A Report of 18 Unrecorded Prokaryotic Species Isolated

Journal of Species Research 9(4):325-338, 2020

A report of 18 unrecorded prokaryotic species isolated from the feces of an Oriental stork (Ciconia boyciana), and from the intestinal tracts of a cobitid fish (Kichulchoia multifasciata) and a Korean splendid dace (Coreoleuciscus splendidus)

† † So-Yeon Lee , Jeong Eun Han , Pil Soo Kim and Jin-Woo Bae

1Department of Biology and Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 02447, Republic of Korea

*Correspondent: [email protected] †These authors contributed equally to this work.

The animal gut is filled with highly diverse microbes associated with host metabolism, physiology, and pathology. However, numerous animal gut microbes have not been cultured or reported. We isolated various bacterial species using culture-dependent approaches during a comprehensive investigation of endangered endemic vertebrate species in the Republic of Korea. A total of 18 unrecorded bacterial species were isolated from the feces of an Oriental stork (Ciconia boyciana), and from the intestinal tracts of a cobitid fish (Kichulchoia multifasciata) and a Korean splendid dace (Coreoleuciscus splendidus). Based on a phylogenetic analysis of 16S rRNA gene sequences, we discovered species belonging to the phyla Actinobacteria (eight species), Firmicutes (seven species), Proteobacteria (two species), and Bacteroidetes (one species). Based on their high 16S rRNA gene sequence similarities (>98.7%) and formation of monophyletic clades with type species, each species was classified into an independent and predefined bacterial species. Gram-stain reaction, colony and cell morphology, basic biochemical characteristics, isolation source, and NIBR IDs for each species are described in the species description section. Keywords: ‌16S rRNA gene sequences, phylogenetic analysis, unreported bacterial species

Introduction gate uncultured or even unculturable bacterial species. Nevertheless, marker gene-based, culture-independent Numerous studies have revealed a pivotal role of the approaches such as NGS cannot provide metabolic func- gut microbial community in host metabolism, physiolo- tional information of bacterial species (Shigematsu et gy, and pathology (Tremaroli and Bäckhed, 2012). Sym- al., 2009). In addition to NGS and bioinformatics analy- biotic bacteria help metabolize indigestible nutrients, sis tools, culture-dependent approaches, including isola- protect hosts from opportunistic pathogens (Hooper and tion and cultivation, can be used to obtain a comprehen- Gordon, 2001), help regulate inflammation and (Round sive understanding of the complex biochemical charac- and Mazmanian, 2009) insulin sensitivity, and even af- teristics and physiology of the isolated bacterial species. fect the progression of neurological disorders (Wu et al., However, many microbes have not been cultured under 2017). There is even speculation that the activity and laboratory conditions (Dave et al., 2012; Fodor et al., composition of gut microbiota are related to all patho- 2012; Nayfach et al., 2019). logical or physiological conditions (Cani, 2017). Given In 2018, we collected the feces of an Oriental stork the importance of the gut microbiota, they have been (Ciconia boyciana) and the intestinal tracts of a cobitid called ‘a forgotten organ’ (O’Hara and Shanahan, 2006). fish (Kichulchoia multifasciata) and a Korean splendid Next-generation sequencing (NGS) technologies and dace (Coreoleuciscus splendidus). These vertebrate spe- bioinformatics analysis tools have undergone remark- cies are endemic to the Republic of Korea (Choe et al., able advances and offer alternative methods to investi- 2016; Kim and Park, 2017). We isolated a number of 326 JOURNAL OF SPECIES RESEARCH Vol. 9, No. 4 bacterial species from these indigenous samples, includ- Morphological and Biochemical analysis ing 18 unreported bacterial species in the Republic of We observed cells under a LIBRA 120 transmission Korea. We present the detailed descriptions of these 18 electron microscope (Carl Zeiss) to describe morpholog- bacterial species belonging to the phyla Actinobacteria, ical characteristics. Gram-stain reactions were evaluated Firmicutes, Proteobacteria, and Bacteroidetes. using a Gram staining kit (bioMerieux). For biochemical analysis, 71 carbon source utilization assays were

Materials and Methods performed using Biolog GEN III MicroPlate system

(BiOLOG) (dextrin, d-maltose, d-trehalose, d-cellobi- Strain isolation and characterization ose, gentiobiose, sucrose, d-turanose, stachyose, d-raf finose, α-d-lactose, d-melibiose, β-methyl-d-gluco- The feces of C. boyciana were collected at the Seoul side, d-salicin, N-acetyl-d-glucosamine, N-acetyl-β- Grand Park Zoo (N 37°25ʹ22.3ʺ E 127°1ʹ17.8ʺ). Intesti- d-mannosamine, N-acetyl-d-galactosamine, N-acetyl- nal tracts of K. multifasciata and C. splendidus were col- neuraminic acid, α-d d d lected from streams in Gimcheon-si, Gyeongsangbuk-do -glucose, -mannose, -fructose, (N 36°05ʹ54.9ʺ E 128°07ʹ32.1ʺ), and Gapyeong-gun, d-galactose, 3-methyl glucose, d-fucose, l-fucose, l-rhamnose, inosine, d-sorbitol, d-mannitol, d-arabitol, Gyeonggi-do (N 37°51ʹ55.3ʺ E 127°21ʹ00.9ʺ), Republic of Korea, respectively. All feces and intestinal samples myo-inositol, glycerol, d-glucose-6-PO4, d-fructose-6- PO4, d-aspartic acid, gelatin, glycyl-l-proline, l-alanine, were homogenized in sterile phosphate buffered saline l-arginine, l-aspartic acid, l-glutamic acid, l-histidine, and serially diluted. Samples were spread onto three dif- l-pyroglutamic acid, l-serine, pectin, d-galacturonic ferent culture media (marine agar, tryptic soy agar, and l-galactonic acid lactone, d-gluconic acid, d-glu- Reasoner’s 2A agar; BD Difco) and incubated at 10°C acid, curonic acid, glucuronamide, mucin acid, quinic acid, and 30°C. We repeated several sub-cultivations using d-saccharic acid, p-hydroxy-phenylacetic acid, methyl the streak-plating method to obtain pure isolates. pyruvate, d-lactic acid methyl ester, l-lactic acid, citric α d l Analysis of 16S rRNA genes acid, -keto-glutaric acid, -malic acid, -malic acid, bromo-succinic acid, Tween 40, γ-amino-butyric acid, Genomic DNA was extracted using an UltraClean α-hydroxy-butyric acid, β-hydroxy-d,l-butyric acid, Microbial DNA Isolation Kit (MoBio) and prepared as α-keto-butyric acid, acetoacetic acid, propionic acid, described previously (Lee et al., 2019). Extracted DNA acetic acid, and formic acid) and 23 chemical sensitivity was amplified using a PCR premix (iNtron Biotechnolo- assays (pH 6, pH 5, 1% NaCl, 4% NaCl, 8% NaCl, 1% gy) and two universal bacteria-specific primers, Bac 27F sodium lactate, fusidic acid, d-serine, troleandomycin, (5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ) and Bac 1492R rifamycin SV, minocycline, lincomycin, guanidine HCl, (5ʹ-GGYTACCTTGTTACGACTT-3ʹ) (Lane, 1991). niaproof 4, vancomycin, tetrazolium violet, tetrazolium Near-full-length 16S rRNA genes were sequenced using blue, nalidixic acid, lithium chloride, potassium tellurite, a PRISM 3730xl DNA analyzer (Applied Biosystem) and aztreonam, sodium butyrate, and sodium bromate). the sequence fragments were assembled using SeqMan 5.0 program (DNASTAR). The genomic DNA G+C content was determined using high-performance liquid chroma- Results and Discussion tography (Mesbah et al., 1989). The most closely related taxa were determined by comparing to bacterial type Based on the phylogenetic analyses using 16S rRNA strains from the EzBioCloud database (Yoon et al., 2017). gene sequences, 18 strains (designated H11M7, H11M9, H23M10, H21T1, H13T1, H43T7, H21T20, H23M25, Phylogenetic analysis H23M9, S13R1, H13R26, H11M5, H11M15, H11R12, H21T7, H11R21, M13M1, and H23T21) were assigned The 16S rRNA gene sequences of the isolates were to the phyla Actinobacteria, Firmicutes, Proteobacteria, aligned to closely related species using CLUSTAL W and Bacteroidetes. The isolated strains were confirmed software (Thompson et al., 1994). To determine the as chemoheterotrophic, and rod or coccoid-shaped bac- phylogenetic position of the isolates, phylogenetic con- teria (Fig. 1). Detailed morphological and biochemical sensus trees were constructed using MEGA version 7 properties of these isolated strains are described in the (Kumar et al., 2016) with neighbor-joining (NJ) (Saitou description section. and Nei, 1987), maximum-likelihood (ML) (Felsenstein, 1981), and maximum-parsimony (MP) (Kluge and Far- Strains assigned to the phylum Actinobacteria ris, 1969) tree-construction algorithms. To confirm the robustness of the phylogenetic trees, 1000 bootstrap rep- Phylogenetic consensus tree concluded that eight stra licates were performed. ins (H11M7, H11M9, H23M10, H21T1, H13T1, H43T7, November 2020 Lee et al. Unrecorded gut bacterial species from Korean endemic vertebrates 327

Fig. 1. Transmission electron micrographs of the strains isolated in this study. The arrows indicate flagella. Strains: 1, H11M7; 2, H11M9; 3, H23M10; 4, H21T1; 5, H13T1; 6, H43T7; 7, H21T20; 8, H23M25; 9, H23M9; 10, S13R1; 11, H13R26; 12, H11M5; 13, H11M15; 14, H11R12; 15, H21T7; 16, H11R21; 17, M13M1; 18, H23T21.

H21T20, and H23M25) were all assigned to the class 99.9%) for H23M25 (Table 1). Actinobacteria. Strains H11M7, H11M9, H23M10, These strains were all Gram-stain positive and rod- H21T1, H13T1, and H43T7 were assigned to the order shaped bacteria, except for strain H13T1 that was coc- Micrococcales, and strains H21T20 and H23M25 were coid-shaped (Fig. 1). assigned to the order Corynebacteriales (Fig. 2). The most closely related strains were Glutamicibacter pro Strains assigned to the phylum Firmicutes tophormiae DSM 20168T (X80745; 100%) for H11M7, Glutamicibacter creatinolyticus JCM 19049T (D88211; Phylogenetic analysis using 16S rRNA gene sequenc- 99.6%) for H11M9, Microbacterium keratanolyticum es revealed that seven strains (H23M9, S13R1, H13R26, IFO 13309T (AB004717; 99.8%) H23M10, Microbacte H11M5, H11M15, H11R12, and H21T7) were all as- rium hydrocarbonoxydans NBRC 103074T (AJ698726; signed to the class Bacilli. Strain H21T7 was assigned 99.9%) for H21T1, Janibacter limosus DSM 11140T to the order Lactobacillales and the other strains were (Y08539; 100%) H13T1, Jonesia denitrificans DSM assigned to the order Bacillales (Fig. 3). The most phy- 20603T (NR_074806; 100%) H43T7, Corynebacterium logenetically related species to strains were Jeotgalicoc casei LMG S-19264T (AF267152; 99%) for H21T20, cus aerolatus MPA-33T (GU295939; 99.9%) for H23 and Dietzia aurantiaca CCUG 35676T (FR821260; M9, Bacillus pacificus EB422T (KJ812450; 100%) for 328 JOURNAL OF SPECIES RESEARCH Vol. 9, No. 4 ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) Isolation source Ciconia boyciana Ciconia boyciana Ciconia boyciana Ciconia boyciana Ciconia boyciana Ciconia boyciana Ciconia boyciana Ciconia boyciana Ciconia boyciana Coreoleuciscus splendidus Coreoleuciscus Ciconia boyciana Ciconia boyciana Ciconia boyciana Ciconia boyciana Ciconia boyciana Ciconia boyciana Kichulchoia multifasciata Ciconia boyciana Oriental stork ( Oriental stork ( Oriental stork ( Oriental stork ( Oriental stork ( Oriental stork ( Oriental stork ( Oriental stork ( Oriental stork ( Korean splendid dace ( Oriental stork ( Oriental stork ( Oriental stork ( Oriental stork ( Oriental stork ( Oriental stork ( Cobitid fish ( Oriental stork (

) and a Korean splendid (%) 100 100 100 100 100 100 100 100 100 100 100 99.93 99.79 99.93 99.86 99.85 99.79 99.43 Similarity number Accession X80745 D88211 AB004717 AJ698726 Y08539 NR_074806 AF267152 FR821260 GU295939 KJ812450 AB271740 NR_113350 AB009939 AJ320272 AJ877015 KM486054 FJ589031 NR_118144 Kichulchoia multifasciata Kichulchoia (

equorum casei Most closely related species Closest type strain Glutamicibacter protophormiae Glutamicibacter creatinolyticus Microbacterium keratanolyticum Microbacterium Microbacterium hydrocarbonoxydans Microbacterium Janibacter limosus Jonesia denitrificans Corynebacterium casei Dietzia aurantiaca Jeotgalicoccus aerolatus Bacillus pacificus Kurthia zopfii Staphylococcus xylosus Staphylococcus equorum subsp. Staphylococcus succinus subsp. Enterococcus aquimarinus Enterococcus Psychrobacter ciconiae Psychrobacter Shewanella xiamenensis Sphingobacterium mizutaii number Accession MK253322 MK253323 MK253315 MK253327 MK253316 MK253318 MK253328 MK253319 MK253314 MT055843 MK253317 MK253321 MK253324 MK253325 MK253329 MK253326 MK253332 MK253331 ) and from intestinal tracts of a cobitid fish NIBR ID NIBRBAC000502712 NIBRBAC000502713 NIBRBAC000502706 NIBRBAC000502717 NIBRBAC000502707 NIBRBAC000502709 NIBRBAC000502718 NIBRBAC000502710 NIBRBAC000502705 NIBRBAC000502725 NIBRBAC000502708 NIBRBAC000502711 NIBRBAC000502714 NIBRBAC000502715 NIBRBAC000502719 NIBRBAC000502716 NIBRBAC000502722 NIBRBAC000502721 ID Strain H11M7 H11M9 H23M10 H21T1 H13T1 H43T7 H21T20 H23M25 H23M9 S13R1 H13R26 H11M5 H11M15 H11R12 H21T7 H11R21 M13M1 H23T21 Ciconia boyciana Ciconia (

Glutamicibacter Genus Glutamicibacter Microbacterium Microbacterium Janibacter Jonesia Corynebacterium Dietzia Jeotgalicoccus Bacillus Kurthia Staphylococcus Staphylococcus Staphylococcus Enterococcus Psychrobacter Shewanella Sphingobacterium Micrococcaceae Family Micrococcaceae Microbacteriaceae Microbacteriaceae Intrasporangiaceae Jonesiaceae Corynebacteriaceae Dietziaceae Staphylococcaceae Bacillaceae Planococcaceae Staphylococcaceae Staphylococcaceae Staphylococcaceae Enterococcaceae Moraxellaceae Shewanellaceae Sphingobacteriaceae Micrococcales Order Micrococcales Micrococcales Micrococcales Micrococcales Micrococcales Corynebacteriales Corynebacteriales Bacillales Bacillales Bacillales Bacillales Bacillales Bacillales Lactobacillales Pseudomonadales Alteromonadales Sphingobacteriales ) and their taxonomic affiliations. Actinobacteria Actinobacteria Class Actinobacteria Actinobacteria Actinobacteria Actinobacteria Actinobacteria Actinobacteria Bacilli Bacilli Bacilli Bacilli Bacilli Bacilli Bacilli Gammaproteobacteria Gammaproteobacteria Sphingobacteriia Summary of the isolated strains from the feces of an Oriental stork of an Oriental feces strains from the isolated of the Summary Coreoleuciscus splendidus Coreoleuciscus (

Actinobacteria Actinobacteria Table 1. Table Phylum dace Actinobacteria Actinobacteria Actinobacteria Actinobacteria Actinobacteria Actinobacteria Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Proteobacteria Proteobacteria Bacteroidetes November 2020 Lee et al. Unrecorded gut bacterial species from Korean endemic vertebrates 329

Fig. 2. Phylogenetic tree based on 16S rRNA gene sequence comparisons, showing the relationship between the eight strains belonging to the phylum Actinobacteria isolated in this study and the notable type species from each genus. The tree was reconstructed using neighbor joining (NJ), maximum likelihood (ML), and maximum parsimony (MP) algorithms. Filled diamonds indicate corresponding branches present in the phylogenetic tree generated using the three different tree construction methods. Numbers at the nodes represent bootstrap values of more than 70% are shown, based on 1000 replicates (NJ/MP/ML). Akkermansia muciniphila ATCC BAA-835T (NR_074436.1) was used as the outgroup. The bar indicates 0.02 accumulated substitutions per nucleotide.

S13R1, Kurthia zopfii NBRC 101529T (AB271740; Strains assigned to the phylum Bacteroidetes 100%) for H13R26, Staphylococcus xylosus CCM 2738T Strain H23T21 was assigned to the class Flavobac (NR_113350; 100%) for H11M5, Staphylococcus equo teria and the order Flavobacteriales (Fig. 5). The most rum subsp. equorum ATCC 43958T (AB009939; 100%) closely related species to strain H23T21 was Sphingo for H11M15, Staphylococcus succinus subsp. casei bacterium mizutaii ATCC 33299T (NR_118144; 100%) SB72T (AJ320272; 100%) for H11R12, and Enterococ (Table 1). cus aquimarinus DSM 17690T (AJ877015; 99.6%) for This strain was Gram-stain negative and rod-shaped H21T7 (Table 1). (Fig. 1). These strains were all Gram-stain positive and rod- shaped bacteria, except for strains H11M15, H11R12, Description of Glutamicibacter protophormiae H11M7 and H21T7 that were coccoid-shaped (Fig. 1). Cells are Gram-stain positive, non-flagellated, and rod- Strains assigned to the phylum Proteobacteria shaped. Colonies are circular, raised, entire, and white colored after 2 days of incubation on MA at 10℃. Pos- Phylogenetic tree showed that strains H11R21, and itive for N-acetyl neuraminic acid, l-fucose, sodium M13M1 were assigned to the class Gammaproteobac lactate, l-glutamic acid, l-pyroglutamic acid, guani- teria. Strain H11R21 and M13M1 were members of the dine HCl, tetrazolium blue, l-lactic acid, citric acid, α- order Pseudomonadales and Alteromonadales, respec- keto-glutaric acid, l-malic acid, bromo-succinic acid, tively (Fig. 4). The most closely related taxa of strain nalidixic acid, lithium chloride, potassium tellurite, T H11R21 was Psychrobacter ciconiae 176/10 (KM4860 aztreonam, sodium butyrate, and sodium bromate in 54; 100%) and of M13M1 was Shewanella xiamenensis Biolog GEN III; but negative for dextrin, d-maltose, T S4 (FJ589031; 99.4%) (Table 1). d-trehalose, d-cellobiose, gentiobiose, sucrose, d-tura-

Both strains were Gram-stain negative and rod-shaped nose, stachyose, d-raffinose, α-d-lactose, d-melibiose, (Fig. 1). β-methyl-d-glucoside, d-salicin, N-acetyl-d-glucos- 330 JOURNAL OF SPECIES RESEARCH Vol. 9, No. 4

Fig. 3. Phylogenetic tree based on 16S rRNA gene sequence comparisons, showing the relationship between the seven strains belonging to the phylum Firmicutes isolated in this study and the notable type species from each genus. The tree was reconstructed using neighbor joining (NJ), maximum likelihood (ML), and maximum parsimony (MP) algorithms. Filled diamonds indicate corresponding branches present in the phylogenetic tree generated using the three different tree construction methods. Numbers at the nodes represent bootstrap values of more than 70% are shown, based on 1000 replicates (NJ/MP/ML). Limnochorda pilosa HC45T (AP014924) was used as the outgroup. The bar indicates 0.02 accumulated substitutions per nucleotide.

amine, N-acetyl-β-d-mannosamine, N-acetyl-d-galac- rod-shaped. Colonies are circular, raised, entire, and yel- tosamine, α-d-glucose, d-mannose, d-fructose, d-galac- low-colored after 2 days of incubation on MA at 10℃. tose, 3-methyl glucose, d-fucose, l-rhamnose, inosine, Positive for dextrin, gentiobiose, sucrose, d-turanose, d-sorbitol, d-mannitol, d-arabitol, myo-inositol, glycer- stachyose, α-d-lactose, d-melibiose, β-methyl-d-gluco- ol, d-glucose, d-fructose-6-PO4, d-aspartic acid, d-ser- side, d-salicin, N-acetyl-d-glucosamine, N-acetyl-β-d- ine, troleandomycin, gelatin, l-arginine, l-aspartic acid, mannosamine, N-acetyl-d-galactosamine, N-acetylneur- l-histidine, l-serine, lincomycin, pectin, d-galacturonic aminic acid, α-d-glucose, d-mannose, d-fructose, d-ga- acid, l-galactonic acid lactone, d-gluconic acid, d-glu- lactose, 3-methyl glucose, d-fucose, l-fucose, l-rham- curonic acid, glucuronamide, mucin acid, quinic acid, nose, inosine, sodium lactate, fusidic acid, d-serine, d-saccharic acid, vancomycin, p-hydroxy-phenylacetic d-sorbitol, d-mannitol, d-arabitol, myo-inositol, glycer- acid, methyl pyruvate, d-lactic acid, d-malic acid, Tween ol, d-glucose, d-aspartic acid, d-fructose-6-PO4, trole- 40, γ-amino-butyric acid, β-hydroxy-d,l-butyric acid, andomycin, rifamycin SV, glycyl-l-proline, l-alanine, α-hydroxy-butyric acid, α-keto-butyric acid, acetoacetic l-arginine, l-aspartic acid, l-glutamic acid, l-histidine, acid, propionic acid, acetic acid, formic acid, pH 5, 8% l-pyroglutamic acid, l-serine, lincomycin, guanidine NaCl, fusidic acid, rifamycin SV, minocycline, niaproof HCl, pectin, d-galacturonic acid, l-galactonic acid lac- 4, and tetrazolium violet. tone, d-gluconic acid, d-glucuronic acid, glucuronamide,

Strain H11M7 (=NIBRBAC000502712) was isolated mucin acid, quinic acid, d-saccharic acid, vancomycin, from the feces of a stork (Ciconia boyciana) from Seoul tetrazolium violet, tetrazolium blue, methyl pyruvate, Grand Park Zoo, Gwacheon-si, Gyeonggi-do, Korea. d-lactic acid, l-lactic acid, citric acid, α-keto-glutaric The DNA G+C content of the type strain is 31.9 mol%. acid, d-malic acid, l-malic acid, bromo-succinic acid, nalidixic acid, lithium chloride, α-hydroxy-butyric acid, Description of Glutamicibacter creatinolyticus H11M9 α-keto-butyric acid, acetoacetic acid, acetic acid, aztreonam, sodium butyrate, and sodium bromate in Bi- Cells are Gram-stain positive, non-flagellated, and olog GEN III; but negative for d-maltose, d-trehalose, November 2020 Lee et al. Unrecorded gut bacterial species from Korean endemic vertebrates 331

Fig. 4. Phylogenetic tree based on 16S rRNA gene sequence comparisons, showing the relationship between the two strains belonging to the phylum Proteobacteria isolated in this study and the notable type species from each family. The tree was reconstructed using neighbor joining (NJ), maximum likelihood (ML), and maximum parsimony (MP) algorithms. Filled diamonds indicate corresponding branches present in the phylogenetic tree generated using the three different tree construction methods. Numbers at the nodes represent bootstrap values of more than 70% are shown, based on 1000 replicates (NJ/MP/ML). Bacteroides fragilis NCTC 9343T (NR_074784.2) was used as the outgroup. The bar indicates 0.05 accumulated substitutions per nucleotide.

d-raffinose, gelatin, p-hydroxy-phenylacetic acid, pH 5, 6-PO4, d-serine, gelatin, glycyl-l-proline, l-alanine, minocycline, niaproof 4, potassium tellurite, Tween 40, l-arginine, l-aspartic acid, l-glutamic acid, l-histidine, γ-amino-butyric acid, β-hydroxy-d,l-butyric acid, pro- l-pyroglutamic acid, l-serine, guanidine HCl, pectin, pionic acid, and formic acid. d-gluconic acid, d-saccharic acid, tetrazolium blue,

Strain H11M9 (=NIBRBAC000502713) was isolated p-hydroxy-phenylacetic acid, methyl pyruvate, d-lactic from the feces of a stork (Ciconia boyciana) from Seoul acid, l-lactic acid, citric acid, α-keto-glutaric acid, d-ma- Grand Park Zoo, Gwacheon-si, Gyeonggi-do, Korea. lic acid, l-malic acid, nalidixic acid, lithium chloride, The DNA G+C content of the type strain is 63.8 mol%. potassium tellurite, Tween 40, α-hydroxy-butyric acid, α-keto-butyric acid, acetoacetic acid, propionic acid, Description of Microbacterium keratanolyticum acetic acid, aztreonam, sodium butyrate, and sodium H23M10 bromate in Biolog GEN III; but negative for stachyose, d-raffinose, N-acetyl-d-galactosamine, d-aspartic acid, Cells are Gram-stain positive, non-flagellated, and troleandomycin, lincomycin, d-galacturonic acid, l-ga- rod-shaped. Colonies are circular, raised, entire, and yel- lactonic acid lactone, d-glucuronic acid, glucuronamide, low colored after 2 days of incubation on MA at 25℃. mucin acid, quinic acid, vancomycin, bromo-succinic Positive for dextrin, d-maltose, d-trehalose, d-cellobiose, acid, γ-amino-butyric acid, β-hydroxy-d,l-butyric acid, gentiobiose, sucrose, d-turanose, α-d-lactose, d-melibi- formic acid, pH 5, 8% NaCl, rifamycin SV, minocycline, ose, β-methyl-d-glucoside, d-salicin, N-acetyl-d-glucos- and niaproof 4. amine, N-acetyl-β-d-mannosamine, N-acetylneuraminic Strain H23M10 (=NIBRBAC000502706) was isolat acid, α-d-glucose, d-mannose, d-fructose, d-galactose, ed from the feces of a stork (Ciconia boyciana) from 3-methyl glucose, d-fucose, l-fucose, l-rhamnose, ino- Seoul Grand Park Zoo, Gwacheon-si, Gyeonggi-do, sine, sodium lactate, fusidic acid, d-sorbitol, d-mannitol, Korea. The DNA G+C content of the type strain is 67.3 d-arabitol, myo-inositol, glycerol, d-glucose, d-fructose- mol%. 332 JOURNAL OF SPECIES RESEARCH Vol. 9, No. 4

Fig. 5. Phylogenetic tree based on 16S rRNA gene sequence comparisons, showing the relationship between strain H23T21 and the notable type species from the family Sphingobacteriaceae. The tree was reconstructed using neighbor joining (NJ), maximum likelihood (ML), and maximum parsimony (MP) algorithms. Filled diamonds indicate corresponding branches present in the phylogenetic tree generated using the three different tree construction methods. Numbers at the nodes represent bootstrap values of more than 70% are shown, based on 1000 replicates (NJ/MP/ML). Filobacterium rodentium SMR-CT (LC055729) was used as the outgroup. The bar indicates 0.05 accumulated substitutions per nucleotide.

Description of Microbacterium hydrocarbonoxydans methyl pyruvate, d-lactic acid, l-lactic acid, citric acid, H21T1 α-keto-glutaric acid, d-malic acid, l-malic acid, bromo-succinic acid, nalidixic acid, lithium chloride, po- Cells are Gram-stain positive, flagellated, and rod- tassium tellurite, Tween 40, γ-amino-butyric acid, β-hy- shaped. Colonies are circular, raised, entire, and white- droxy-d,l-butyric acid, α-hydroxy-butyric acid, α-ke- colored after 2 days of incubation on TSA at 25℃. Pos- to-butyric acid, acetoacetic acid, propionic acid, acetic itive for dextrin, d-maltose, d-trehalose, d-cellobiose, acid, aztreonam, sodium butyrate, and sodium bromate gentiobiose, sucrose, d-turanose, stachyose, d-raffinose, in Biolog GEN III; but negative for formic acid, pH 5, α-d-lactose, d-melibiose, β-methyl-d-glucoside, d-sal- minocycline, and niaproof 4. icin, N-acetyl-d-glucosamine, N-acetyl-β-d-man- Strain H21T1 (=NIBRBAC000502717) was isolated nosamine, N-acetyl-d-galactosamine, N-acetylneuramin- from the feces of a stork (Ciconia boyciana) from Seoul ic acid, α-d-glucose, d-mannose, d-fructose, d-galactose, Grand Park Zoo, Gwacheon-si, Gyeonggi-do, Korea. 3-methyl glucose, d-fucose, l-fucose, l-rhamnose, ino- The DNA G+C content of the type strain is 66.5 mol%. sine, sodium lactate, fusidic acid, d-sorbitol, d-mannitol, d-arabitol, myo-inositol, glycerol, d-glucose, d-fructose- Description of Janibacter limosus H13T1 6-PO4, d-aspartic acid, d-serine, troleandomycin, rifamycin SV, gelatin, glycyl-l-proline, l-alanine, l-arginine, Cells are Gram-stain positive, non-flagellated, and coc- l-aspartic acid, l-glutamic acid, l-histidine, l-pyroglu- coid-shaped. Colonies are circular, raised, entire, and tamic acid, l-serine, lincomycin, guanidine HCl, pectin, cream colored after 2 days of incubation on TSA at 10℃. d-galacturonic acid, l-galactonic acid lactone, d-glucon- Positive for d-maltose, d-trehalose, gentiobiose, su- ic acid, d-glucuronic acid, glucuronamide, mucin acid, crose, d-turanose, N-acetyl-d-glucosamine, α-d-glucose, quinic acid, d-saccharic acid, vancomycin, tetrazolium sodium lactate, rifamycin SV, d-gluconic acid, tetrazoli- violet, tetrazolium blue, p-hydroxy-phenylacetic acid, um blue, l-malic acid, nalidixic acid, lithium chloride, November 2020 Lee et al. Unrecorded gut bacterial species from Korean endemic vertebrates 333

Tween 40, β-hydroxy-d,l-butyric acid, α-hydroxy-bu- lic acid, bromo-succinic acid, Tween 40, γ-amino-butyric tyric acid, α-keto-butyric acid, propionic acid, acetic acid, β-hydroxy-d,l-butyric acid, propionic acid, formic acid, aztreonam, and sodium butyrate in Biolog GEN acid, pH 5, rifamycin SV, niaproof 4, and sodium bro- III; but negative for dextrin, d-cellobiose, stachyose, mate. d-raffinose, α-d-lactose, d-melibiose, β-methyl-d-glu- Strain H43T7 (=NIBRBAC000502709) was isolated coside, d-salicin, N-acetyl-β-d-mannosamine, N-ace- from the feces of a stork (Ciconia boyciana) from Seoul tyl-d-galactosamine, N-acetylneuraminic acid, d-man- Grand Park Zoo, Gwacheon-si, Gyeonggi-do, Korea. nose, d-fructose, d-galactose, 3-methyl glucose, d-fu- The DNA G+C content of the type strain is 57.9 mol%. cose, l-fucose, l-rhamnose, inosine, d-sorbitol, d-mannitol, d-arabitol, myo-inositol, glycerol, d-glucose, d-fruc- Description of Corynebacterium casei H21T20 tose-6-PO4, d-aspartic acid, d-serine, troleandomycin, Cells are Gram-stain positive, flagellated, and rod- gelatin, glycyl-l-proline, l-alanine, l-arginine, l-aspartic shaped. Colonies are circular, raised, entire, and yel- acid, l-glutamic acid, l-histidine, l-pyroglutamic acid, low-colored after 2 days of incubation on TSA at 25℃. l-serine, lincomycin, d-galacturonic acid, l-galactonic Positive for dextrin, d-trehalose, d-cellobiose, gentiobi- acid lactone, d-glucuronic acid, glucuronamide, mucin ose, sucrose, d-turanose, stachyose, d-raffinose, α-d-lac- acid, quinic acid, d-saccharic acid, vancomycin, p-hy- tose, d-melibiose, β-methyl-d-glucoside, d-salicin, droxy-phenylacetic acid, methyl pyruvate, d-lactic acid, N-acetyl-d-glucosamine, N-acetyl-β-d-mannosamine, l-lactic acid, citric acid, α-keto-glutaric acid, d-malic N-acetyl-d-galactosamine, N-acetylneuraminic acid, acid, bromo-succinic acid, γ-amino-butyric acid, ace- α-d-glucose, d-mannose, d-fructose, d-galactose, 3-meth- toacetic acid, formic acid, pH 5, 8% NaCl, fusidic acid, yl glucose, d-fucose, l-fucose, l-rhamnose, inosine, sodi- minocycline, guanidine HCl, niaproof 4, tetrazolium vi- um lactate, fusidic acid, d-serine, d-mannitol, d-arabitol, olet, potassium tellurite, and sodium bromate. myo-inositol, glycerol, d-glucose, d-fructose-6-PO4, d-as- Strain H13T1 (=NIBRBAC000502707) was isolated partic acid, troleandomycin, rifamycin SV, minocycline, from the feces of a stork (Ciconia boyciana) from Seoul gelatin, glycyl-l-proline, l-alanine, l-arginine, l-aspartic Grand Park Zoo, Gwacheon-si, Gyeonggi-do, Korea. acid, l-glutamic acid, l-histidine, l-pyroglutamic acid, The DNA G+C content of the type strain is 68.7 mol%. l-serine, lincomycin, guanidine HCl, d-galacturonic acid, l-galactonic acid lactone, d-gluconic acid, d-glucuronic Description of Jonesia denitrificans H43T7 acid, glucuronamide, mucin acid, quinic acid, d-saccharic Cells are Gram-stain positive, flagellated, and rod- acid, vancomycin, tetrazolium violet, tetrazolium blue, shaped. Colonies are circular, raised, entire, and white p-hydroxy-phenylacetic acid, methyl pyruvate, d-lactic l α d colored after 2 days of incubation on TSA at 30℃. Pos- acid, -lactic acid, citric acid, -keto-glutaric acid, -malic acid, l-malic acid, bromo-succinic acid, nalidixic acid, itive for dextrin, d-maltose, d-trehalose, d-cellobiose, lithium chloride, potassium tellurite, Tween 40, γ-ami- gentiobiose, sucrose, d-raffinose, d-turanose, α-d-lactose, no-butyric acid, α-hydroxy-butyric acid, α-keto-butyric d-melibiose, d-salicin, N-acetyl-d-glucosamine, α-d-glu- acid, β-hydroxy-d,l-butyric acid, acetoacetic acid, propi- cose, d-mannose, d-fructose, d-galactose, inosine, sodium onic acid, acetic acid, formic acid, aztreonam, sodium bu- lactate, fusidic acid, d-sorbitol, glycerol, d-fructose-6- PO4, troleandomycin, minocycline, lincomycin, guan- tyrate, and sodium bromate in Biolog GEN III; but negative for d-maltose, d-sorbitol, pectin, pH 5, and niaproof 4. idine HCl, pectin, d-gluconic acid, d-glucuronic acid, = glucuronamide, mucin acid, vancomycin, tetrazolium Strain H21T20 ( NIBRBAC000502718) was isolated Ciconia boyciana violet, tetrazolium blue, methyl pyruvate, nalidixic acid, from the feces of a stork ( ) from Seoul α Grand Park Zoo, Gwacheon-si, Gyeonggi-do, Korea. lithium chloride, potassium tellurite, -hydroxy-butyric + acid, α-keto-butyric acid, acetoacetic acid, acetic acid, The DNA G C content of the type strain is 61.9 mol%. aztreonam, and sodium butyrate in Biolog GEN III; but Description of Dietzia aurantiaca H23M25 negative for stachyose, β-methyl-d-glucoside, N-acetyl- β-d-mannosamine, N-acetyl-d-galactosamine, N-acetyl- Cells are Gram-stain positive, non-flagellated, and rod- neuraminic acid, 3-methyl glucose, d-fucose, l-fucose, shaped. Colonies are circular, raised, entire, and cream l-rhamnose, d-mannitol, d-arabitol, myo-inositol, d-glu- colored after 2 days of incubation on MA at 25℃. Posi- cose, d-aspartic acid, d-serine, gelatin, glycyl-l-proline, tive for d-trehalose, d-fructose, sodium lactate, d-serine, l-alanine, l-arginine, l-aspartic acid, l-glutamic acid, tetrazolium blue, nalidixic acid, lithium chloride, potas- l-histidine, l-pyroglutamic acid, l-serine, d-galacturonic sium tellurite, Tween 40, β-hydroxy-d,l-butyric acid, ac- acid, l-galactonic acid lactone, quinic acid, d-saccharic etoacetic acid, propionic acid, and acetic acid in Biolog acid, p-hydroxy-phenylacetic acid, d-lactic acid, l-lactic GEN III; but negative for dextrin, d-maltose, d-cellobiose, acid, citric acid, α-keto-glutaric acid, d-malic acid, l-ma- gentiobiose, sucrose, d-turanose, stachyose, d-raffinose, 334 JOURNAL OF SPECIES RESEARCH Vol. 9, No. 4

α-d-lactose, d-melibiose, β-methyl-d-glucoside, d-sali- HC, niaproof 4, and sodium bromate. cin, N-acetyl-d-glucosamine, N-acetyl-β-d-mannosamine, Strain H23M9 (=NIBRBAC000502705) was isolated

N-acetyl-d-galactosamine, N-acetylneuraminic acid, from the feces of a stork (Ciconia boyciana) from Seoul α-d-glucose, d-mannose, d-galactose, 3-methyl glu- Grand Park Zoo, Gwacheon-si, Gyeonggi-do, Korea. cose, d-fucose, l-fucose, l-rhamnose, inosine, d-sorbitol, The DNA G+C content of the type strain is 39.7 mol%. d-mannitol, d-arabitol, myo-inositol, glycerol, d-glucose, d-fructose-6-PO4, d-aspartic acid, troleandomycin, gel- Description of Bacillus pacificus S13R1 atin, glycyl-l-proline, l-alanine, l-arginine, l-aspartic Cells are Gram-stain positive, flagellated, and rod- acid, l-glutamic acid, l-histidine, l-pyroglutamic acid, shaped. Colonies are circular, raised, entire, and white l-serine, lincomycin, pectin, d-galacturonic acid, l-ga- colored after 2 days of incubation on R2A at 10℃. Pos- lactonic acid lactone, d-gluconic acid, d-glucuronic acid, itive for sodium lactate, d-fructose, rifamycin SV, gel- glucuronamide, mucin acid, quinic acid, d-saccharic acid, atin, glycyl-l-proline, guanidine HCl, tetrazolium blue, vancomycin, p-hydroxy-phenylacetic acid, methyl pyru- potassium tellurite, acetoacetic acid, propionic acid, vate, d-lactic acid, l-lactic acid, citric acid, α-keto-glutar- aztreonam, and sodium butyrate in Biolog GEN III; but ic acid, d-malic acid, l-malic acid, bromo-succinic acid, negative for dextrin, d-maltose, d-trehalose, d-cellobiose, γ-amino-butyric acid, α-hydroxy-butyric acid, α-keto-bu- gentiobiose, sucrose, d-turanose, stachyose, d-raffinose, tyric acid, formic acid, aztreonam, pH 5, fusidic acid, α-d-lactose, d-melibiose, β-methyl-d-glucoside, d-sali- rifamycin SV, minocycline, guanidine HCl, niaproof 4, cin, N-acetyl-d-glucosamine, N-acetyl-β-d-mannosamine, tetrazolium violet, sodium butyrate, and sodium bromate. N-acetyl-d-galactosamine, N-acetylneuraminic acid, Strain H23M25 ( =NIBRBAC000502710) was iso- α-d-glucose, d-mannose, d-fructose-6-PO4, d-galactose, lated from the feces of a stork (Ciconia boyciana) from 3-methyl glucose, d-fucose, l-fucose, l-rhamnose, ino- Seoul Grand Park Zoo, Gwacheon-si, Gyeonggi-do, sine, d-sorbitol, d-mannitol, d-arabitol, myo-inositol, Korea. The DNA G+C content of the type strain is 67.9 glycerol, d-glucose, d-aspartic acid, d-serine, troleando- mol%. mycin, l-alanine, l-arginine, l-aspartic acid, l-glutamic acid, l-histidine, l-pyroglutamic acid, l-serine, lincomy- Description of Jeotgalicoccus aerolatus H23M9 cin, pectin, d-galacturonic acid, l-galactonic acid lactone, Cells are Gram-stain positive, non-flagellated, and rod- d-gluconic acid, d-glucuronic acid, glucuronamide, mucin shaped. Colonies are circular, raised, entire, and cream acid, quinic acid, d-saccharic acid, vancomycin, p-hy- colored after 2 days of incubation on MA at 25℃. Posi- droxy-phenylacetic acid, methyl pyruvate, d-lactic acid, l-lactic acid, citric acid, α-keto-glutaric acid, d-malic tive for d-cellobiose, gentiobiose, β-methyl-d-glucoside, acid, l-malic acid, bromo-succinic acid, nalidixic acid, d-salicin, N-acetyl-d-glucosamine, N-acetyl-β-d-man- Tween 40, γ-amino-butyric acid, β-hydroxy-d,l-butyric nosamine, N-acetyl-d-galactosamine, N-acetylneuramin- acid, α-hydroxy-butyric acid, α-keto-butyric acid, acetic ic acid, α-d-glucose, d-fucose, l-fucose, sodium lactate, acid, formic acid, pH 5, 4% NaCl, 8% NaCl, fusidic acid, d-sorbitol, d-arabitol, d-aspartic acid, troleandomycin, minocycline, niaproof 4, tetrazolium violet, lithium chlo- minocycline, l-glutamic acid, l-histidine, glucuronamide, tetrazolium violet, tetrazolium blue, α-keto-glutaric acid, ride, and sodium bromate. Strain S13R1 ( =NIBRBAC000502725) was isolated d-malic acid, nalidixic acid, lithium chloride, potassium Co tellurite, acetoacetic acid, acetic acid, aztreonam, and so- from the intestinal tract of a Korean splendid dace ( reoleuciscus splendidus dium butyrate in Biolog GEN III; but negative for dextrin, ) from Gapyeong-gun, Gyeong- gi-do, Korea. The DNA G+C content of the type strain d-maltose, d-trehalose, sucrose, d-turanose, stachyose, is 43.6 mol%. d-raffinose, α-d-lactose, d-melibiose, d-mannose, d-fructose, d-galactose, 3-methyl glucose, l-rhamnose, inosine, Description of Kurthia zopfii H13R26 d-mannitol, myo-inositol, glycerol, d-glucose, d-fructose-6-PO4, d-serine, gelatin, glycyl-l-proline, l-alanine, Cells are Gram-stain positive, flagellated, and rod- l-arginine, l-aspartic acid, l-pyroglutamic acid, l-serine, shaped. Colonies are circular, raised, entire, and cream lincomycin, pectin, d-galacturonic acid, l-galactonic colored after 2 days of incubation on TSA at 10℃. Pos- acid lactone, d-gluconic acid, d-glucuronic acid, mucin itive for sodium lactate, l-glutamic acid, guanidine HCl, acid, quinic acid, d-saccharic acid, vancomycin, p-hy- tetrazolium blue, l-lactic acid, nalidixic acid, lithium droxy-phenylacetic acid, methyl pyruvate, d-lactic acid, chloride, potassium tellurite, acetoacetic acid, acetic acid, l-lactic acid, citric acid, l-malic acid, bromo-succinic formic acid, aztreonam, and sodium butyrate in Biolog acid, Tween 40, γ-amino-butyric acid, β-hydroxy-d,l-bu- GEN III; but negative for dextrin, d-maltose, d-trehalose, tyric acid, α-hydroxy-butyric acid, α-keto-butyric acid, d-cellobiose, gentiobiose, sucrose, d-turanose, stachyose, formic acid, pH 5, fusidic acid, rifamycin SV, guanidine d-raffinose, α-d-lactose, d-melibiose, β-methyl-d-gluco- November 2020 Lee et al. Unrecorded gut bacterial species from Korean endemic vertebrates 335

side, d-salicin, N-acetyl-d-glucosamine, N-acetyl-β-d- γ-amino-butyric acid, minocycline, and niaproof 4. mannosamine, N-acetyl-d-galactosamine, N-acetylneur- Strain H11M5 (=NIBRBAC000502711) was isolated aminic acid, α-d-glucose, d-mannose, d-fructose, d-galac- from the feces of a stork (Ciconia boyciana) from Seoul tose, 3-methyl glucose, d-fucose, l-fucose, l-rhamnose, Grand Park Zoo, Gwacheon-si, Gyeonggi-do, Korea. inosine, d-sorbitol, d-mannitol, d-arabitol, myo-inositol, The DNA G+C content of the type strain is 31.9 mol%. glycerol, d-glucose, d-fructose-6-PO4, d-aspartic acid, d-serine, troleandomycin, gelatin, glycyl-l-proline, l-al- Description of Staphylococcus equorum subsp. anine, l-arginine, l-aspartic acid, l-histidine, l-pyroglu- equorum H11M15 tamic acid, l-serine, lincomycin, pectin, d-galacturonic Cells are Gram-stain positive, flagellated, and coc- acid, l-galactonic acid lactone, d-gluconic acid, d-glucu- coid-shaped. Colonies are circular, raised, entire, and ronic acid, glucuronamide, mucin acid, quinic acid, d-sac- cream-colored after 2 days of incubation on MA at 10℃. charic acid, vancomycin, p-hydroxy-phenylacetic acid, Positive for dextrin, d-maltose, d-trehalose, d-cellobiose, methyl pyruvate, d-lactic acid, citric acid, α-keto-glutaric gentiobiose, sucrose, d-turanose, stachyose, d-raffinose, acid, d-malic acid, l-malic acid, bromo-succinic acid, α-d-lactose, d-melibiose, β-methyl-d-glucoside, d-sali- Tween 40, γ-amino-butyric acid, β-hydroxy-d,l-butyric cin, N-acetyl-d-glucosamine, N-acetyl-β-d-mannosamine, acid, α-hydroxy-butyric acid, α-keto-butyric acid, propi- N-acetyl-d-galactosamine, N-acetylneuraminic acid, onic acid, pH 5, 4% NaCl, 8% NaCl, fusidic acid, rifam- α-d-glucose, d-mannose, d-fructose, d-galactose, 3-meth- ycin SV, minocycline, niaproof 4, tetrazolium violet, and yl glucose, d-fucose, l-fucose, l-rhamnose, inosine, sodi- sodium bromate. um lactate, fusidic acid, d-sorbitol, d-mannitol, d-arabi- Strain H13R26 ( =NIBRBAC000502708) was iso- tol, myo-inositol, glycerol, d-glucose, d-fructose-6-PO4, lated from the feces of a stork (Ciconia boyciana) from d-aspartic acid, d-serine, troleandomycin, rifamycin SV, Seoul Grand Park Zoo, Gwacheon-si, Gyeonggi-do, gelatin, glycyl-l-proline, l-alanine, l-arginine, l-aspartic Korea. The DNA G+C content of the type strain is 36.9 acid, l-glutamic acid, l-histidine, l-pyroglutamic acid, mol%. l-serine, lincomycin, guanidine HCl, pectin, d-galacturonic acid, l-galactonic acid lactone, d-gluconic acid, Description of Staphylococcus xylosus H11M5 d-glucuronic acid, glucuronamide, mucin acid, quinic Cells are Gram-stain positive, non-flagellated, rod- acid, d-saccharic acid, vancomycin, tetrazolium violet, shaped, motile, and spore forming. Colonies are circular, tetrazolium blue, methyl pyruvate, d-lactic acid, l-lac- α d raised, entire, and white colored after 2 days of incu- tic acid, citric acid, -keto-glutaric acid, -malic acid, l-malic acid, bromo-succinic acid, nalidixic acid, lithium bation on MA at 10℃. Positive for dextrin, d-maltose, chloride, potassium tellurite, Tween 40, γ-amino-butyric d-trehalose, d-cellobiose, gentiobiose, sucrose, d-tura- acid, β-hydroxy-d,l-butyric acid, α-hydroxy-butyric acid, nose, stachyose, d-raffinose, α-d-lactose, d-melibiose, α-keto-butyric acid, acetoacetic acid, propionic acid, ace- β-methyl-d-glucoside, d-salicin, N-acetyl-d-glucosamine, tic acid, aztreonam, sodium butyrate and sodium bromate N-acetyl-β-d-mannosamine, N-acetyl-d-galactosamine, in Biolog GEN III; but negative for p-hydroxy-pheny- N-acetylneuraminic acid, α-d-glucose, d-mannose, d-fruc- lacetic acid, pH 5, minocycline, and niaproof 4. tose, d-galactose, 3-methyl glucose, d-fucose, l-fucose, Strain H11M15 (=NIBRBAC000502714) was isolated l-rhamnose, inosine, sodium lactate, fusidic acid, d-ser- from the feces of a stork (Ciconia boyciana) from Seoul ine, d-sorbitol, d-mannitol, d-arabitol, myo-inositol, glyc- Grand Park Zoo, Gwacheon-si, Gyeonggi-do, Korea. The erol, d-glucose, d-fructose-6-PO4, d-aspartic acid, gelatin, DNA G+C content of the type strain is 33.2 mol%. glycyl-l-proline, l-alanine, l-arginine, l-aspartic acid, l-glutamic acid, l-histidine, l-pyroglutamic acid, l-serine, Description of Staphylococcus succinus subsp. casei lincomycin, guanidine HCl, pectin, d-galacturonic acid, H11R12 l-galactonic acid lactone, d-gluconic acid, d-glucuronic acid, glucuronamide, mucin acid, quinic acid, d-sac- Cells are Gram-stain positive, non-flagellated, and charic acid, vancomycin, tetrazolium violet, tetrazolium coccoid-shaped. Colonies are circular, raised, entire, and blue, methyl pyruvate, d-lactic acid, l-lactic acid, citric white-colored after 2 days of incubation on R2A at 10℃. acid, α-keto-glutaric acid, d-malic acid, l-malic acid, Positive for dextrin, d-maltose, d-trehalose, d-cellobiose, bromo-succinic acid, nalidixic acid, lithium chloride, po- gentiobiose, sucrose, d-turanose, d-raffinose, α-d-lactose, tassium tellurite, Tween 40, β-hydroxy-d,l-butyric acid, d-melibiose, β-methyl-d-glucoside, d-salicin, N-ace- α-hydroxy-butyric acid, α-keto-butyric acid, acetoacetic tyl-d-glucosamine, N-acetylneuraminic acid, α-d-glucose, acid, propionic acid, acetic acid, formic acid, aztreonam, d-mannose, d-fructose, d-galactose, 3-methyl glucose, sodium butyrate, and sodium bromate in Biolog GEN III; d-fucose, l-fucose, l-rhamnose, sodium lactate, d-serine, but negative for stachyose, p-hydroxy-phenylacetic acid, d-sorbitol, d-mannitol, d-arabitol, myo-inositol, glycer- 336 JOURNAL OF SPECIES RESEARCH Vol. 9, No. 4

ol, d-glucose, d-fructose-6-PO4, d-aspartic acid, gelatin, from the feces of a stork (Ciconia boyciana) from Seoul glycyl-l-proline, l-alanine, l-arginine, l-aspartic acid, Grand Park Zoo, Gwacheon-si, Gyeonggi-do, Korea. l-glutamic acid, l-histidine, l-pyroglutamic acid, l-ser- The DNA G+C content of the type strain is 39.4 mol%. ine, guanidine HCl, pectin, d-galacturonic acid, l-galactonic acid lactone, d-gluconic acid, d-glucuronic acid, Description of Psychrobacter ciconiae H11R21 glucuronamide, mucin acid, quinic acid, d-saccharic acid, Cells are Gram-stain positive, flagellated, and rod- tetrazolium blue, methyl pyruvate, d-lactic acid, l-lac- shaped. Colonies are circular, raised, entire, and tic acid, citric acid, α-keto-glutaric acid, d-malic acid, white-colored after 2 days of incubation on MA at 10℃. l-malic acid, bromo-succinic acid, nalidixic acid, lithium Positive for sodium lactate, l-glutamic acid, l-histidine, chloride, potassium tellurite, Tween 40, γ-amino-butyr- l-pyroglutamic acid, l-serine, lincomycin, tetrazolium vi- ic acid, β-hydroxy-d,l-butyric acid, α-hydroxy-butyric olet, l-lactic acid, α-keto-glutaric acid, lithium chloride, acid, α-keto-butyric acid, acetoacetic acid, propionic acid, β-hydroxy-d,l-butyric acid, propionic acid, acetic acid, acetic acid, formic acid, aztreonam, sodium butyrate, and sodium bromate in Biolog GEN III; but negative for and sodium bromate in Biolog GEN III; but negative for dextrin, d-maltose, d-trehalose, d-cellobiose, gentiobiose, stachyose, N-acetyl-β-d-mannosamine, N-acetyl-d-galac- sucrose, d-turanose, stachyose, d-raffinose, α-d-lactose, tosamine, inosine, troleandomycin, lincomycin, vancomy- d-melibiose, β-methyl-d-glucoside, d-salicin, N-ace- cin, p-hydroxy-phenylacetic acid, fusidic acid, rifamycin tyl-d-glucosamine, N-acetyl-β-d-mannosamine, N-ace- SV, minocycline, niaproof 4, and tetrazolium violet. tyl-d-galactosamine, N-acetylneuraminic acid, α-d-glu- Strain H11R12 (=NIBRBAC000502715) was isolated cose, d-mannose, d-fructose, d-galactose, 3-methyl glu- from the feces of a stork (Ciconia boyciana) from Seoul cose, d-fucose, l-fucose, l-rhamnose, inosine, d-sorbitol, Grand Park Zoo, Gwacheon-si, Gyeonggi-do, Korea. The d-mannitol, d-arabitol, myo-inositol, glycerol, d-glucose, DNA G+C content of the type strain is 32.7 mol%. d-fructose-6-PO4, d-aspartic acid, d-serine, troleandomycin, gelatin, glycyl-l-proline, l-alanine, l-arginine, l-as- Description of Enterococcus aquimarinus H21T7 partic acid, pectin, d-galacturonic acid, l-galactonic acid lactone, d-glucuronic acid, glucuronamide, quinic acid, Cells are Gram-stain positive, non-flagellated, and d-saccharic acid, vancomycin, p-hydroxy-phenylacetic coccoid-shaped. Colonies are circular, raised, entire, and acid, methyl pyruvate, d-lactic acid, citric acid, d-malic cream-colored after 2 days of incubation on TSA at 25℃. acid, l-malic acid, bromo-succinic acid, nalidixic acid, Positive for dextrin, sucrose, α-d-lactose, d-melibiose, Tween 40, γ-amino-butyric acid, α-hydroxy-butyric acid, β-methyl-d-glucoside, d-salicin, N-acetyl-d-glucosamine, α-keto-butyric acid, acetoacetic acid, formic acid, aztreo- N-acetyl-β-d-mannosamine, N-acetyl-d-galactosamine, nam, pH 5, 8% NaCl, fusidic acid, rifamycin SV, minocy- N-acetylneuraminic acid, α-d-glucose, d-mannose, d-fruc- cline, guanidine HCl, niaproof 4, and sodium butyrate. tose, d-galactose, 3-methyl glucose, d-fucose, l-fucose, Strain H11R21 (=NIBRBAC000502716) was isolated l-rhamnose, inosine, sodium lactate, fusidic acid, d-ser- from the feces of a stork (Ciconia boyciana) from Seoul ine, d-mannitol, d-arabitol, myo-inositol, glycerol, d-glu- Grand Park Zoo, Gwacheon-si, Gyeonggi-do, Korea. cose, d-fructose-6-PO4, d-aspartic acid, troleandomycin, The DNA G+C content of the type strain is 44.7 mol%. rifamycin SV, minocycline, glycyl-l-proline, l-alanine, l-arginine, l-aspartic acid, l-glutamic acid, l-histidine, Description of Shewanella xiamenensis M13M1 l-pyroglutamic acid, l-serine, lincomycin, guanidine HCl, pectin, d-galacturonic acid, l-galactonic acid lactone, Cells are Gram-stain positive, non-flagellated, and d-gluconic acid, d-glucuronic acid, glucuronamide, mucin rod-shaped. Colonies are circular, raised, entire, and acid, quinic acid, d-saccharic acid, vancomycin, tetra- cream-colored after 2 days of incubation on MA at 10℃. zolium violet, tetrazolium blue, methyl pyruvate, d-lactic Positive for dextrin, d-mannose, d-maltose, gentiobiose, acid, l-lactic acid, citric acid, α-keto-glutaric acid, d-ma- sucrose, d-melibiose, N-acetyl-β-d-mannosamine, d-fruc- lic acid, l-malic acid, bromo-succinic acid, nalidixic acid, tose, d-galactose, 3-methyl glucose, d-fucose, l-fucose, lithium chloride, potassium tellurite, aztreonam, sodium l-rhamnose, inosine, sodium lactate, fusidic acid, d-fruc- butyrate, and sodium bromate in Biolog GEN III; but tose-6-PO4, troleandomycin, glycyl-l-proline, l-glutamic negative for d-maltose, d-trehalose, d-cellobiose, gentio- acid, lincomycin, guanidine HCl, niaproof 4, pectin, d-ga- biose, d-turanose, stachyose, d-raffinose, d-sorbitol, gela- lacturonic acid, l-galactonic acid lactone, d-glucuronic tin, p-hydroxy-phenylacetic acid, Tween 40, γ-amino-bu- acid, glucuronamide, vancomycin, tetrazolium violet, tyric acid, β-hydroxy-d,l-butyric acid, α-hydroxy-butyric tetrazolium blue, methyl pyruvate, d-lactic acid, l-lactic acid, α-keto-butyric acid, acetoacetic acid, propionic acid, acid, l-malic acid, lithium chloride, potassium tellur- acetic acid, formic acid, and niaproof 4. ite, acetoacetic acid, acetic acid, and aztreonam in Bio-

Strain H21T7 (=NIBRBAC000502719) was isolated log GEN III; but negative for d-trehalose, d-cellobiose, November 2020 Lee et al. Unrecorded gut bacterial species from Korean endemic vertebrates 337 d-turanose, stachyose, d-raffinose, α-d-lactose, β-meth- Acknowledgements yl-d-glucoside, d-salicin, N-acetyl-d-galactosamine, N-acetylneuraminic acid, α-d-glucose, d-sorbitol, d-man- We thank all the staffs of Seoul Grand Park Zoo for nitol, d-arabitol, myo-inositol, glycerol, d-glucose, d-as- their cooperation, especially J. R. Roh and S. D. Park, for partic acid, d-serine, gelatin, l-alanine, l-arginine, l-as- providing research permits to conduct our work, and H. E. partic acid, l-histidine, l-pyroglutamic acid, l-serine, Shim for providing the avian feces. This work was sup- d-gluconic acid, mucin acid, quinic acid, d-saccharic acid, ported by grants from the National Institute of Biological p-hydroxy-phenylacetic acid, citric acid, α-keto-glutaric Resources (NIBR), funded by the Ministry of Environ- acid, d-malic acid, bromo-succinic acid, nalidixic acid, ment (MOE) of the Republic of Korea (NIBR201801106 Tween 40, γ-amino-butyric acid, β-hydroxy-d,l-butyric and NIBR201902111). acid, α-hydroxy-butyric acid, α-keto-butyric acid, propionic acid, formic acid, pH 5, 4% NaCl, 8% NaCl, rifamycin SV, minocycline, sodium butyrate, N-acetyl-d-glucosamine, and sodium bromate. References Strain M13M1 (=NIBRBAC000502722) was isolated Cani, P.D. 2017. Gut microbiota-at the intersection of every- from the intestinal tract of a cobitid fish (Niwaella multi thing? Nature Reviews Gastroenterology & Hepatology fasciata) from Gimcheon-si, Gyeongsangbuk-do, Korea. 14:321. + The DNA G C content of the type strain is 42.7 mol%. Choe, S., D. Lee, H. 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