Ana L. Diéguez

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)).

Pseudomonas baetica sp. nov., a fish pathogen isolated from wedge sole, Dicologlossa cuneata (Moreau)

Five Gram-negative bacterial isolates, recovered from an outbreak that occurred in March 2006 in Huelva, Spain, affecting adult diseased cultured wedge sole [Dicologlossa cuneata (Moreau)], were characterized phenotypically and genotypically in order to clarify their taxonomic position. On the basis of 16S rRNA gene sequence analysis, the isolates were included in the genus Pseudomonas, within the Pseudomonas fluorescens-related species group, their closest relatives being the Pseudomonas jessenii and Pseudomonas koreensis subgroups. The highest sequence similarities were recorded with the type strains of Pseudomonas reinekei, P. moorei, P. umsongensis, P. jessenii and P. mohnii (99.4-99.3 % similarity). Sequence analysis of the housekeeping genes gyrB and rpoD clearly differentiated the isolates from currently described Pseudomonas species, the highest sequence similarities recorded to type strains being below 95 % for both genes. Phylogenetic analysis using concatenated sequences of the three genes showed Pseudomonas moraviensis DSM 16007T and P. koreensis DSM 16610T as the closest reference strains. DNA-DNA hybridization assays with related strains confirmed that these isolates belong to a novel species of the genus Pseudomonas, for which the name Pseudomonas baetica sp. nov. is proposed. The type strain is strain a390T (=CECT 7720T=LMG 25716T). The novel species could be easily distinguished from phylogenetically related species by several phenotypic characteristics, including gelatin hydrolysis, acid production from glucose and growth at 6 % NaCl. Virulence assays revealed that the novel species is pathogenic for wedge sole.

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.

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).

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.

Evaluation of different species-specific PCR protocols for the detection of Vibrio tapetis

In this study the specificity and sensitivity of three primer pairs, Jvt1-Jvt2, VtF-VtR and VtKF-VtKR, for the detection of Vibrio tapetis were evaluated in parallel using 23 V. tapetis strains isolated from different mollusc and fish species and with different geographical origin, as well as 29 representatives of related Vibrio species. The three primer pairs amplified all the V. tapetis strains, regardless of their host or geographical origin. However, with primer sets VtF-VtR and VtKF-VtKR amplification products of the expected size were obtained from chromosomal DNA of some of the non-V. tapetis bacteria tested. The sensitivity of the three PCR detection methods was also different. The detection limit obtained with primer pairs Jvt1-Jvt2 and VtF-VtR was between 1 and 10 pg DNA/PCR tube (2-20 bacterial cells per reaction). The primer set VtKF-VtKR showed a reduction of sensitivity in at least one order of magnitude. The results were highly reproducible with all primer sets when using the same thermal cycler, although some differences were observed in the results obtained in different PCR machines. Based on the findings reported here, we propose the Jvt1-Jvt2 PCR protocol as the most adequate for an accurate detection of V. tapetis in diagnostic pathology as well as in epidemiological studies of this clam pathogen.

Evaluation of different culture media for the isolation and growth of the fastidious Vibrio tapetis, the causative agent of brown ring disease

Thirteen culture media were evaluated at two temperatures for the growth and isolation of Vibrio tapetis. The bacterium showed similar growth dynamics at 15 °C or 25 °C, being faster at 15 °C regardless the general media employed. Best growth of V. tapetis was obtained on Agar Seawater (ASWT) (1.7 × 10(6)cfu/ml), Mannitol Marine Agar (MMA) (2.6 × 10(6)cfu/ml), and Mannitol Trypticase Soy Agar (MTSA-1) (1.9 × 10(6)cfu/ml), being slightly lower on Marine Agar (MA) (5.0 × 10(5)cfu/ml). Growth was poor on TCBS and nule in the other media containing bile salts, indicating their inhibitory effect on the V. tapetis growth. Recovery of V. tapetis from mixed Vibrio populations, differing in acid production from sucrose and mannitol, was only possible using the selective medium MMA at both temperatures. The use of ASWT or MA at 15 °C for the routinary growth of V. tapetis, and MMA for isolation of V. tapetis from bivalve samples is recommended.

Arcobacter haliotis Tanaka et al. 2017 is a later heterotypic synonym of Arcobacter lekithochrous Diéguez et al. 2017

The draft whole-genome sequence of Arcobacter haliotis strain LMG 28652T was obtained and compared against the type strain of Arcobacter lekithochrous LFT 1.7T. High similarity was found between the two strains, showing average nucleotide identity and in silico DNA-DNA hybridization values of 98.40 and 86.10 %, respectively. These values indicated that both genomes belonged to the same species, confirming the evidences derived from the phylogenetic analysis performed with the 16S rRNA gene and the concatenated sequences of five housekeeping genes. In addition, the metabolic, physiological and chemotaxonomic features of A. haliotis LMG 28652T were shown to be congruent with those of A. lekithochrous. We conclude that Arcobacter haliotis Tanaka et al. 2017 is a later heterotypic synonym of Arcobacter lekithochrousDiéguez et al. 2017.