isolate a255 was also carried out; however this strain was unable to induce disease in fry and juvenile Wedge sole.

Vibrio breoganii sp. nov., a non-motile, alginolytic, marine bacterium within the Vibrio halioticoli clade

Seven non-motile, facultatively anaerobic, alginolytic marine bacteria were isolated from the cultured clams Ruditapes philippinarum and Ruditapes decussatus. Phylogenetic analysis based on 16S rRNA gene sequences showed that these marine bacteria were closely related to the recently described species Vibrio comitans, Vibrio rarus and Vibrio inusitatus (>or=99.0 % sequence similarity). Phylogenetic analysis based on the housekeeping genes rpoA, recA and atpA grouped the isolates together and allocated them to the Vibrio halioticoli species group. Amplified fragment length polymorphism DNA fingerprinting also grouped them together and enabled them to be differentiated from recognized species of the V. halioticoli clade. DNA-DNA hybridizations showed that the isolates belonged to a novel species; phenotypic features such as the ability to grow at 4 degrees C and in the presence of 6 % NaCl also enabled them to be separated from other species. The DNA G+C content of RD 15.11(T) is 44.4 mol%. The genotypic and phenotypic data showed that the isolates represent a novel species in the V. halioticoli clade. The name Vibrio breoganii sp. nov. is proposed, with RD 15.11(T) (=CECT 7222(T) =LMG 23858(T)) as the type strain.

The use of multiple typing methods allows a more accurate molecular characterization of Vibrio parahaemolyticus strains isolated from the Italian Adriatic Sea.

Vibrio parahaemolyticus is a natural inhabitant of marine environments and constitutes part of the autochthonous microbial communities, but is also associated with human gastroenteritis, wound infections and septicemia. Recently, a number of clinical cases of infection due to ingestion of seafood contaminated with V. parahaemolyticus and potentially pandemic marine strains isolated from water and plankton have been reported in Europe. To identify the source of virulent strains and to analyze the possible persistence, in time and space, of particular clones, the molecular typing of Vibrio strains is of high epidemiological interest. In this study, we applied pulsed-field gel electrophoresis and two PCR-based techniques (enterobacterial repetitive intergenic consensus- and repetitive extragenic palindromic-PCR) to establish the DNA fingerprints for the analysis of genetic variability among the environmental V. parahaemolyticus strains isolated in the area of the Venetian Lagoon. A temporal distribution of the environmental strains in the studied geographical area and, in some cases, a strong association between a certain genetic profile and a specific source have been evidenced. A number of genetic clusters/clones seem to persist over time, reappearing in the marine environment for subsequent months and also at a 1-year gap. The use of multiple typing methods allowed a more accurate characterization of the environmental strain genetic profiles and the identification of clones hardly revealed through common techniques.