Aide Lasa

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 toranzoniae sp. nov., a new member of the Splendidus clade in the genus Vibrio

Four motile facultative anaerobic marine isolates (Vb 10.8(T) [CECT 7225(T), CAIM 1869(T)], CMJ 9.4 [CECT 8091, CAIM 1870], CMJ 9.11 and Cmf 13.9), were obtained from cultured clams (Venerupis philippinarum and Venerupis decussata) in Galicia (NW Spain). These isolates were studied by a polyphasic approach, including a phylogenetic analysis based on sequences of 16S rRNA and five housekeeping genes atpA, recA, pyrH, rpoA and rpoD, that supported their inclusion in the Splendidus clade of the genus Vibrio, forming a well-defined group separated from the others species of the clade. DNA-DNA hybridizations with the type strains of species showing more than 98.5% 16S rRNA gene sequence similarity rendered values of hybridization below 60%. These isolates could be differentiated from the closest relatives on the basis of several phenotypic and chemotaxonomic features. These results demonstr8ated that the strains constitute a novel specie of the genus for which the name Vibrio toranzoniae sp. nov. is proposed, with Vb 10.8(T) (=CECT 7225(T)CAIM 1869(T)) as the type strain.

Description of Lacinutrix venerupis sp. nov.: a novel bacterium associated with reared clams.

A group of five Gram-negative strains isolated from clams (Venerupis decussata and Venerupis philippinarum) in Galicia (NW Spain) were analyzed using a polyphasic taxonomic approach. The strains were non-motile, strictly aerobic rods, and formed yellow-pigmented colonies. Based on 16S rRNA gene sequence similarity, the clam isolates belonged to the Lacinutrix genus, within the Flavobacteriaceae family, and showed the highest sequence similarities with Lacinutrix mariniflava (97.0%) and Lacinutrix algicola (96.9%). The DNA G+C content of the designated type strain Cmf 20.8(T) was 34.1%, which was within the range of the other species in the genus (29.0-37.0%). The main cellular fatty acids of the type strain Cmf 20.8(T) were iso-C15:0 3-OH, iso-C15:1 G, iso-C15:0 and iso-C17:0 3-OH. The DNA-DNA analysis value of strain Cmf 20.8(T) with L. mariniflava AKS292(T) was 51% and with L. algicola AKS293(T) it was 56%. Phenotypic data showed that the isolates could be differentiated easily from the other species of the genus. On the basis of these results, the strains represent a novel species of the genus Lacinutrix, for which the name Lacinutrix venerupis sp. nov. is proposed, with Cmf 20.8(T) (=CECT 8573(T)=DSM 28755(T)) as the designated type strain.

Isolation and identification of Vibrio toranzoniae associated with diseased red conger eel (Genypterus chilensis) farmed in Chile

The present study deals with the first isolation of Vibrio toranzoniae from cultured red conger eel (Genypterus chilensis). During the summer season of 2011, mortalities were observed in young red conger eel at one aquaculture experimental rearing system in Quintay, Valparaiso, Chile. The microbiological analysis of the diseased fish resulted in the isolation of three dominant and representative isolates, designated as R.17, R.18 and R.19, which were obtained from gill, fin and external lesions from three different fish, respectively. All isolates were identified as V. toranzoniae by means of a polyphasic taxonomic approach, including phenotypic characterization, sequencing of 16S rRNA and housekeeping genes, and DNA-DNA hybridization. Inoculation of a representative strain (R18) in turbot as model fish species demonstrated the pathogenic potential for fish of the Chilean isolates. Results obtained indicate that the geographical and host distribution of V. toranzoniae is wider than expected, and that this species may have negative incidence in the culture of marine organisms.

Vibrio tapetis isolated from vesicular skin lesions in Dover sole (Solea solea)

Vibrio tapetis is primarily known as the causative agent for brown ring disease in bivalves, although it has been isolated from cultivated fish during mortalities on farms. Here we describe the first isolation of V. tapetis from wild-caught and subsequently captive-held Dover sole Solea solea. Pathological features consisted of multifocal circular greyish-white skin discolourations evolving into vesicular lesions and subsequent ulcerations on the pigmented side. On the non-pigmented side, multiple circular lesions-white at the center and red at the edges-were evident. Histological examination of the vesicular lesions revealed dermal fluid-filled spaces, collagen tissue necrosis and a mixed inflammatory infiltrate, with large numbers of small rod-shaped bacteria. In the deep skin lesions, loss of scales and dermal connective tissue, with degeneration and fragmentation of the myofibres bordering the ulceration, were noted. Serotyping, DNA-DNA hybridization and REP- and ERIC-PCR techniques showed that the retrieved isolates displayed a profile similar to the representative strain of genotype/serotype O2 which originally was isolated from carpet-shell clam Venerupis decussata and to which isolates obtained from wedge sole Dicologoglossa cuneata were also closely related.

Isolation of Vibrio tapetis from two native fish species (Genypterus chilensis and Paralichthys adspersus) reared in Chile and description of Vibrio tapetis subsp. quintayensis subsp. nov

A group of seven Chilean isolates presumptively belonging to Vibrio tapetis was isolated from diseased fine flounders (Paralichthys adspersus) and red conger eel (Genypterus chilensis) experimentally reared in Quintay (Chile). All isolates were confirmed as members of V. tapetis on the basis of matrix-assisted laser desorption ionization time-of-flight MS, 16S rRNA gene sequencing, DNA-DNA hybridization values and G+C content. The ERIC-PCR and REP-PCR patterns were homogeneous among those isolates recovered from the same host (red conger or fine flounders), but distinct from the type strains V. tapetis subsp. tapetis CECT 4600T and V. tapetis subsp. britannicus CECT 8161T. On the basis of atpA, rpoA, rpoD, recA and pyrH gene sequence similarities (99.7-100 %) and clustering in the phylogenetic trees, the red conger isolates (Q20, Q047, Q48 and Q50) were confirmed as representing V. tapetis subsp. tapetis. However, they differed from V. tapetis subsp. tapetis CECT 4600T in their lipase, alpha quimiotripsin and non-acid phosphatase production. On the other hand, the fine flounder isolates (QL-9T, QL-35 and QL-41) showed rpoD, recA and pyrH gene sequence similarities ranging from 91.6 to 97.7 % with the type strains of the two V. tapetis subspecies (CECT 4600T and CECT 8161T) and consistently clustered together as an independent phylogenetic line within V. tapetis. Moreover, they could be differentiated phenotypically from strains CECT 4600T and CECT 8161T by nine and three different biochemical tests, respectively. In conclusion, the presence of V. tapetis in diseased red conger eel and fine flounder was demonstrated, extending the known host range and geographical location for this pathogen. Furthermore, this study demonstrates that the three isolates from fine flounder represent a novel subdivision within V. tapetis, for which the name V. tapetis subsp. quintayensis subsp. nov. is proposed and with QL-9T (=CECT 8851T=LMG 28759T) as the type strain. Although QL-9T was isolated from kidney of diseased fine flounder specimens, the challenge assays showed that it was non-pathogenic for this species.

Genome sequence of three Psychrobacter sp. strains with potential applications in bioremediation

To date, the genus Psychrobacter consists of 37 recognized species isolated from different sources, however they are more frequently found in cold and other non-polar environments of low water activity. Some strains belonging to the genus have shown different enzymatic activities with potential applications in bioremediation or food industry. In the present study, the whole genome sequences of three Psychrobacter-like strains (C 20.9, Cmf 22.2 and Rd 27.2) isolated from reared clams in Galicia (Spain) are described. The sequenced genomes resulted in an assembly size of 3,143,782 bp for C 20.9 isolate, 3,168,467 bp for Cmf 22.2 isolate and 3,028,386 bp for Rd 27.2 isolate. Among the identified coding sequences of the genomes, mercury detoxification and biogeochemistry genes were found, as well as genes related to heavy metals and antibiotic resistance. Also virulence-related features were identified such as the siderophore vibrioferrin or an aerobactin-like siderophore. The phylogenetic analysis of the 16S rRNA gene suggested that these strains may represent novel species of the Psychrobacter genus. The genome sequences of the Psychrobacter sp. strains have been deposited at DDBJ/EMBL/GenBank under the accession numbers MRYA00000000 (Cmf 22.2), MRYB00000000 (Rd 27.2) and MRYC00000000 (C 20.9), and the sequences could be found at the site https://www.ncbi.nlm.nih.gov/bioproject/PRJNA353858.

Marinomonas gallaica sp. nov. and Marinomonas atlantica sp. nov., isolated from reared clams (Ruditapes decussatus).

Three Gram-negative bacterial strains (Cmf 17.2T, Rd 20.33 and Cmf 18.22T) isolated from reared clams in Galicia were subjected to a taxonomic study, based on genetic and phenotypic characterization. Analysis of the 16S rRNA gene allowed the identification of the strains as members of the genus Marinomonas, sharing the highest similarity with Marinomonas aquimarina CECT 5080T (97.8 %-98.5 % 16S rRNA gene sequence similarity). Phylogenetic analysis of the sequences showed that the three isolates formed two different groups distantly related to their closest relative, M. aquimarina. DNA-DNA hybridizations were performed to confirm the taxonomic position and the results were below the recommended threshold for species delimitation, specifically 44.5 % (Cmf 17.2T with M. aquimarina CECT 5080T) and 55 % (Cmf 18.22Twith M. aquimarina CECT 5080T). Furthermore, the average nucleotide identity (ANIb, ANIm and OrthoANI) and in silico estimated DNA-DNA reassociation values among Cmf 17.2T, Cmf 18.22T and M. aquimarina CECT 5080T were in all cases below the respective threshold for species differentiation. The estimated G+C content of the genomic DNA was found to be 45.3 % (Cmf 17.2T) and 44.6 % (Cmf 18.22T). The principal fatty acids of the strains were found to be summed feature 3 (C16 : 1 ω7c/C16 : 1ω6c), summed feature 8 (C18 : 1ω7c/C18 : 1ω6c), C16 : 0, C12 : 0 and C10 : 0 3-OH. The results obtained on the characterization of the clam isolates indicate that they represent two novel species of the genus Marinomonas, for which the names Marinomonas gallaica sp. nov. (type strain Cmf 17.2T=CECT 9049T=LMG 29243T) and Marinomonas atlantica sp. nov. (type strain Cmf 18.22T=CECT 9050T=LMG 29244T) are proposed.

Draft Genome Sequence of Vibrio toranzoniae Strain CECT 7225T

ABSTRACT Vibrio toranzoniae (CECT 7225T) was isolated from healthy reared carpet shell clams in Galicia (Northwest Spain). In addition, this species has been recently identified as a potential pathogen of red conger eel in Chile. The draft genome sequence has 4.5 Mbp, a G+C content of 43.9%, and >3,800 protein-coding genes.

Kiloniella majae sp. nov., isolated from spider crab (Maja brachydactyla) and pullet carpet shell clam (Venerupis pullastra)☆

Ten Gram-negative, rod-shaped and motile bacterial strains were isolated from spider crab (M27.10, M27.11a, F36.1, F36.4, M56.1, F76.17b, M146.1, M166.3 and M166.6) and pullet carpet shell clam (SBRF 1.10) collected in the coast of Galicia. Analyses of the 16S rRNA genes showed that the strains belong to the genus Kiloniella and have high similarity with the species Kiloniella spongiae (99.44-99.86%) and Kiloniella litopenaei (99.0-99.5%). Strains M56.1T (=CECT 9195, =LMG 29925), M146.1 (=CECT 9193, =LMG 29926) and SBRF 1.10 (=CECT 9194, =LMG 29927) were selected on the basis of genotyping by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Phylogenetic analysis based on concatenated sequences of the genes gyrB, ftsZ, rpoD and mreB showed that the isolates form a differentiated branch within the genus Kiloniella. Moreover, the average nucleotide identity (ANIm, ANIb and OrthoANI) and in silico estimated DNA-DNA reassociation values between selected Galician isolates and Kiloniella species were below the established cut-off for species deliniation. The results obtained in the genetic and phenotypical analyses indicate that the isolates represent a new species of the genus Kiloniella, for which the name Kiloniella majae sp. nov. is proposed with strain M56.1T (=CECT 9195T, =LMG 29925T) as the type strain.