Development of strain-specific PCR primers for the identification of Fusobacterium nucleatum subsp. fusiforme ATCC 51190T and subsp. vincentii ATCC 49256T

The objective of this study was to develop the strain-specific PCR primers for Fusobacterium nucleatum subsp. fusiforme ATCC 51190^T and F. nucleatum subsp. vincentii ATCC 49256^T based on the nucleotide sequence of the Fs17 and Fv35 DNA probes, respectively. The strain specificity was tested against 10 type strains of Fusobacterium spp. or subsp., 21 clinical isolates of F. nucleatum from Koreans, and five type strains of distinct Fusobacterium species. Primer sensitivity was determined by testing serial dilutions (4 ng–4 fg) of the purified genomic DNA from each of the type strains. PCR showed that two pairs of PCR primers, Fs17-F14/Fs17-R14 and Fv35-F1/Fv35-R1 primers, could produce strain-specific amplicons from F. nucleatum subsp. fusiforme ATCC 51190^T and F. nucleatum subsp. vincentii ATCC 49256^T, respectively. The two PCR primer sets could detect as little as 0.4 pg or 4 pg of the genomic DNA of each target strain. These results suggest that the two sets of PCR primers could be used to identify F. nucleatum subsp. fusiforme ATCC 51190^T and F. nucleatum subsp. vincentii ATCC 49256^T, particularly for ascertaining the authenticity of the strain.     

Rahnella victoriana sp. nov., Rahnella bruchi sp. nov., Rahnella woolbedingensis sp. nov., classification of Rahnella genomospecies 2 and 3 as Rahnella variigena sp. nov. and Rahnella inusitata sp. nov., respectively and emended description of the genus Rahnella

Isolations from oak symptomatic of Acute Oak Decline, alder and walnut log tissue, and buprestid beetles in 2009–2012 yielded 32 Gram-negative bacterial strains showing highest gyrB sequence similarity to Rahnella aquatilis and Ewingella americana. Multilocus sequence analysis (using partial gyrB, rpoB, infB and atpD gene sequences) delineated the strains into six MLSA groups. Two MLSA groups contained reference strains of Rahnella genomospecies 2 and 3, three groups clustered within the Rahnella clade with no known type or reference strains and the last group contained the type strain of E. americana. DNA–DNA relatedness assays using both the microplate and fluorometric methods, confirmed that each of the five Rahnella MLSA groups formed separate taxa. Rahnella genomospecies 2 and 3 were previously not formally described due to a lack of distinguishing phenotypic characteristics. In the present study, all five Rahnella MLSA groups were phenotypically differentiated from each other and from R. aquatilis. Therefore we propose to classify the strains from symptomatic oak, alder and walnut and buprestid beetles as: Rahnella victoriana sp. nov. (type strain FRB 225^T = LMG 27717^T = DSM 27397^T), Rahnella variigena sp. nov. (previously Rahnella genomosp. 2, type strain CIP 105588^T = LMG 27711^T), Rahnella inusitata sp. nov. (previously Rahnella genomosp. 3, type strain DSM 30078^T = LMG 2640^T), Rahnella bruchi sp. nov. (type strain FRB 226^T = LMG 27718^T = DSM 27398^T) and Rahnella woolbedingensis sp. nov. (type strain FRB 227^T = LMG 27719^T = DSM 27399^T).     

Characterization of Bifidobacterium species in feaces of the Egyptian fruit bat: Description of B. vespertilionis sp. nov. and B. rousetti sp. nov.

Fifteen bifidobacterial strains were obtained from faeces of Rousettus aegyptiacus; after grouping them by RAPD PCR only eight were selected and characterized. Analysis of 16S rRNA and of five housekeeping (hsp60, rpoB, clpC, dnaJ, dna G) genes revealed that these eight strains were classified into five clusters: Cluster I (RST 8 and RST 16^T), Cluster II (RST 9^T and RST 27), Cluster III (RST 7 and RST 11), Cluster IV (RST 19), Cluster V (RST 17) were closest to Bifidobacterium avesanii DSM 100685^T (96.3%), Bifidobacterium callitrichos DSM 23973^T (99.2% and 99.7%), Bifidobacterium tissieri DSM 100201^T (99.7 and 99.2%), Bifidobacterium reuteri DSM 23975 ^T (98.9%) and Bifidobacterium myosotis DSM 100196^T (99.3%), respectively. Strains in Cluster I and strain RST 9 in Cluster II could not be placed within any recognized species while the other ones were identified as known species. The average nucleotide identity values between two novel strains, RST 16^T and RST 9^T and their closest relatives were lower than 79% and 89%, respectively. In silico DNA–DNA hybridization values for those closest relatives were 32.5 and 42.1%, respectively. Phenotypic and genotypic tests demonstrated that strains in Cluster I and RST 9^T in Cluster II represent two novel species for which the names Bifidobacterium vespertilionis sp. nov. (RST 16^T = BCRC 81138^T = NBRC 113380^T = DSM 106025^T ; RST 8 = BCRC 81135 = NBRC 113377) and Bifidobacterium rousetti sp. nov. (RST 9^T = BCRC 81136^T = NBRC 113378^T = DSM 106027^T) are proposed.     

Marinitoga lauensis sp. nov., a novel deep-sea hydrothermal vent thermophilic anaerobic heterotroph with a prophage

A novel moderately thermophilic, heterotrophic anaerobe, designated strain LG1^T, was isolated from the Mariner deep-sea hydrothermal vent field along the Eastern Lau Spreading Center and Valu Fa Ridge. Cells of strain LG1^T were motile rods, occurring singly or in pairs, 0.6 μm in width and 1.2 μm in length. The strain LG1^T grew between 40 and 70 °C (optimum 50–55 °C), at a pH between 5 and 8 (optimum pH 6.5) and with 7.5–50 g L⁻¹ NaCl (optimum 30 g L⁻¹). Sulfur, cystine and thiosulfate were reduced to sulfide, and cell yield was improved in the presence of cystine. Strain LG1^T was an organotroph able to use a variety of organic compounds. Phylogenetic analysis based on 16S rRNA gene sequence comparisons indicated that strain LG1^T was affiliated to the genus Marinitoga within the order Petrotogales. It shared 95.34–96.31% 16S rRNA gene sequence similarity with strains of other Marinitoga species, and is most closely related to Marinitoga okinawensis. Genome analysis revealed the presence of a prophage sharing high sequence homology with the viruses MPV1, MCV1 and MCV2 hosted by Marinitoga strains. Based on the data from the phylogenetic analyses and the physiological properties of the novel isolate, we propose that strain LG1^T is a representative of a novel species, for which the name Marinitoga lauensis sp. nov. is proposed; the type strain is LG1^T (=DSM 106824 = JCM 32613).     

Bifidobacterium primatium sp. nov., Bifidobacterium scaligerum sp. nov., Bifidobacterium felsineum sp. nov. and Bifidobacterium simiarum sp. nov.: Four novel taxa isolated from the faeces of the cotton top tamarin (Saguinus oedipus) and the emperor tamarin (Saguinus imperator)

Four novel Gram-stain-positive, non spore forming and fructose-6-phosphate phosphoketolase-positive strains were isolated from the faeces of a cotton top tamarin (Saguinus oedipus) and an emperor tamarin (Saguinus imperator). Phylogenetic analyses based on 16S rRNA revealed that bifidobacterial strains TRE 1^T exhibit close phylogenetic relatedness to Bifidobacterium catulorum DSM 103154 (96.0%) and Bifidobacterium tissieri DSM 100201 (96.0%); TRE D^T and TRE H^T were closely related to Bifidobacterium longum subsp. longum ATCC 15708^T with similarity values of 97.4% and 97.5%, respectively; TRI 7^T was closely related to Bifidobacterium tissieri DSM 100201 (96.0%). The Average Nucleotide Identity (ANI) and in silico DDH (isDDH) analysis with closest neighbour supported an independent phylogenetic position of all strains with values ranged from 74 to 85% for ANI and from 24 to 28% for isDDH. DNA base composition of the four strains was in the range of 58.3–63.5 mol% G + C. Based on the phylogenetic, genotypic and phenotypic data, the strains TRE 1^T, TRE D^T, TRE H^T and TRI 7^T clearly represent four novel taxa within the genus Bifidobacterium for which the names Bifidobacterium primatium sp. nov. (type strain TRE 1^T = DSM 100687^T = JCM 30945^T), Bifidobacterium scaligerum sp. nov. (type strain TRE D^T = DSM 103140^T = JCM 31792^T), Bifidobacterium felsineum sp. nov. (type strain TRE H^T = DSM 103139^T = JCM 31789^T) and Bifidobacterium simiarum sp. nov. (type strain TRI 7^T = DSM 103153^T = JCM 31793) are proposed.     

Tenuifilum thalassicum gen. nov., sp. nov., a novel moderate thermophilic anaerobic bacterium from a Kunashir Island shallow hot spring representing a new family Tenuifilaceae fam. nov. in the class Bacteroidia

A novel anaerobic moderately thermophilic bacterium, designated strain 38H-str^T, was isolated from a 12 m deep hot spring of the Kunashir Island shore. Gram-negative cells were non-spore-forming, motile, straight or curved filamentous rods, occasionally forming loops and knots. The strain grew at 20–65 °C and pH range of 4.0–9.0 with an optimum at 50 °C and pH 6.5–7.0. Strain 38H-str^T required 0.5–2.5% NaCl (1.5% is an optimum) for growth. It was a chemoorganoheterotroph, growing on carbohydrates (starch, pullulan, alginate, laminarin, beta-glucan) or peptide mixtures and proteins (peptone, tryptone, gelatin, and α- or β- keratins). Major products of glucose fermentation were acetate, hydrogen, and carbon dioxide. Major cellular fatty acids were iso- and anteiso-C_15:0. Phosphatidylethanolamine, an unidentified phospholipid, and three unidentified polar lipids were detected in cellular lipids fractions. The quinone was MK-7. The size of complete genome of strain 38H-str^T was 3.2 Mb; DNA G+C content was 38.3 mol%. According to 16S rRNA gene sequence and conserved protein sequences phylogenies, strain 38H-str^T represented a deeply branched lineage near the root of the class Bacteroidia. Based on phylogenetic analysis and phenotypic features the novel isolate was assigned to a novel family within the order Bacteroidales for which the name Tenuifilaceae fam. nov. is proposed. Strain 38H-str^T (=DSM 100343^T =VKM B-2964^T) represents the first genus and species Tenuifilum thalassicum gen. nov., sp. nov.     

Lacipirellula parvula gen. nov., sp. nov., representing a lineage of planctomycetes widespread in low-oxygen habitats,description of the family Lacipirellulaceae fam. nov. and proposal of the orders Pirellulales ord. nov., Gemmatales ord. nov. and Isosphaerales ord. nov.

Pirellula-like planctomycetes are ubiquitous aquatic bacteria, which are often detected in anoxic or micro-oxic habitats. By contrast, the taxonomically described representatives of these bacteria, with very few exceptions, are strict aerobes. Here, we report the isolation and characterization of the facultatively anaerobic planctomycete, strain PX69^T, which was isolated from a boreal lake. Its 16S rRNA gene sequence is affiliated with the Pirellula-related Pir4 clade, which is dominated by environmental sequences retrieved from a variety of low-oxygen habitats. Strain PX69^T was represented by ellipsoidal cells that multiplied by budding and grew on sugars, some polysaccharides and glycerol. Anaerobic growth occurred by means of fermentation. Strain PX69^T grew at pH 5.5–7.5 and at temperatures between 10 and 30 °C. The major fatty acids were C18:1ω9c, C16:0 and C16:1ω7c; the major intact polar lipid was dimethylphosphatidylethanolamine. The complete genome of strain PX69^T was 6.92 Mb in size; DNA G + C content was 61.7 mol%. Among characterized planctomycetes, the highest 16S rRNA gene similarity (90.4%) was observed with ‘Bythopirellula goksoyri’ Pr1d, a planctomycete from deep-sea sediments. We propose to classify PX69^T as a novel genus and species, Lacipirellula parvula gen. nov., sp. nov.; the type strain is strain PX69^T (=KCTC 72398^T = CECT 9826^T = VKM B-3335^T). This genus is placed in a novel family, Lacipirellulaceae fam. nov., which belongs to the order Pirellulales ord. nov. Based on the results of comparative genome analysis, we also suggest establishment of the orders Gemmatales ord. nov. and Isosphaerales ord. nov. as well as an emendation of the order Planctomycetales.     

Polyphasic characterization of two novel Lactobacillus spp. isolated from blown salami packages: Description of Lactobacillus halodurans sp. nov. and Lactobacillus salsicarnum sp. nov.

Microbiota analysis of blown pack spoiled salami revealed five distinguishable Lactobacillus isolates we could not assign to a known species. Two of the isolates (TMW 1.2172^T and TMW 1.1920) are rod-shaped, whilst three isolates (TMW 1.2098^T, TMW 1.2118 and TMW 1.2188) appear coccus shaped or as short rods. All isolates are Gram-stain positive, facultative anaerobic, catalase and oxidase negative, non-motile and non-sporulating. Phylogenetic analysis of the 16S rRNA, dnaK, pheS and rpoA gene sequences revealed two distinct lineages within the genus Lactobacillus (L.). The isolates are members of the Lactobacillus alimentarius group with Lactobacillus ginsenosidimutans DSM 24154^T (99.4% 16S similarity), Lactobacillus versmoldensis DSM 14857^T (97.9%) and Lactobacillus furfuricola DSM 27174^T (97.7%) as phylogenetic closest related species and L. alimentarius DSM 20249^T (97.7%) and Lactobacillus paralimentarius DSM 13961^T (97.5%) as closest relatives, respectively. Average Nucleotide Identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the isolates and their close related type strains are lower than 80% and 25%, respectively. For both designated type strains, the peptidoglycan type is A4α l-Lys-d-Asp and the major fatty acids are C_16:0, C_18:1ω9c and summed feature 7. Based on phylogenetic, phenotypic and chemotaxonomic analysis we demonstrated that the investigated isolates belong to two novel Lactobacillus species for which we propose the names Lactobacillus salsicarnum with the type strain TMW 1.2098^T = DSM 109451^T = LMG 31401^T and Lactobacillus halodurans with the type strain TMW 1.2172^T = DSM 109452^T = LMG 31402^T.     

Alkaliphilus serpentinus sp. nov. and Alkaliphilus pronyensis sp. nov., two novel anaerobic alkaliphilic species isolated from the serpentinite-hosted Prony Bay Hydrothermal Field (New Caledonia)

Two novel anaerobic alkaliphilic strains, designated as LacT^T and LacV^T, were isolated from the Prony Bay Hydrothermal Field (PBHF, New Caledonia). Cells were motile, Gram-positive, terminal endospore-forming rods, displaying a straight to curved morphology during the exponential phase. Strains LacT^T and LacV^T were mesophilic (optimum 30 °C), moderately alkaliphilic (optimum pH 8.2 and 8.7, respectively) and halotolerant (optimum 2% and 2.5% NaCl, respectively). Both strains were able to ferment yeast extract, peptone and casamino acids, but only strain LacT^T could use sugars (glucose, maltose and sucrose). Both strains disproportionated crotonate into acetate and butyrate. Phylogenetic analysis revealed that strains LacT^T and LacV^T shared 96.4% 16S rRNA gene sequence identity and were most closely related to A. peptidifermentans Z-7036, A. namsaraevii X-07-2 and A. hydrothermalis FatMR1 (95.7%–96.3%). Their genome size was of 3.29 Mb for strain LacT^T and 3.06 Mb for strain LacV^T with a G + C content of 36.0 and 33.9 mol%, respectively. The ANI value between both strains was 73.2 %. Finally, strains LacT^T (=DSM 100337 = JCM 30643) and LacV^T (=DSM 100017 = JCM 30644) are proposed as two novel species of the genus Alkaliphilus, order Clostridiales, phylum Firmicutes, Alkaliphilus serpentinus sp. nov. and Alkaliphilus pronyensis sp. nov., respectively. The genomes of the three Alkaliphilus species isolated from PBHF were consistently detected in the PBHF chimney metagenomes, although at very low abundance, but not significantly in the metagenomes of other serpentinizing systems (marine or terrestrial) worldwide, suggesting they represent indigenous members of the PBHF microbial ecosystem.     

Description of a new anaerobic thermophilic bacterium,Thermoanaerobacterium butyriciformans sp. nov.

Strain USBA-019^T, an anaerobic and thermophilic strain, was identified as a new member of the genus Thermoanaerobacterium. USBA-019^T cells are gram-positive, strictly anaerobic, thermophilic, chemoorganotrophic, moderately acidophilic, non-motile, endospore-forming, slightly curved, and rod-shaped. Cells measure 0.4 × 3.0–7.0 μm. Optimal growth occurs at 50–55 °C (35–65 °C). Optimum pH is 5.0–5.5 (4.0–8.5). Thiosulfate, elemental sulfur and nitrate were utilized as electron acceptors. Fermentation of glucose, lactose, cellobiose, galactose, arabinose, xylose, starch and xylan primarily produced acetate and butyrate. Xylan, starch and cellobiose produced ethanol and starch, cellobiose, galactose, arabinose and mannose produced lactic acid. Phylogenetic analyses based on 16S rRNA gene sequence comparison and genomic relatedness indices show the close relation of USBA-019^T to Thermoanaerobacterium thermostercoris and Thermoanaerobacterium aotearoense (similarity value: 99%). Hybridization of USBA-019^T, Th. thermostercoris DSM22141^T and Th. aotearoense DMS10170^T found DNA–DNA relatedness of 33.2% and 18.2%, respectively. Based on phenotypic, chemotaxonomic and phylogenetic evidence, along with low identity at whole genome level, USBA-019^T is a novel species of the genus Thermoanaerobacterium which we propose to name Thermoanaerobacterium butyriciformans sp. nov. The type strain is USBA-019^T (=CMPUJ U-019^T = DSM 101588^T).     

Flavobacterium circumlabens sp. nov. and Flavobacterium cupreum sp. nov., two psychrotrophic species isolated from Antarctic environmental samples

A taxonomic study of 24 Gram-stain-negative rod-shaped bacteria originating from the Antarctic environment is described. Phylogenetic analysis using 16S rRNA gene sequencing differentiated isolated strains into two groups belonging to the genus Flavobacterium. Group I (n = 20) was closest to Flavobacterium aquidurense WB 1.1-56^T (98.3% 16S rRNA gene sequence similarity) while group II (n = 4) showed Flavobacterium hydatis DSM 2063^T as its nearest neighbour (98.5–98.9% 16S rRNA gene sequence similarity). Despite high 16S rRNA gene sequence similarity, these two groups represented two distinct novel species as shown by phenotypic traits and low genomic relatedness assessed by rep-PCR fingerprinting, DNA-DNA hybridization and whole-genome sequencing. Common to representative strains of both groups were the presence of major menaquinone MK-6 and sym-homospermidine as the major polyamine. Common major fatty acids were C_15:0 iso, C_15:1 iso G, C_15:0 iso 3-OH, C_17:0 iso 3OH and Summed Feature 3 (C_16:1 ω7c/C_16:1 ω6c). Strain CCM 8828^T (group I) contained phosphatidylethanolamine, three unidentified lipids lacking a functional group, three unidentified aminolipids and single unidentified glycolipid in the polar lipid profile. Strain CCM 8825^T (group II) contained phosphatidylethanolamine, eight unidentified lipids lacking a functional group, three unidentified aminolipids and two unidentified glycolipids in the polar lipid profile. These characteristics corresponded to characteristics of the genus Flavobacterium. The obtained results showed that the analysed strains represent novel species of the genus Flavobacterium, for which the names Flavobacterium circumlabens sp. nov. (type strain CCM 8828^T = P5626^T = LMG 30617^T) and Flavobacterium cupreum sp. nov. (type strain CCM 8825^T = P2683^T = LMG 30614^T) are proposed.     

Rhizobium cauense sp. nov., isolated from root nodules of the herbaceous legume Kummerowia stipulacea grown in campus lawn soil

Three bacterial isolates (CCBAU 101002^T, CCBAU 101000 and CCBAU 101001) originating from root nodules of the herbaceous legume Kummerowia stipulacea grown in the campus lawn of China Agricultural University were characterized with a polyphasic taxonomic approach. Comparative 16S rRNA gene sequence analysis showed that the isolates shared 99.85–99.92% sequence similarities and had the highest similarities to the type strains of Rhizobium mesoamericanum (99.31%), R. endophyticum (98.54%), R. tibeticum (98.38%) and R. grahamii (98.23%). Sequence similarity of four concatenated housekeeping genes (atpD, glnII, recA and rpoB) between CCBAU 101002^T and its closest neighbor (R. grahamii) was 92.05%. DNA–DNA hybridization values between strain CCBAU 101002^T and the four type strains of the most closely related Rhizobium species were less than 28.4 ± 0.8%. The G + C mol% of the genomic DNA for strain CCBAU 101002^T was 58.5% (Tm). The major respiratory quinone was ubiquinone (Q-10). Summed feature 8 (18:1ω7cis/18:1ω6cis) and 16:0 were the predominant fatty acids. Strain CCBAU 101002^T contained phosphatidylcholine and phosphatidylethanolamine as major polar lipids, and phosphatidylglycerol and cardiolipin as minor ones. No glycolipid was detected. Unlike other strains, this novel species could utilize dulcite or sodium pyruvate as sole carbon sources and it was resistant to 2% (w/v) NaCl. On the basis of the polyphasic study, a new species Rhizobium cauense sp. nov. is proposed, with CCBAU 101002^T (=LMG 26832^T = HAMBI 3288^T) as the type strain.     

Desulfamplus magnetovallimortis gen. nov., sp. nov., a magnetotactic bacterium from a brackish desert spring able to biomineralize greigite and magnetite,that represents a novel lineage in the Desulfobacteraceae

A magnetotactic bacterium, designated strain BW-1^T, was isolated from a brackish spring in Death Valley National Park (California, USA) and cultivated in axenic culture. The Gram-negative cells of strain BW-1^T are relatively large and rod-shaped and possess a single polar flagellum (monotrichous). This strain is the first magnetotactic bacterium isolated in axenic culture capable of producing greigite and/or magnetite nanocrystals aligned in one or more chains per cell. Strain BW-1^T is an obligate anaerobe that grows chemoorganoheterotrophically while reducing sulfate as a terminal electron acceptor. Optimal growth occurred at pH 7.0 and 28 °C with fumarate as electron donor and carbon source. Based on its genome sequence, the G + C content is 40.72 mol %. Phylogenomic and phylogenetic analyses indicate that strain BW-1^T belongs to the Desulfobacteraceae family within the Deltaproteobacteria class. Based on average amino acid identity, strain BW-1^T can be considered as a novel species of a new genus, for which the name Desulfamplus magnetovallimortis is proposed. The type strain of D. magnetovallimortis is BW-1^T (JCM 18010^T–DSM 103535^T).     

Sphingomonas pokkalii sp. nov., a novel plant associated rhizobacterium isolated from a saline tolerant pokkali rice and its draft genome analysis

Three strains L3B27^T, 3CNBAF, L1A4 isolated from a brackish cultivated pokkali rice rhizosphere were characterised using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA and recA gene sequences revealed that these strains were highly similar among each other and formed a separate monophyletic cluster within the genus Sphingomonas with Sphingomonas pituitosa DSM 13101^T, Sphingomonas azotifigens DSM 18530^T and Sphingomonas trueperi DSM 7225^T as their closest relatives sharing 97.9–98.3% 16S rRNA similarity and 91.3–94.0% recA similarity values, respectively. The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA–DNA hybridisation (dDDH) values between L3B27^T (representative of the novel strains) and its phylogenetically closest Sphingomonas species were well below the established cut-off <94% (ANI/AAI) and <70% (dDDH) for species delineation. Further, the novel strains can be distinguished from its closest relatives based on several phenotypic traits. Thus, based on the polyphasic approach, we describe a novel Sphingomonas species for which the name Sphingomonas pokkalii sp. nov (type strain L3B27^T = KCTC 42098^T = MCC 3001^T) is proposed. In addition, the novel strains were characterised for their plant associated properties and found to possess several phenotypic traits which probably explain its plant associated lifestyle. This was further confirmed by the presence of several plant associated gene features in the genome of L3B27^T. Also, we could identify gene features which may likely involve in brackish water adaptation. Thus, this study provides first insights into the plant associated lifestyle, genome and taxonomy of a novel brackish adapted plant associated Sphingomonas.     

Vagococcus teuberi sp. nov., isolated from the Malian artisanal sour milk fènè

Ten bacterial isolates belonging to the genus Vagococcus were obtained from Malian sour milk fènè produced from spontaneously fermented cow milk. However, these isolates could not be assigned to a species upon initial comparative 16S rRNA gene sequence analysis and were therefore further characterized. Rep-PCR fingerprinting of the isolates yielded four strain clusters represented by strains CG-21^T (=DSM 21459^T), 24CA, CM21 and 9H. Sequence identity of the 16S rRNA gene of DSM 21459^T to its closest relative species Vagococcus penaei was 97.9%. Among the four rep strain clusters, DSM 21459^T and 24CA shared highest 16S rRNA gene sequence identity of 99.6% while CM21 and 9H shared 98.6–98.8% with DSM 21459^T and V. penaei CD276^T. DSM 21459^T and 24CA were thus subjected to a polyphasic typing approach. The genome of DSM 21459^T featured a G + C content of 34.1 mol% for a 2.17-bp chromosome and a 15-kbp plasmid. Average nucleotide identity (ANI) of DSM 21459^T to Vagococcus fluvialis bH819, V. penaei CD276^T were 72.88%, 72.63%, respectively. DNA–DNA hybridization (DDH) similarities of strain DSM 21459^T to other Vagococcus species were <42.0%. ANI and DDH findings strongly supported the 16S rRNA gene phylogenetic tree delineations. The fatty acid patterns of DSM 21459^T was palmitic acid (C _16:0, 24.5%), oleic acid (C _18:1-ω9c, 32.8%), stearic acid (C _18:0, 18.9%). General physiological characterization of DSM 21459^T and 24CA were consistent with those of the genus Vagococcus. Strain DSM 21459^T and further strains are therefore considered to belong to a novel species, for which the nomenclature Vagococcus teuberi sp. nov. is proposed. The type strain is named CG-21^T (=DSM 21459^T and LMG 24695^T).     

Acetobacter oryzifermentans sp. nov., isolated from Korean traditional vinegar and reclassification of the type strains of Acetobacter pasteurianus subsp. ascendens (Henneberg 1898) and Acetobacter pasteurianus subsp. paradoxus (Frateur 1950) as Acetobacter ascendens sp. nov., comb. nov.

Twelve Acetobacter pasteurianus-related strains with publicly available genomes in GenBank shared high 16S rRNA gene sequence similarity (>99.59%), but average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) values and multilocus sequence- and genome-based relatedness analyses suggested that they were divided into four different phylogenetic lineages. Relatedness analyses based on multilocus sequences, 1,194 core genes and whole-cell MALDI-TOF profiles supported that strains LMG 1590^T and LMG 1591 (previously classified as the type strains of A. pasteurianus subsp. ascendens and paradoxus, respectively) and strain SLV-7^T do not belong to A. pasteurianus. Strain SLV-7^T, isolated from Korean traditional vinegar, shared low ANI (<91.0%) and in silico DDH (44.2%) values with all other Acetobacter type strains analyzed in this study, indicating that strain SLV-7^T represents a new Acetobacter species. The phenotypic and chemotaxonomic analyses confirmed these results and therefore a new species named Acetobacter oryzifermentans sp. nov. is proposed with SLV-7^T (= KACC 19301^T = JCM 31096^T) as the type strain. Strains LMG 1590^T and LMG 1591 shared high ANI (99.4%) and in silico DDH (96.0%) values between them, but shared low ANI (<92.3%) and in silico DDH (<49.0%) values with other type strains analyzed in this study, indicating that strains LMG 1590^T and LMG 1591 should be reclassified into a new single species that should be named Acetobacter ascendens sp. nov., comb. nov., with LMD 51.1^T (= LMG 1590^T = NCCB 51001^T) as its type strain.     

Effects of temperature,salinity and their interaction on growth of benthic dinoflagellates Ostreopsis spp. from Thailand

Benthic dinoflagellates Ostreopsis spp. are known as producers of palytoxin and its analogs, resulting occasionally in human health problems worldwide. Although distribution of Ostreopsis spp. along the Thai coasts has been reported, little is known about their growth characteristics. To discuss the bloom dynamics of Ostreopsis spp. in Thailand, first we tested four kinds of media to optimize growth conditions and then clarified the effects of temperature, salinity and temperature–salinity interaction on the growth of strains of the O. cf. ovata Thailand subclade, O. cf. ovata South China Sea subclade, Ostreopsis sp. 6 and Ostreopsis sp. 7. We showed that the f/2 medium was a suitable medium which gave the highest cell yields for all the strains tested. The strains of the O. cf. ovata Thailand subclade, O. cf. ovata South China Sea subclade and Ostreopsis sp. 6 grew in the temperature range 20–32.5 °C, whereas the strain of Ostreopsis sp. 7 grew in 20–30 °C. The semi-optimal temperature ranges (≧80% of the maximal growth rate) for the former three strains were 22.7–27.4 °C, 27.9–30.8 °C and 23.5–26.4 °C, respectively, whereas that of the latter strain was 23–27.2 °C. The optimal temperature for the O. cf. ovata South China Sea subclade was 30 °C, whereas for the others it was 25 °C. All the Ostreopsis strains tested could grow in a salinity range of 20–40. The semi-optimal salinities for the O. cf. ovata Thailand subclade, O. cf. ovata South China Sea subclade Ostreopsis sp. 6 and Ostreopsis sp. 7 were 28.7–35, 23.8–30.8, 29.8–36 and 28–36, respectively. The optimal salinities for the O. cf. ovata Thailand subclade and O. cf. ovata South China Sea subclade were 30 and 25, respectively, whereas for Ostreopsis sp. 6 and Ostreopsis sp. 7 it was 35. In this study, our results suggested that the optimal and tolerable temperature–salinity conditions differ among the Thai Ostreopsis species/clades/subclades. Tolerances of the O. cf. ovata Thailand subclade, O. cf. ovata South China Sea subclade and Ostreopsis sp. 6 to the high temperature of 32.5 °C may allow these organisms to be distributed in the tropical areas, where the water temperature often reaches >30 °C.     

Avrilella dinanensis gen. nov., sp. nov., a novel bacterium of the family Flavobacteriaceae isolated from human blood

Polyphasic taxonomic analysis was performed on a novel bacterium, designated UR159^T, isolated in 2016 from human blood of a septic patient hospitalized in France. Preliminary 16S rRNA gene sequence-based phylogenetic analysis indicated that strain UR159^T belonged to the family Flavobacteriaceae, forming a distinct phyletic line distantly related (<94% sequence similarity) to known species of the family. Further phenotypic, chemotaxonomic and genomic analyses were performed. Cells were non-motile, oxidase-negative, catalase-positive Gram-negative rods. It was strictly aerobic yielding yellow-pigmented colonies, and was metabolically rather inert. Major fatty acids were iso-branched fatty acids, predominantly iso-C_15:0 (55.5%) and iso-C_17:1ω9c (8.8%). Whole genome sequencing revealed a 2.3-Mbp genome encoding a total of 2262 putative genes with a genomic DNA G + C content at 37.6 mol%. The average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) values between strain UR159^T and the most closely related members of the Flavobacteriaceae family were <75% and <39%, respectively, much below the established cut-offs for ANI (<95–96%) and isDDH (<70%) for species and genus delineation. Average Amino Acid Identity (AAI) percentages were also estimated and were lower than 65% (cut-off proposed for genus delineation for uncultivated prokaryotes) in all cases, except for F. marinum that was just at the limit (65.1%). Based on these findings, we propose it as a new genus and species, Avrilella dinanensis gen. nov., sp. nov. (type strain UR159^T = CIP 111616^T = DSM 105483^T).     

Description of Maribellus sediminis sp. nov., a marine nitrogen-fixing bacterium isolated from sediment of cordgrass and mangrove

Two marine bacterial strains designated Y2-1-60^T and GM1-28 were isolated from sediments of cordgrass and mangrove along the Luoyang estuary in Quanzhou Bay, China, respectively. Both strains were Gram-staining-negative, straight rod-shaped, non-flagellum, facultatively anaerobic, nitrogen-fixing, and did not contain carotenoid pigment. Catalase activities were found to be weak positive and oxidase activities negative. The 16S rRNA gene sequences of the two strains were identical and had maximum similarity of 98.0% with Maribellus luteus XSD2^T, and of <94.5% with other species. ANI value (96.9%) and DDH estimate (71.5%) between the two strains supported that they belonged to the same species. ANI value and DDH estimate between the two strains and M. luteus XSD2^T was 74.3% and 19.4%, respectively, indicating that they represent a novel species. Phylogenetic analysis based on 16S rRNA gene and phylogenomic analysis indicated that strains Y2-1-60^T and GM1-28 formed a monophyletic branch within the genus Maribellus. The respiratory quinone was menaquinone MK-7. The major fatty acid (>10%) consisted of iso-C_15:0, and iso-C_17:0 3-OH. The polar lipids consisted of phosphatidylethanolamine and several unidentified lipids. The genomic G + C contents were 41.9–42.0 mol%. Gene annotation revealed that strains Y2-1-60^T and GM1-28 contained a set of nif gene cluster (nifHDKENB) responsible for nitrogen fixation. Based on the above characteristics, strains Y2-1-60^T and GM1-28 represent a novel species within the genus Maribellus. Thus, Maribellus sediminis sp. nov. is proposed with type strain Y2-1-60^T (=MCCC 1K04285^T = KCTC 72884^T), isolated from cordgrass sediment and strain GM1-28 (=MCCC 1K04384 = KCTC 72880), isolated from mangrove sediment.