OrthoANI Values Research Articles

Lentibacillus cibarius sp. nov., isolated from kimchi, a Korean fermented food.

Two bacterial strains designated NKC220-2T and NKC851-2 were isolated from commercial kimchi from different areas in Korea. The strains were Gram-positive, aerobic, oxidaseand catalase-positive, rod-shaped, spore-forming, non-motile, and halophilic bacteria. Both strains grew without NaCl, unlike type species in the genus Lentibacillus. The optimal pH for growth was 8.0, higher than that of the type species in the genus Lentibacillus, although growth was observed at pH 5.5-9.0. 16S rRNA gene sequence-based phylogenetic analysis indicated that the two strains (99.3-99.9% similarity) are grouped within the genus Lentibacillus and most closely related to Lentibacillus juripiscarius IS40-3T (97.4-97.6% similarity) isolated from fish sauce in Thailand. OrthoANI value between two novel strains and Lentibacillus lipolyticus SSKP1-9T (79.5-79.6% similarity) was far lower than the species demarcation threshold. Comparative genomic analysis displayed differences between the two strains as well as among other strains belonging to Lentibacillus. Furthermore, each isolate had strain-specific groups of orthologous genes based on pangenome analysis. Genomic G + C contents of strains NKC-220-2T and NKC851-2 were 41.9 and 42.2 mol%, respectively. The strains contained meso-diaminopimelic acid in their cell walls, and the major menaquinone was menaquinone-7. Phosphatidylglycerol, diphosphatidylglycerol, and an unidentified glycolipid, aminophospholipid, and phospholipid were the major polar lipid components of both strains. The major cellular fatty acids of the strains were anteiso-C15:0 and anteiso-C17:0. Based on phenotypic, genomic, phylogenetic, and chemotaxonomic features, strains NKC220-2T and NKC851-2 represent novel species of the genus Lentibacillus, for which the name Lentibacillus cibarius sp. nov. is proposed. The type strain is NKC220-2T (= KACC 21232T = JCM 33390T).

Thermococcus indicus sp. nov., a Fe(III)-reducing hyperthermophilic archaeon isolated from the Onnuri Vent Field of the Central Indian Ocean ridge.

A strictly anaerobic, dissimilatory Fe(III)-reducing hyperthermophilic archaeon, designated as strain IOH1T, was isolated from a new deep-sea hydrothermal vent (Onnuri Vent Field) area in the Central Indian Ocean ridge. Strain IOH1T showed > 99% 16S rRNA gene sequence similarity with Thermococcus celericrescens TS2T (99.4%) and T. siculi DSM 12349T (99.2%). Additional three species T. barossii SHCK-94T (99.0%), T. celer Vu13T (98.8%), and T. piezophilus (98.6%) showed > 98.6% of 16S rRNA gene sequence similarity, however, the maximum OrthoANI value is 89.8% for the genome of T. celericrescens TS2T. Strain IOH1T cells are coccoid, 1.2-1.8 μm in diameter, and motile by flagella. Growth was at 70-82°C (optimum 80°C), pH 5.4-8.0 (optimum pH 6.0) with 2-4% (optimum 3%) NaCl. Growth of strain IOH1T was enhanced by starch, pyruvate, D(+)-maltose and maltodextrin as a carbon sources, and elemental sulfur as an electron acceptor; clearly different from those of related species T. celecrescens DSM 17994T and T. siculi DSM 12349T. Strain IOH1T, T. celercrescence DSM 17994T, and T. siculi DSM 12349T reduced soluble Fe(III)-citrate present in the medium, whereas the amount of total cellular proteins increased with the concomitant accumulation of Fe(II). We determined a circular chromosome of 2,234 kb with an extra-chromosomal archaeal plasmid, pTI1, of 7.7 kb and predicted 2,425 genes. The DNA G + C content was 54.9 mol%. Based on physiological properties, phylogenetic, and genome analysis, we proposed that strain IOH1T (= KCTC 15844T = JCM 39077T) is assigned to a new species in the genus Thermococcus and named Thermococcus indicus sp. nov.

Streptomyces lasalocidi sp. nov. (formerly 'Streptomyces lasaliensis'), an actinomycete isolated from soil which produces the polyether antibiotic lasalocid.

Strain ATCC 31180T was isolated from soil collected in Hyde Park, Massachusetts (USA), and found to produce the polyether antibiotic lasalocid. The name 'Streptomyces lasaliensis' has been in common use since 1974, without a recognized taxonomic description. The most closely related type cultures determined by rRNA gene sequence similarity were Streptomyces longwoodensis DSM 41677T (100 %) and Streptomyces galbus DSM 40089T (100 %). OrthoANI values with S. longwoodensis and S. galbus were 95.50 and 94.41 %, respectively. Chemotaxonomic characteristics supported inclusion within the genus Streptomyces. The cell wall peptidoglycan contained ll-diaminopimelic acid, and the major whole-cell sugars were glucose and ribose. Polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylglycerol, one unidentified lipid and one unidentified glycolipid. The major menaquinones detected were MK9(H4), MK9(H6) and MK9(H8). The major cellular fatty acids were anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0, iso-C15 : 0 and anteiso-C17 : 1. Its DNA had a G+C content of 72.6 %. Differentiation of ATCC 31180T from the closely related species was evident from digital DNA-DNA hybridization values of 61.80 and 56.90 % for S. longwoodensis and S. galbus respectively. Significant differences were seen in the polyphasic phenotypic analyses. ATCC 31180T produced lasalocid, grew from 10 to 45 °C, pH4-8 and in the presence of 0-10 % NaCl, 0.01 % NaN3 and 1 % phenol. Melanin was produced; H2S and indole were not. Nitrate was not reduced. Spore chains were retinaculum-apertum and spore surfaces were smooth. Spore colour, mycelia colour and soluble pigment production were medium-dependent. The proposed name is Streptomyces lasalocidi sp. nov.; the type strain being ATCC 31180T (=NRRL 3382T=DSM 46487T).

Description of Azoarcus nasutitermitis sp. nov. and Azoarcus rhizosphaerae sp. nov., two nitrogen-fixing species isolated from termite nest and rhizosphere of Ficus religiosa.

A polyphasic taxonomic approach was used to characterise two presumably novel bacteria, designated strains CC-YHH838T and CC-YHH848T isolated from termite nest and rhizosphere of Ficus religiosa, respectively. These two nitrogen-fixing strains were observed to be Gram-staining-negative, aerobic rod, and colonies were yellowish in color. Growth of strains was observed at 20-37°C, pH 7-8, and in the presence of 1-2% NaCl. Phylogenetic analyses based on 16S rRNA genes revealed a distinct taxonomic position attained by strain CC-YHH838T and CC-YHH848T associated with Thauera hydrothermalis (97.1% sequence identity), and formed a separate branch with Azoarcus indigens (95.4%), Aromatoleum aromaticum (96.2%), and lower sequence similarity to other species. The calculation of OrthoANI values pointed out strains CC-YHH838T and CC-YHH848T gave 78.9% and 79.8% compared to Thauera hydrothermalis, respectively. The major fatty acids (> 5%) were C16:0, C17:0 cyclo, C10:0 3-OH, C16:1ω7c/C16:1ω6c and C18:1ω7c/C18:1ω6c. The polar lipid profile comprised phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and unidentified aminophospholipid and phospholipids; the predominant polyamines were putrescine and spermidine. The predominant respiratory system was ubiquinone (Q-8) and the DNA G + C contents were 61.4 ± 0.1mol% and 60.2 ± 1.3mol%, respectively. Based on the phylogenetic and polyphasic comparisons, strains CC-YHH838T and CC-YHH848T are proposed to represent two novel species within the genus Azoarcus in the family Rhodocyclaceae, for which the name Azoarcus nasutitermitis sp. nov. (type strain CC-YHH838T = BCRC 81059T = JCM 32001T) and Azoarcus rhizosphaerae sp. nov. (type strain CC-YHH848T = BCRC 81060T = JCM 32002T) were proposed.

Identification and Genome Analysis of Vibrio coralliilyticus Causing Mortality of Pacific Oyster (Crassostrea gigas) Larvae.

Vibrio coralliilyticus is known as a coral pathogen that also infects marine bivalve larvae worldwide. It is considered to be one of the major constraints in artificial marine bivalve seed production as it causes mortality. In this study, we first isolated and characterized a high virulent of V. coralliilyticus designated as SNUTY-1 that was the cause of Pacific oyster larvae mortality in Korea. In the pathogenicity test, exposure to 2.14 × 105 CFU/mL for 24 h caused mortality to 88.65 ± 2.4% of the tested healthy Pacific oyster larvae. SNUTY-1 showed anti-microbial resistance to β-lactams, such as penicillins, cephalosporins, and carbapenems. We sequenced and assembled the complete genome of SNUTY-1 (5,842,676 bp), consisting of two chromosomes (Chr I and Chr II) and two plasmids (pSNUTY1 and pSNUTY2). The COG functional analysis confirmed that Chr I had more genes associated with basic cellular functions in comparison to Chr II. The results of the phylogenetic trees based on OrthoANI values indicated that the SNUTY-1 was closely related to V. coralliilyticus strains. SNUTY-1 had a unique plasmid (pSNUTY2), which could mean that the Korean isolate is different from other sequenced V. coralliilyticus strains from different geographical origins. Toxic proteins such as cytolysin/hemolysin and extracellular metalloprotease genes were encoded on Chr I and Chr II of SNUTY-1. These data facilitate the control of V. coralliilyticus infections in aquaculture by providing valuable insights into the biodiversity of this organism and valuable information for the study of virulence factors.

Genome sequence-based criteria for demarcation and definition of species in the genus Rickettsia.

Over recent years, genomic information has increasingly been used for prokaryotic species definition and classification. Genome sequence-based alternatives to the gold standard DNA-DNA hybridization (DDH) relatedness have been developed, notably average nucleotide identity (ANI), which is one of the most useful measurements for species delineation in the genomic era. However, the strictly intracellar lifestyle, the few measurable phenotypic properties and the low level of genetic heterogeneity made the current standard genomic criteria for bacterial species definition inapplicable to Rickettsia species. We evaluated a range of whole genome sequence (WGS)-based taxonomic parameters to develop guidelines for the classification of Rickettsia isolates at genus and species levels. By comparing the degree of similarity of 74 WGSs from 31 Rickettsia species and 61 WGSs from members of three closely related genera also belonging to the order Rickettsiales (Orientia, 11 genomes; Ehrlichia, 22 genomes; and Anaplasma, 28 genomes) using digital DDH (dDDh) and ANI by orthology (OrthoANI) parameters, we demonstrated that WGS-based taxonomic information, which is easy to obtain and use, can serve for reliable classification of isolates within the Rickettsia genus and species. To be classified as a member of the genus Rickettsia, a bacterial isolate should exhibit OrthoANI values with any Rickettsia species with a validly published name of ≥83.63 %. To be classified as a new Rickettsia species, an isolate should not exhibit more than any of the following degrees of genomic relatedness levels with the most closely related species: >92.30 and >99.19 % for the dDDH and OrthoANI values, respectively. When applied to four rickettsial isolates of uncertain status, the above-described thresholds enabled their classification as new species in one case. Thus, we propose WGS-based guidelines to efficiently delineate Rickettsia species, with OrthoANI and dDDH being the most accurate for classification at the genus and species levels, respectively.

Development of a Kribbella-specific isolation medium and description of Kribbella capetownensis sp. nov. and Kribbella speibonae sp. nov., isolated from soil.

Two actinobacterial strains were isolated from samples collected from the University of Cape Town, South Africa. A third actinobacterial strain was isolated from soil collected in the town of Stellenbosch, South Africa, using a newly-developed Kribbella-selective medium. Analysis of the 16S rRNA genes showed that the three strains belonged to the genus Kribbella. A multilocus sequence analysis using the concatenated gene sequences of the gyrB, rpoB, relA, recA and atpD genes showed that strains YM55T and SK5 were most closely related to the type strains of Kribbella sindirgiensis and Kribbella soli, while strain YM53T was most closely related to the type strain of Kribbella pittospori. Digital DNA-DNA hybridisation and Average Nucleotide Identity (ANI) analyses showed that strains YM55T and SK5 belong to the same genomic species (OrthoANI value = 98.4%), but are distinct from the genomic species represented by the type strains of K. sindirgiensis (OrthoANI values < 95.6%) and K. soli (OrthoANI values < 91.4%). Strain YM53T is distinct from the genomic species represented by the type strain of K. pittospori (OrthoANI value = 94.0%). Phenotypic comparisons showed that strains YM55T and SK5 are distinct from the type strains of K. sindirgiensis and K. soli and that strain YM53T is distinct from the type strain of K. pittospori. Strains YM53T and YM55T are thus presented as the type strains of novel species, for which the names Kribbella capetownensis sp. nov. (= DSM 29426T = NRRL B-65062T) and Kribbella speibonae sp. nov. (= DSM 29425T = NRRL B-59161T), respectively, are proposed.

Colibacter massiliensis gen. nov. sp. nov., a novel Gram-stain-positive anaerobic diplococcal bacterium, isolated from the human left colon

The gut microbiota is considered to play a key role in human health. As a consequence, deciphering its microbial diversity is mandatory. A polyphasic taxonogenomic strategy based on the combination of phenotypic and genomic analyses was used to characterize a new bacterium, strain Marseille-P2911. This strain was isolated from a left colon sample of a 60-year old man who underwent a colonoscopy for an etiological investigation of iron-deficiency anemia in Marseille, France. On the basis of 16S rRNA sequence comparison, the closest phylogenetic neighbor was Anaeroglobus geminatus (94.59% 16S rRNA gene sequence similarity) within the family Veillonellaceae. Cells were anaerobic, Gram-stain-positive, non-spore-forming, catalase/oxidase negative cocci grouped in pairs. The bacterium was able to grow at 37 °C after 2 days of incubation. Strain Marseille-P2911 exhibited a genome size of 1,715,864-bp with a 50.2% G + C content, and digital DNA-DNA hybridization (dDDH) and OrthoANI values with A. geminatus of only 19.1 ± 4.5% and 74.42%, respectively. The latter value being lower than the threshold for genus delineation (80.5%), we propose the creation of the new genus Colibacter gen. nov., with strain Marseille-P2911T (=DSM 103304 = CSUR P2911) being the type strain of the new species Colibacter massiliensis gen. nov., sp. nov.

Salicibibacter halophilus sp. nov., a moderately halophilic bacterium isolated from kimchi.

A Gram-stain-positive, rod-shaped, alkalitolerant, and halophilic bacterium-designated as strain NKC3-5T-was isolated from kimchi that was collected from the Geumsan area in the Republic of Korea. Cells of isolated strain NKC3-5T were 0.5-0.7 μm wide and 1.4-2.8 μm long. The strain NKC3-5T could grow at up to 20.0% (w/v) NaCl (optimum 10%), pH 6.5-10.0 (optimum pH 9.0), and 25-40°C (optimum 35°C). The cells were able to reduce nitrate under aerobic conditions, which is the first report in the genus Salicibibacter. The genome size and genomic G + C content of strain NKC3-5T were 3,754,174 bp and 45.9 mol%, respectively; it contained 3,630 coding sequences, 16S rRNA genes (six 16S, five 5S, and five 23S), and 59 tRNA genes. Phylogenetic analysis based on 16S rRNA showed that strain NKC3-5T clustered with bacterium Salicibibacter kimchii NKC1-1T, with a similarity of 96.2-97.6%, but formed a distinct branch with other published species of the family Bacillaceae. In addition, OrthoANI value between strain NKC3-5T and Salicibibacter kimchii NKC1-1T was far lower than the species demarcation threshold. Using functional genome annotation, the result found that carbohydrate, amino acid, and vitamin metabolism related genes were highly distributed in the genome of strain NKC3-5T. Comparative genomic analysis revealed that strain NKC3-5T had 716 pan-genome orthologous groups (POGs), dominated with carbohydrate metabolism. Phylogenomic analysis based on the concatenated core POGs revealed that strain NKC3-5T was closely related to Salicibibacter kimchii. The predominant polar lipids were phosphatidylglycerol and two unidentified lipids. Anteiso-C15:0, iso-C17:0, anteiso-C17:0, and iso-C15:0 were the major cellular fatty acids, and menaquinone-7 was the major isoprenoid quinone present in strain NKC3-5T. Cell wall peptidoglycan analysis of strain NKC3-5T showed that meso-diaminopimelic acid was the diagnostic diamino acid. The phephenotypic, genomic, phylogenetic, and chemotaxonomic properties reveal that the strain represents a novel species of the genus Salicibibacter, for which the name Salicibibacter halophilus sp. nov. is proposed, with the type strain NKC3-5T (= KACC 21230T = JCM 33437T).

Cellulomonas algicola sp. nov., an actinobacterium isolated from a freshwater alga.

An actinomycete strain, TKZ-21T, was isolated from a freshwater alga (Chetophoraceae) collected from the Takizawa River, Yamanashi, Japan, and examined using a polyphasic taxonomic approach. Cells were Gram-stain positive, aerobic, non-sporulating, motile, and coccoid or short rod-shaped. The strain grew in the presence of 0-4 % (w/v) NaCl, between pH 6-9.4, and over a temperature range of 15-40 °C, with optimum growth at 30 °C. The peptidoglycan type of strain TKZ-21T was A4β, containing l-ornithine as diagnostic diamino acid and d-glutamic acid as the interpeptide bridge. The predominant menaquinone was MK-9(H4). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, ninhydrin-positive glycolipid, and unidentified phospholipids. The major cellular fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0, and the DNA G+C content was 75.6 mol%. On the basis of 16S rRNA gene sequence analysis, strain TKZ-21T was closely related to Cellulomonas fimi (98.5 % sequence similarity) and Cellulomonas biazotea (98.3 %). The genome orthoANI value between strain TKZ-21T and C. biazotea and C. fimi were 84.7 and 84.2 %, respectively. On the basis of fatty acid and MALDI-TOF MS profile analysis, phylogenetic analyses, genomic analysis, and phenotypic data, it is proposed that the isolate be classified as a representative of a novel species of the genus Cellulomonas, with the name Cellulomonas algicola sp. nov. The type strain is TKZ-21T (=NBRC 112905T=TBRC 8129T).

Genomic metrics analyses indicate that Paenibacillus azotofixans is not a later synonym of Paenibacillus durus

Paenibacillus durusand Paenibacillus azotofixans, both Gram-stain-positive and endospore-forming bacilli, have been considered to be a single species. However, a preliminary computation of their average nucleotide identity (ANI) values suggested that these species are not synonyms. Given this, the taxonomic attributions of these species were evaluated through genomic and phylogenomic approaches. Although the identity of 16S rRNA gene sequences of P. durus DSM 1735T and P. azotofixans ATCC 35681T are above the circumscription species threshold, genomic metrics analyses indicate otherwise. ANI, gANI and OrthoANI values computed from their genome sequences were around 92 %, below the species limits. Digital DNA-DNA hybridization and MUMi estimations also corroborated these observations. In fact, in all metrics, Paenibacillus zanthoxyli JH29T seemed to be more similar to Paenibacillus azotofixans. ATCC 35681T than P. durus DSM 1735T. Phylogenetic analyses based on concatenated core-proteome and concatenated gyrB, recA, recN and rpoB genes confirmed that P. zanthoxyli is the closest Paenibacillus species to P. azotofixans. A review of the phenotypic profiles from these three species revealed that their biochemical repertoires are very similar, although P. azotofixans ATCC 35681T can be differentiated from P. durus DSM1735T in 13 among more than 90 phenotypic traits. Considering phylogenetic and genomic analyses, Paenibacillus azotofixans should be considered as an independent species, and not as a later synonym of Paenibacillus durus.

Kineobactrum sediminis gen. nov., sp. nov., isolated from marine sediment.

A novel Gram-stain-negative, rod-shaped marine bacterium, designated strain F02T, was isolated from a marine saltern in Weihai, PR China. The cells of strain F02T were approximately 0.8-1.0×3.0-4.0 µm and motile by means of a polar flagellum. Strain F02T grew optimally at 33-35 °C, pH 7.5 and in the presence of 3.0 % (w/v) NaCl. Strain F02T showed oxidase- and catalase-positive activities. Phylogenetic analysis of 16S rRNA gene sequences revealed that strain F02T belonged to the family Halieaceae and exhibited 16S rRNA gene sequence similarities of 96.6 and 96.4 % to the type strains of Chromatocurvus halotolerans and Parahaliea mediterranea, respectively. The major cellular fatty acids of strain F02T were C18 : 1ω7c, C16 : 1ω7c, C15 : 0 and C18 : 1ω9c. The major polar lipids of strain F02T were phosphatidylglycerol, phosphatidylethanolamine and one unidentified aminolipid. Strain F02T contained Q-8 as the sole respiratory quinone. The genomic DNA G+C content was 58.4 mol%. The genome sequences of strain F02T and Chromatocurvus halotolerans DSM 23344T had an OrthoANI value of 70.5 %, and the average amino acid identity value between the two genomes was 62.6 %. The sequence similarity value between the rpoB genes of strain F02T and Chromatocurvus halotolerans DSM 23344T was 79.5 %. On the basis of polyphasic analysis, strain F02T represents a novel species in a new genus, for which the name Kineobactrum sediminis gen. nov., sp. nov. is proposed. The type strain is F02T (=KCTC 52616T=MCCC 1H00224T).

Suicoccus acidiformans gen. nov., sp. nov., isolated from a sick pig.

A Gram-stain-positive, non-spore-forming, catalase-positive and facultatively anaerobic coccus, designated ZY16052T, was isolated from mesenteric lymph nodes of a sick piglet in Kunming, Yunnan Province, PR China and its taxonomic position was studied by following a polyphasic approach. Optimal growth was observed at 37 °C, pH 8.0 and 2 % NaCl (w/v) on Columbia agar. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZY16052T formed a separated evolutionary lineage from recognized genera of the family Aerococcaceae and shared low similarity to its closest related species Facklamiasourekii (93.8 %) and Ignavigranum ruoffiae (93.4 %). Phylogenetic analysis based on the housekeeping gene recA indicated that strain ZY16052T represented a deep and distinct evolutionary lineage, and was well separated from all genera in the family Aerococcaceae, with very low sequence similarity(≤73.2 %). Sequence analysis based on the housekeeping gene rpoA indicated that strain ZY16052T shared very low similarity ≤77.0 % to related genera. The genomic OrthoANI values between strain ZY16052T and type species of related genera in the family Aerococcaceae and species in the genus Facklamia were ≤67.77 and ≤68.11 %, respectively. The genomic G+C content was 42.3 mol%. The predominant fatty acids (>5 %) were C16 : 0, C18 : 1ω9c, C14 : 0 and summed feature 5 (C18 : 2ω6,9c and/or C18 : 0 ante). The major polar lipids were digalactosyldiacylglycerol, phosphatidylglycerol, diacylglycerols, triacylglycerol and phosphatidic acid. The peptidoglycan contained the amino acids lysine, glycine, alanine and glutamic acid, which is characteristic of peptidoglycan type A1a. Based on the phylogenetic and phenotypic evidence, we propose that the unknown bacterium be classified as Suicoccus acidiformans gen. nov., sp. nov. The type strain of Suicoccus acidiformans is ZY16052T (=CCTCC AB 2017017T=DSM 105755T).

Alteromonas alba sp. nov., a marine bacterium isolated from seawater of the West Pacific Ocean.

A Gram-stain-negative, strictly aerobic, motile by one polar flagellum, non-spore-forming, rod-shaped bacterium, designated as 190T, was isolated from seawater of the West Pacific Ocean and subjected to a polyphasic taxonomic investigation. Colonies were 1.0-2.0 mm in diameter, smooth, circular, convex and white after growth on marine agar at 30 °C for 24 h. Strain 190T was found to grow at 4-40 °C (optimum, 30 °C), at pH 5.5-10.5 (optimum, pH 6.5) and with 0.5-12.5 % (w/v) NaCl (optimum, 2.0 %). Chemotaxonomic analysis showed the sole respiratory quinone was ubiquinone 8 (Q-8), and the major fatty acids were summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH), C16 : 0 and summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c). The major polar lipids were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), one unidentified aminolipid (AL1) and two unidentified glycolipids (GL1, GL2). The DNA G+C content of strain 190T was 48.7 mol% based on the genome sequence. The comparison of 16S rRNA gene sequence similarities showed that strain 190T was closely related to Alteromonas oceani S35T (99.6 % sequence similarity), A. lipolytica JW12T (98.2 %), A. aestuariivivens JDTF-113T (97.7 %) and A. mediterranea DET (97.5 %); it exhibited 97.0 % or less sequence similarity with the type strains of other species with validly published names. Phylogenetic trees reconstructed with the neighbour-joining, maximum-parsimony and maximum-likelihood methods based on 16S rRNA gene sequences showed that strain 190T constituted a separate branch with A. oceani, A. confluentis, A. aestuariivivens and A. lipolytica in a clade of the genus Alteromonas. OrthoANI values between strain 190T and A. oceani S35T and A. lipolytica JW12T were 93.5 and 77.9 %, respectively, and in silico DNA-DNA hybridization values were 53.8 and 21.2 %, respectively. Differential phenotypic properties, together with phylogenetic distinctiveness, demonstrated that strain 190T is clearly distinct from recognized species of the genus Alteromonas. On the basis of these features, we propose that strain 190T (=MCCC 1K03456T=KCTC 62227T) represents a novel species of the genus Alteromonas with the name Alteromonas alba sp. nov.

Nocardioides silvaticus sp. nov., isolated from forest soil.

A Gram-stain-positive, non-motile, rod-shaped bacterial strain S-34T was isolated from forest soil. According to 16S rRNA gene sequence analysis, strain S-34T was related to Nocardioides members and showed the highest similarities to Nocardioides thalensis NCCP-696T (97.3 %) and Nocardioides panacisoliGsoil 346T (97.0 %), Nocardioides litorisoli X-2T (96.5 %) and Nocardioides immobilis FLL521T (96.4 %). Phylogenetic trees showed that strain S-34T fell within the cluster containing strain S-34T and N. immobilis FLL521T. The levels of DNA-DNA relatedness between strain S-34T and N. thalensis CCTCC AB 2016296T and between strain S-34T and N. panacisoli KCTC 19470T were 50.6 and 58.8 %, respectively. The genome orthoANI value between strain S-34T and N. immobilis CCTCC AB 2017083T was 82.4 %. Strain S-34T had ll-diaminopimelic acid in the cell-wall peptidoglycan, diphosphatidylglycerol, phosphatidylglycerol, four unknown phospholipids and one unknown lipid as the polar lipids, meanquinone-8(H4) as the only respiratory quinone and iso-C16 : 0, C17:1ω8c, C17:1ω6c, C17 : 0 and C17 : 0 10-methyl (tbsa) as the major fatty acids. The genome length of strain S-34T was 4.53 Mb containing 52 contigs and with a DNA G+C content of 71.2 mol%. Strain S-34T could be distinguished from the other Nocardioides members mainly based on the data of phylogenetic analyses, DNA-DNA hybridization, polar lipids and some biochemical differences. Therefore, strain S-34T represents a novel species of the genus Nocardioides, for which the name Nocardioidessilvaticus sp. nov. is proposed. The type strain is S-34T (=KCTC 49137T=CCTCC AB 2018079T).

Halorubrum chaoviator Mancinelli et al. 2009 is a later, heterotypic synonym of Halorubrum ezzemoulense Kharroub et al. 2006. Emended description of Halorubrum ezzemoulense Kharroub et al. 2006.

A polyphasic comparative taxonomic study of Halorubrum ezzemoulense Kharroub et al. 2006, Halorubrum chaoviator Mancinelli et al. 2009 and eight new Halorubrum strains related to these haloarchaeal species was carried out. Multilocus sequence analysis using the five concatenated housekeeping genes atpB, EF-2, glnA, ppsA and rpoB', and phylogenetic analysis based on the 757 core protein sequences obtained from their genomes showed that Hrr. ezzemoulense DSM 17463T, Hrr. chaoviator Halo-G*T (=DSM 19316T) and the eight Halorubrum strains formed a robust cluster, clearly separated from the remaining species of the genus Halorubrum. The orthoANI value and digital DNA-DNA hybridization value, calculated by the Genome-to-Genome Distance Calculator (GGDC), showed percentages among Hrr. ezzemoulense DSM 17463T, Hrr. chaoviator DSM 19316T and the eight Halorubrum strains ranging from 99.4 to 97.9 %, and from 95.0 to 74.2 %, respectively, while these values for those strains and the type strains of the most closely related species of Halorubrum were 88.7-77.4 % and 36.1-22.3 %, respectively. Although some differences were observed, the phenotypic and polar lipid profiles were quite similar for all the strains studied. Overall, these data show that Hrr. ezzemoulense, Hrr. chaoviator and the eight new Halorubrum isolates constitute a single species. Thus, Hrr. chaoviator should be considered as a later, heterotypic synonym of Hrr. ezzemoulense. We propose an emended description of Hrr. ezzemoulense, including the features of Hrr. chaoviator and those of the eight new isolates.

Confluentibacter flavum sp. nov., Isolated from the Saline Lake.

A Gram-stain-negative, rod-shaped, non-motile, bacterial isolate designated 3BT, was isolated from a saline lake, and subjected to a polyphasic taxonomic investigation. The phylogenetic analysis based on 16S rRNA gene sequence clearly showed an allocation to the genus Confluentibacter with similarity ranging from 95.1 to 98%. OrthoANI values between strain 3BT and related strains of Confluentibacter (< 90%) were lower than the threshold value of 95% ANI relatedness recommended for species demarcation. Strain 3BT grew at 4-35°C and pH 6.0-8.0 (optimum, 28°C and pH 6.5) and with 0-3% (w/v) NaCl (optimum, 0.5%). The predominant respiratory quinone was menaquinone-6 (MK-6) and the major fatty acids were iso-C15:0, iso-C15:1 G, iso-C15:0 3-OH, and iso-C17:0 3-OH. The polar lipid profile of strain 3BT comprised phosphatidylethanolamine, one unidentified aminolipid, one aminophospholipid, and three unidentified lipids (L1-3). The DNA G+C content was 33.1mol%. On the basis of morphological, physiological, and chemotaxonomic characteristics, together with the results of phylogenetic analysis, strain 3BT is described as a novel species in genus Confluentibacter, for which the name Confluentibacter flavum sp. nov. (type strain 3BT = CGMCC115960T = KCTC52969T) is proposed.

Genome-based insights into a putative novel Methylomonas species (strain Kb3), isolated from an Indian rice field

Methylomonas is one of the most important and frequently detected genera in aquatic habitats and rice fields. A strain of Methylomonas, Kb3, an aerobic methanotroph, was isolated from a rice field from the Western parts of India. Kb3 cells were composed of short, motile rods and formed pink colored colonies and pink pellicles in liquid. On the basis of 16S rRNA gene, the closest valid species was Methylomonas methanica strain S1 (97.9% sequence similarity). Kb3 also showed close affiliation to “Methylomonas denitrificans” FJG1, a recently described, but not yet valid species (99.1% sequence similarity) and to strain LW13, a freshwater lake Washington isolate (99.5% sequence similarity). To understand the correct taxonomical position and metabolic properties of Kb3, the whole genome was sequenced. Kb3 genome showed a complete methane oxidation pathway, the presence of pMMO and sMMO genes, genes for nitrogen fixation and potential to convert nitrite to nitric oxide and nitric oxide to nitrous oxide. Numerous genes for chemotaxis and motility were detected. Genome comparison of Kb3 with the genomes of Methylomonas methanica S1 and “Methylomonas denitrificans” FJG1 showed ortho-ANI values of 86–87%, much less than 95%, the current cut-off for species. Digital DNA-DNA hybridization also showed values of 41–42%, much lesser than the 70% cut-off, both indicating that Kb3 did not belong to any of these species. Methylomonas LW13 and Methylomonas Kb3 showed close genome level similarities, i.e. OrthoANI value of 97.9% and a DDH of 87.3% and close branching in the phylogenomic tree confirming that Kb3 and LW13 can together form a new species.

Application of OrthoANI combined with MALDI-TOF MS in identification and typing of Campylobacter fetus

Objective Establishing mass spectrum library of a Campylobacter- C.fetus subsp.testudinum for rapid species identification in clinical microbiology laboratory. Methods Illumina second generation sequencing platform 2000/miSeq was used to carry out high flux genome sequencing for strains which were collected to establish mass spectrum library.The analysis oforthologous average nucleotide identity (OrthoANI) between collected strains and reference strains was performed at JAVA 8 operation environment. Then, mass spectrums ofcollected strains andreference strains were acquired using MALDI-TOF MS. And mass spectrum library of C. fetus subsp.testudinum. were established and verified. Results The OrthoANI analysis showed that OrthoANI value of collected strains and reference strain C. fetus subsp.testudinum03-427 was 99.30%-99.96%, while OrthoANI values of collected strains and C. fetus subsp.venerealisNCTC10354 orC.fetus subsp.fetus82-40 were 91.05%-92.26%. With reference to OrthoANI ≥ 95% as basis for determination of same strain, strains which collected to establish mass spectrum library was finally identified as C. fetus subsp.testudinum . The identification accuracy rate of mass spectrum library was 100% (consistent with gene sequencing), and confidence interval was 82.3%-99.9%, identification of same strain is 100% reproducible. Conclusions The new gold standard based on high throughput sequencing and total genome analysis has provided ideal reference value for establishment of mass spectrum library.And accurate and objective reference spectrum of the C.fetus subsp.testudinum provides a platform for rapid diagnosis of fetal Campylobacter infection. (Chin J Lab Med, 2018, 41: 583-588) Key words: Campylobacter fetus; Bacteremia; Genomics; High-throughput nucleotide sequencing; Spectrometry, mass, matrix-assisted laser desorption-ionizatino; Bacterial typing techniques

Novel haloarchaeon Natrinema thermophila having the highest growth temperature among haloarchaea with a large genome size

Environmental temperature is one of the most important factors for the growth and survival of microorganisms. Here we describe a novel extremely halophilic archaeon (haloarchaea) designated as strain CBA1119T isolated from solar salt. Strain CBA1119T had the highest maximum and optimal growth temperatures (66 °C and 55 °C, respectively) and one of the largest genome sizes among haloarchaea (5.1 Mb). It also had the largest number of strain-specific pan-genome orthologous groups and unique pathways among members of the genus Natrinema in the class Halobacteria. A dendrogram based on the presence/absence of genes and a phylogenetic tree constructed based on OrthoANI values highlighted the particularities of strain CBA1119T as compared to other Natrinema species and other haloarchaea members. The large genome of strain CBA1119T may provide information on genes that confer tolerance to extreme environmental conditions, which may lead to the discovery of other thermophilic strains with potential applications in industrial biotechnology.