Parvicella tangerina gen. nov., sp. nov. (Parvicellaceae fam. nov., Flavobacteriales), first cultured representative of the marine clade UBA10066, and Lysobacter luteus sp. nov., from activated sludge of a seawater-processing wastewater treatment plant.

Two strains isolated from a sample of activated sludge that was obtained from a seawater-based wastewater treatment plant on the southeastern Mediterranean coast of Spain have been characterized to achieve their taxonomic classification, since preliminary data suggested they could represent novel taxa. Given the uniqueness of this habitat, as this sort of plants are rare in the world and this one used seawater to process an influent containing intermediate products from amoxicillin synthesis, we also explored their ecology and the annotations of their genomic sequences. Analysis of their 16S rRNA gene sequences revealed that one of them, which was orange-pigmented, was distantly related to Vicingus serpentipes (family Vicingaceae) and to other representatives of neighbouring families in the order Flavobacteriales (class Flavobacteriia) by 88-89 % similarities; while the other strain, which was yellow-pigmented, was a putative new species of Lysobacter (family Xanthomonadaceae, order Xanthomonadales, class Gammaproteobacteria) with Lysobacter arseniciresistens as closest relative (97.3 % 16S rRNA sequence similarity to its type strain). Following a polyphasic taxonomic approach, including a genome-based phylogenetic analysis and a thorough phenotypic characterization, we propose the following novel taxa: Parvicella tangerina gen. nov., sp. nov. (whose type strain is AS29M-1T=CECT 30217T=LMG 32344T), Parvicellaceae fam. nov. (whose type genus is Parvicella), and Lysobacter luteus sp. nov. (whose type strain is AS29MT=CECT 30171T=LMG 32343T).

Flavobacterium difficile sp. nov., isolated from a freshwater waterfall.

A bacterial strain designated KDG-16 T is isolated from a freshwater waterfall in Taiwan and characterized to determine its taxonomic affiliation. Cells of strain KDG-16 T are Gram-stain-negative, strictly aerobic, motile by gliding, rod-shaped and form light yellow colonies. Optimal growth occurs at 20-25 °C, pH 6-7, and with 0% NaCl. Phylogenetic analyses based on 16S rRNA gene sequences and an up-to-date bacterial core gene set reveal that strain KDG-16 T is affiliated with species in the genus Flavobacterium. Analysis of 16S rRNA gene sequences shows that strain KDG-16 T shares the highest similarity with Flavobacterium terrigena DSM 17934 T (97.7%). The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between strain KDG-16 T and the closely related Flavobacterium species are below the cut-off values of 95-96, 90 and 70%, respectively, used for species demarcation. Strain KDG-16 T contains iso-C15:0, iso-C15:1 G and iso-C17:0 3-OH as the predominant fatty acids. The polar lipid profile consists of phosphatidylethanolamine, one uncharacterized aminophospholipid, one uncharacterized phospholipid, two uncharacterized aminolipids and two uncharacterized lipids. The major polyamine is homospermidine. The major isoprenoid quinone is MK-6. Genomic DNA G + C content of strain KDG-16 T is 31.6%. Based on the polyphasic taxonomic data obtained, strain KDG-16 T is considered to represent a novel species in the genus Flavobacterium, for which the name Flavobacterium difficile sp. nov. is proposed. The type strain is KDG-16 T (= BCRC 81194 T = LMG 31332 T).

Flavobacterium undicola sp. nov., isolated from a freshwater lake.

Bacterial strain BBQ-18T, isolated from a freshwater lake in Taiwan, is characterized using a polyphasic taxonomy approach. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicate that strain BBQ-18T forms a phylogenetic lineage in the genus Flavobacterium. Strain BBQ-18T is most closely related to Flavobacterium alvei HR-AYT with 98.5% 16S rRNA gene sequence similarity. Strain BBQ-18T shows 70.5-89.5% average nucleotide identity and 13.7-38.2% digital DNA-DNA hybridization identity with the type strains of other closely related Flavobacterium species. The strain is Gram-stain negative, strictly aerobic, motile by gliding, rod shaped and formed yellow colonies. Optimal growth occurs at 25 °C, pH 6, and in the absence of NaCl. Strain BBQ-18T contains iso-C15:0, summed feature 3 (C16:1ω6c and/or C16:1ω7c) and anteiso-C15:0 as the predominant fatty acids. The polar lipid profile consists of phosphatidylethanolamine, four uncharacterized aminophospholipids and two uncharacterized phospholipids. The major polyamine is homospermidine. The major isoprenoid quinone is MK-6. The DNA G+C content of the genomic DNA is 33.8%. Differential phenotypic properties, together with the phylogenetic inference, demonstrate that strain BBQ-18T should be classified as a novel species of the genus Flavobacterium, for which the name Flavobacterium undicola sp. nov. is proposed. The type strain is BBQ-18T (= BCRC 81050T = LMG 30052T = KCTC 52810T).

Flavobacterium ichthyis sp. nov., isolated from a fish pond.

Bacterial strain NST-5T, isolated from a fish pond in Taiwan, was characterized using a polyphasic taxonomy approach. Results of phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicated that strain NST-5T formed a phylogenetic lineage in the genus Flavobacterium. Analysis of 16S rRNA gene sequences showed that strain NST-5T showed the highest similarity to Flavobacterium enshiense DK69T (94.9 %), Flavobacterium ahnfeltiae 10Alg 130T (94.8 %) and Flavobacterium vireti THG-SM1T (94.8 %). Strain NST-5T showed 68.9-72.5% average nucleotide identity and 19.1-23.7% digital DNA-DNA hybridization identity with the type strains of other close related Flavobacterium species. Cells of the strain were Gram-stain-negative, strictly aerobic, motile by gliding, rod-shaped and formed yellow colonies. Optimal growth occurred at 30 °C, pH 7 and with 0.5% NaCl. Strain NST-5T contained iso-C15:0, C15:0 and iso-C16:0 as the predominant fatty acids. The major hydroxyl fatty acids were iso-C16:0 3-OH and iso-C17:0 3-OH. The polar lipid profile consisted of phosphatidylethanolamine, three uncharacterized aminophospholipids, two uncharacterized phospholipids and one uncharacterized aminolipid. The major polyamine was homospermidine. The major isoprenoid quinone was MK-6. The DNA G+C content of the genomic DNA was 35.5 mol%. Differential phenotypic properties, together with the phylogenetic inference, demonstrate that strain NST-5T should be classified as a novel species of the genus Flavobacterium, for which the name Flavobacterium ichthyis sp. nov. is proposed. The type strain is NST-5T (=BCRC 81198T=LMG 31341T).

Description of Gelidibacter japonicus sp. nov., isolated from the Inland Sea (Setonaikai) in Japan.

A novel Gelidibacter strain, JCM 31967T, was isolated from seawater collected from the Inland Sea (Setonaikai) in Japan. It was characterized as a Gram-negative, halophilic, oxidase-negative, catalase-positive, aerobic, nonmotile, but gliding, rod-shaped bacterium without flagella. Based on 16S rDNA gene identity, strain JCM 31967T is closely related to Gelidibacter mesophilus (DSM 14095T, 96.6% identity), G. gilvus (IC158T, 96.4%), G. algens (DSM 12408T, 96.1%), G. sediminis (S11-41T, 94.7%), and G. salicanalis (IC162T, 94.7%). The G + C content of strain JCM 31967T DNA was found to be 39.1%. The average nucleotide identity (ANI) values of JCM 31967T against G. algens DSM 12408T and G. mesophilus DSM 14095T were 79.1% and 80.9%, respectively. Strain JCM 31967T phenotypically differed from the closest related Gelidibacter species in its utilization of methyl α-D-mannopyranoside, methyl α-D-glucopyranoside, and D-ribose and in its lack of utilization of L-arginine and D-arabinose. It was further differentiated based on its fatty acid composition, specifically properties of C18:0 and C20:2 ω6c, 9c, which were significantly different from those of G. algens, G. gilvus, G. mesophilus, G. salicanalis, and G. sediminis type strains. Overall, the results of DNA-DNA hybridization and physiological and biochemical analyses differentiated strain JCM 31967T from a previously described species of Gelidibacter. Based on these polyphasic taxonomic findings, it was concluded that strain JCM 31967T is a novel Gelidibacter species, for which the name Gelidibacter japonicus sp. nov. is proposed, with JCM 31967T (= LMG 30063T) as the type strain.

Amniculibacterium aquaticum gen. nov., sp. nov., a new member of the family Flavobacteriaceae isolated from a stream.

Strain KYPW7T, isolated from the Funglin Stream in Taiwan, was characterized using a polyphasic taxonomy approach. Cells of strain KYPW7T were Gram-stain-negative, aerobic, non-spore forming, non-motile rods and formed white colonies. Growth occurred at 15-30 °C (optimum 25 °C), at pH 6-8 (optimum pH 6.5) and with 0-1% NaCl (optimum 0%). Phylogenetic analyses based on 16S rRNA and coding sequences of 92 protein clusters showed that strain KYPW7T represents a novel genus in the family Flavobacteriaceae. The 16S rRNA gene sequence of strain KYPW7T was related to the species of the genera Chryseobacterium (91.8-96.0% sequence similarity), Bergeyella (95.1-95.8%), Cloacibacterium (94.5-95.7%), Daejeonia (95.6%) and Riemerella (94.0-95.0%). Strain KYPW7T showed less than 72% average nucleotide identity and less than 24% digital DNA-DNA hybridization identity compared to the type strains of related genera within the family Flavobacteriaceae. The predominant fatty acids were iso-C15:0 and iso-C17:0 3-OH. The major isoprenoid quinone was MK-6 and the DNA G + C content was 36.8 mol%. The polar lipids had phosphatidylethanolamine, three uncharacterized aminophospholipids and an uncharacterized phospholipid. The polyamines contained homospermidine, putrescine and spermidine. On the basis of the genotypic and phenotypic data, strain KYPW7T represents a novel species of a new genus in the family Flavobacteriaceae, for which the name Amniculibacterium aquaticum gen. nov., sp. nov. is proposed. The type strain is KYPW7T (= BCRC 81123T = LMG 30598T = KCTC 62512T).

Muricauda sediminis sp. nov., isolated from western Pacific Ocean sediment.

A Gram-stain-negative bacterium, designated strain 40Bstr401T, was isolated from a sediment sample collected from the western Pacific Ocean. Analysis of its 16S rRNA gene sequence revealed that strain 40Bstr401T belongs to the genus Muricauda and is closely related to type strains Muricauda antarctica Ar-22T (98.2 %), Muricauda taeanensis 105T (98.2 %) and Muricauda beolgyonensis BB-My12T (97.4 %). The average nucleotide identity values for 40Bstr401T with M. antarctica Ar-22T and M. taeanensis 105T are 79.3 % and 78.8 %, respectively. The in silico DNA-DNA hybridization values between strain 40Bstr401T and M. antarctica Ar-22T and M. taeanensis 105T are 26.7 and 26.6 %, respectively. The major isoprenoid quinone of 40Bstr401T is MK-6, and iso-C17 : 0 3-OH and iso-C15 : 0 are the dominant cellular fatty acids. The major polar lipids are phosphatidylethanolamine, four unidentified amino lipids and two unidentified lipids. The G+C content of the genomic DNA is 42.9 mol%. Its phylogenetic distinctiveness and chemotaxonomic differences, together with the phenotypic properties observed in this study, indicate that strain 40Bstr401T can be differentiated from closely related species. Therefore, we propose strain 40Bstr401T represents a novel species in the genus Muricauda, for which the name Muricauda sediminis sp. nov. is suggested. The type strain is 40Bstr401T (=MCCC 1K04568T=KCTC 82139T).

Flavobacterium sufflavum sp. nov., isolated from a freshwater lake.

A bacterial strain, BBQ-12T, was isolated from a freshwater lake in Taiwan. The strain was Gram-stain-negative, strictly aerobic, motile by gliding, rod-shaped and formed yellow colonies. Optimal growth occurred at 25 °C, pH 6 and in the absence of NaCl. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicated that strain BBQ-12T formed a phylogenetic lineage in the genus Flavobacterium. Strain BBQ-12T was most closely related to Flavobacterium fluminis 3R17T with 98.1 % 16S rRNA gene sequence similarity. Strain BBQ-12T showed 74.4-83.1 % average nucleotide identity and 16.0-21.8 % digital DNA-DNA hybridization identity with the type strains of other closely related Flavobacterium species. Strain BBQ-12T contained iso-C15 : 0, summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) and anteiso-C15 : 0 as the predominant fatty acids. The polar lipid profile consisted of phosphatidylethanolamine, four uncharacterized aminophospholipids and three uncharacterized phospholipids. The major polyamine was homospermidine. The major isoprenoid quinone was MK-6. The DNA G+C content of the genomic DNA was 34.2 mol%. Differential phenotypic properties, together with the phylogenetic inference, demonstrate that strain BBQ-12T should be classified as a novel species of the genus Flavobacterium, for which the name Flavobacteriumsufflavum sp. nov. is proposed. The type strain is BBQ-12T (=BCRC 81049T=LMG 30051T=KCTC 52809T).

Flavobacterium lotistagni sp. nov. and Flavobacterium celericrescens sp. nov., isolated from freshwater habitats.

This study presents taxonomic descriptions of strains CYK-4T and TWA-26T isolated from freshwater habitats in Taiwan. Both strains were Gram-stain-negative, strictly aerobic, motile by gliding and rod-shaped. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicated that both strains belonged to the genus Flavobacterium. Analysis of 16S rRNA gene sequences showed that strains CYK-4T and TWA-26T shared 92.7 % sequence similarity and were most closely related to Flavobacterium ovatum W201ET (95.6 %) and Flavobacterium aquaticum JC164T (96.7 %), respectively. Both strains shared common chemotaxonomic characteristics comprising MK-6 as the main isoprenoid quinone, iso-C15 : 0 and iso-C15 : 1 G as the predominant fatty acids, phosphatidylethanolamine as the principal polar lipid, and homospermidine as the major polyamine. The DNA G+C contents of strains CYK-4T and TWA-26T were 41.5 and 31.8 mol%, respectively. The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between these two novel isolates and their closest relatives were below the cut-off values of 95-96, 90 and 70 %, respectively, used for species demarcation. On the basis of phenotypic and genotypic properties and phylogenetic inference, both strains should be classified as novel species within the genus Flavobacterium, for which the names Flavobacterium lotistagni sp. nov. (type strain CYK-4T=BCRC 81192T=LMG 31330T) and Flavobacterium celericrescens sp. nov. (type strain TWA-26T=BCRC 81200T=LMG 31333T) are proposed.

Flavobacterium piscinae sp. nov., isolated from a fish pond.

Strain ICH-30T was isolated from a freshwater fish pond in Taiwan. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicated that strain ICH-30T formed a phylogenetic lineage in the genus Flavobacterium. Strain ICH-30T was most closely related to Flavobacteriumlacus NP180T with 97.0 % 16S rRNA gene sequence similarity. Strain ICH-30T showed 70.0-83.1 % average nucleotide identity and 16.5-23.7 % digital DNA-DNA hybridization identity with the type strains of other closely related Flavobacterium species. Cells of strain ICH-30T were Gram-stain-negative, strictly aerobic, non-motile, rod-shaped and formed dark orange colonies. Optimal growth occurred at 20-30 °C, pH 8-9 and 0-0.5 % NaCl. Strain ICH-30T contained iso-C15 : 1 G and iso-C15 : 0 as the predominant fatty acids. The major hydroxyl fatty acids were iso-C17 : 0 3-OH, iso-C16 : 0 3-OH and iso-C15 : 0 3-OH. The polar lipid profile consisted of phosphatidylethanolamine, six uncharacterized aminophospholipids, one uncharacterized phospholipid and two uncharacterized lipids. The major polyamine was homospermidine. The only isoprenoid quinone was MK-6. The DNA G+C content of the genomic DNA was 34.3 mol%. On the basis of the phylogenetic inference and phenotypic data, strain ICH-30T should be classified as a novel species, for which the name Flavobacterium piscinae sp. nov. is proposed. The type strain is ICH-30T (=BCRC 81122T=LMG 30579T=KCTC 62513T).

Flavobacterium niveum sp. nov., isolated from a freshwater creek.

Strain TAPW14T was isolated from a freshwater creek in Taiwan. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain TAPW14T belonged to the genus Flavobacterium and was most closely related to Flavobacterium akiainvivens IK-1T (96.6 % sequence identity) and Flavobacterium hauense BX12T (96.0 %) and less than 96 % sequence similarity to other members of the genus. Cells of strain TAPW14T were Gram-negative, strictly aerobic, motile by gliding, rod-shaped and formed white colonies. Optimal growth occurred at 20 °C, pH 7 and in the presence of 0.5 % NaCl. Strain TAPW14T contained summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), iso-C15 : 0 and C16 : 0 as the predominant fatty acids. The polar lipid profile consisted of phosphatidylethanolamine, three uncharacterized aminophospholipids, one uncharacterized phospholipid and one uncharacterized lipid. The major polyamine was homospermidine. The major isoprenoid quinone was MK-6. The DNA G+C content of the genomic DNA was 46.0 mol%. On the basis of the phylogenetic inference and phenotypic data, strain TAPW14T should be classified as a novel species, for which the name Flavobacteriumniveum sp. nov. is proposed. The type strain is TAPW14T (=BCRC 81055T=LMG 30057T=KCTC 52808T).

Atypical flavobacteria recovered from diseased fish in the Western United States.

Flavobacterial diseases, caused by bacteria in the order Flavobacteriales, are responsible for devastating losses in farmed and wild fish populations worldwide. The genera Flavobacterium (Family Flavobacteriaceae) and Chryseobacterium (Weeksellaceae) encompass the most well-known agents of fish disease in the order, but the full extent of piscine-pathogenic species within these diverse groups is unresolved, and likely underappreciated. To identify emerging agents of flavobacterial disease in US aquaculture, 183 presumptive Flavobacterium and Chryseobacterium isolates were collected from clinically affected fish representing 19 host types, from across six western states. Isolates were characterized by 16S rRNA gene sequencing and phylogenetic analysis using the gyrB gene. Antimicrobial susceptibility profiles were compared between representatives from each major phylogenetic clade. Of the isolates, 52 were identified as Chryseobacterium species and 131 as Flavobacterium. The majority of Chryseobacterium isolates fell into six clades (A-F) consisting of ≥ 5 fish isolates with ≥ 70% bootstrap support, and Flavobacterium into nine (A-I). Phylogenetic clades showed distinct patterns in antimicrobial susceptibility. Two Chryseobacterium clades (F & G), and four Flavobacterium clades (B, G-I) had comparably high minimal inhibitory concentrations (MICs) for 11/18 antimicrobials tested. Multiple clades in both genera exhibited MICs surpassing the established F. psychrophilum breakpoints for oxytetracycline and florfenicol, indicating potential resistance to two of the three antimicrobials approved for use in finfish aquaculture. Further work to investigate the virulence and antigenic diversity of these genetic groups will improve our understanding of flavobacterial disease, with applications for treatment and vaccination strategies.

Out From the Shadows - Resolution of the Taxonomy of the Family Cryomorphaceae.

The family Cryomorphaceae for many years has been a poorly defined taxonomic group within the order Flavobacteriales, phylum Bacteroidetes. Members of the Cryomorphaceae, apparently consisting of multiple-family level clades, have been mostly but not exclusively detected in saline ecosystems. The problems with the taxonomy of this group have stemmed from inadequate resolution of taxonomic groups using 16S rRNA gene sequences, sparse numbers of cultivated taxa, and limited phenotypic distinctiveness. The Genome Tiaxonomc Database (GTDB), which is based on normalized taxonomic ranks includes Cryomorphaceae as containing the genera Owenweeksia and Schleiferia. This is at odds with the official taxonomy that places these genera in the family Schleiferiaceae. The other Cryomorphaceae affiliated species have even more uncertain taxonomic positions including Cryomorpha ignava. To clarify the taxonomy of Cryomorphaceae, genomes were generated for all type strains of the family Cryomorphaceae lacking such data. The GTDB-toolkit (GTDB-tk) was used to place taxa in the GTDB, which revealed novelty at the family level for some of these type strains. 16S rRNA gene sequences and concatenated protein sequences were used to further evaluate the taxonomy of the order Flavobacteriales. From the data, the GTDB enabled successful clarification of the taxonomy of the family Cryomorphaceae. A number of placeholder families were given Latinized names. It is proposed that the family Cryomorphaceae is emended to include only the species Cryomorpha ignava. The family Schleiferiaceae is emended to account for the expansion of its membership. Luteibaculum oceani represents a new family designated Luteibaculaceae fam. nov. Vicingus serpentipes is the representative of Vicingaceae fam. nov. while Salibacter halophilus represents Salibacteraceae fam. nov.

Comparative genome analysis of the Flavobacteriales bacterium strain UJ101, isolated from the gut of Atergatis reticulatus.

Here we report the comparative genomic analysis of strain UJ101 with 15 strains from the family Flavobacteriaceae, using the CGExplorer program. Flavobacteriales bacterium strain UJ101 was isolated from a xanthid crab, Atergatis reticulatus, from the East Sea near Korea. The complete genome of strain UJ101 is a 3,074,209 bp, single, circular chromosome with 30.74% GC content. While the UJ101 genome contains a number of annotated genes for many metabolic pathways, such as the Embden-Meyerhof pathway, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and the glyoxylate cycle, genes for the Entner-Douddoroff pathway are not found in the UJ101 genome. Overall, carbon fixation processes were absent but nitrate reduction and denitrification pathways were conserved. The UJ101 genome was compared to genomes from other marine animals (three invertebrate strains and 5 fish strains) and other marine animal- derived genera. Notable results by genome comparisons showed that UJ101 is capable of denitrification and nitrate reduction, and that biotin-thiamine pathway participation varies among marine bacteria; fish-dwelling bacteria, freeliving bacteria, invertebrate-dwelling bacteria, and strain UJ101. Pan-genome analysis of the 16 strains in this study included 7,220 non-redundant genes that covered 62% of the pan-genome. A core-genome of 994 genes was present and consisted of 8% of the genes from the pan-genome. Strain UJ101 is a symbiotic hetero-organotroph isolated from xanthid crab, and is a metabolic generalist with nitrate-reducing abilities but without the ability to synthesize biotin. There is a general tendency of UJ101 and some fish pathogens to prefer thiamine-dependent glycolysis to gluconeogenesis. Biotin and thiamine auxotrophy or prototrophy may be used as important markers in microbial community studies.