INTRODUCTION

The composition of microbial flora depends on external environmental factors, like pH, temperature, seasonality, humidity, and nutrient availability, in the regions in which they are found (Fierer and Jackson 2006), and ecological environments (i.e., habitats) are also impacted by microbes. Eukaryotic hosts are important habitats for microbes, and each has a unique and intimate interaction with microbes. Host-associated bacteria can be divided into two major groups: autochthonous (able to colonize within the host) and allochthonous (considered to be free-living). Many symbiotic bacteria (autochthonous) form specific and, on occasion, obligate interactions with hosts, while other symbiotic bacteria (allochthonous) show less dependent associations. The latter can multiply freely in the environment while also colonizing plant or animal hosts when an opportunity arises (Sullam et al. 2012). Animal intestines and plant roots, where complex metabolic processes occur that are necessary for growth and health, contain the most abundant and diverse symbiotic bacteria. Host-associated bacteria play critical roles in nutritional provisioning, metabolic homeostasis, and immune defense.

The bacterial communities in the intestines of aquatic animals are much denser than those of terrestrial animals, as water is an ideal medium for bacterial growth (Banerjee and Ray 2017) and is a reservoir for bacteria. The development of high-throughput sequencing technology has allowed for analyses of the intestinal bacterial compositions of numerous fish species in an effort to increase production and conserve endangered species. The results suggest that the bacterial population of fish intestines may vary by host species, nutritional status, age (life cycle), surrounding water type, and other environmental conditions (Sullam et al. 2012;Wong and Rawls 2012;Llewellyn et al. 2014;Ringø et al. 2015;Liu et al. 2016). Conventional culture-based methods are limited by the required culture conditions and types of culture media, particularly for fastidious and obligate anaerobes. However, the cultivation of bacterial strains is still essential because the ecological role of bacteria in natural environments and the potential industrial applications of bacterial resources can only be revealed after successful cultivation and characterization.

During the investigation of host associated bacteria from 2017 to 2019, we collected fish species that have unique aquatic habitats or are associated with human lifestyles. A large number of bacterial strains were isolated from the intestines of diverse fish species and freshwater clams collected from different geographic locations, including those with freshwater and brackish water. A total of 42 unrecorded species are among the host-associated bacterial strains described here.

MATERIALS AND METHODS

1. Sampling and bacterial isolation

A total of nine fish species and one freshwater clam species were collected from freshwater and brackish water environments (Table 1). Each intestine of fish and the entire clam insides samples were processed separately by spreading onto the following agar media after homogenization and serial dilution: Reasoner’s 2A agar (R2A), Lactobacillus selection agar (LBS), eosine methylene blue agar (EMB), International Streptomyces Project 4 agar (ISP 4), marine agar (MA), trypticase soy agar (TSA), nutrient agar (NA), and 1/10 medium dilution agar of each. The intestinal tissues of three fish species (Hypomesus nipponensis, Micropterus salmoides, and Silurus asotus) were also used for isolation via a single-cell sorting method using a flow cytometer (FACS Aria II, Becton Dickinson) to remove fast-growing and culture-dominant bacteria. After aerobic incubation at 15-30°C for 7-14 days, the bacterial strains were purified by subculturing a single colony onto fresh media. The pure cultures were preserved at -80°C in 20% (w/v) glycerol.

2. Identification and characterization of unrecorded species

PCR amplification with universal primers 27F and 1492R and 16S rRNA gene sequencing with primers 518F and 800R were performed using standard procedures for bacterial identification. The 16S rRNA gene sequences of the bacterial strains were compared with those of other bacterial strains using the EZ Biocloud server (Yoon et al. 2017). Bacterial strains showing 98.7% or higher sequence similarity with those of validly published species that had not previously been reported in Korea were designated as unrecorded bacterial species. For the phylogenetic analyses, the alignment of the 16S rRNA gene sequences obtained from the isolated strains and those of closely related strains was carried out using EZ Editor (Jeon et al. 2014). Phylogenetic trees were constructed using the neighbor- joining (Saitou and Nei 1987), maximum parsimony (Fitch 1971), and maximum likelihood (Felsenstein 1981) methods, and evolutionary distances were calculated using the Jukes and Cantor one-parameter model (Jukes and Cantor 1969) with the MEGA package ver. 7.0 (Kumar et al. 2016). Bootstrap analysis was carried out using 1,000 resampled datasets (Felsenstein 1985), which were also used to test the topology of the phylogenetic tree.

The colony morphology of the strains was observed on agar plates with a magnifying glass after the cells had grown to stationary phase. Cellular morphology and size were examined by transmission electron microscopy ( JEM- 1400Plus, JEOL, Japan or HT7700, Hitachi, Japan). Gram staining was performed using a Gram staining kit (Sigma, USA), and oxidase activity was determined using 1% (w/ v) tetramethyl ρ-phenylenediamine. Biochemical characteristics were tested using API-20NE galleries (bioMérieux, France) according to the manufacturer’s instructions.

RESULTS AND DISCUSSION

The fast-growing and culture-dominant bacteria isolated from fish intestines and clams belonged to the genera Aeromonas (freshwater) and Shewanella (brackish water). These two genera are common and abundant in aquatic environments, and this finding suggests that the host-associated bacterial strains isolated in this study originated from water as allochthonous bacteria. We used a cell sorter to obtain numerous bacterial strains because culture dominance restricted the isolation of diverse bacterial strains. Most of the strains obtained in this way belonged to the genus Pseudomonas (class Gammaproteobacteria), as well as diverse genera of the class Actinobacteria; the strains were primarily isolated from the fish species Hypomesus nipponensis and Micropterus salmoides, respectively. In particular, strains belonging to the genus Pseudomonas were classified to 28 different species and included six unrecorded species. These results indicate that isolation of host-associated bacteria using a cell sorter can increase the diversity of identified bacterial strains and could assist in overcoming the problem of culture dominance.

Based on the comparison of the 16S rRNA gene sequences and phylogenetic analyses, a total of 42 strains were assigned to four phyla, six classes, 17 orders, 26 families, and 32 genera. The taxonomic affiliation and identification results are summarized in Table 2.

A phylogenetic tree of the 19 unrecorded Gram-positive species assigned to the phyla Actinobacteria (ten species) and Firmicutes (nine species) is shown in Figure 1. At the genus level, these 19 unrecorded species belong to 17 genera: Corynebacterium, Mycobacteroides, Mycolicibacterium, Gordonia, Williamsia, Medestobacter, Brachybacterium, Sanguibacter, Arthrobacter, and Mycolicibacterium of the class Actinobacteria and Fictibacillus (two species), Psychrobacillus, Cohnella, Paenibacillus (two species), Rummeliibacillus, Enterococcus, and Vagococcus of the class Bacilli. The phylogenetic positions of the 23 unrecorded Gram-negative strains assigned to the phyla Bacteroidetes and Proteobacteria are shown in Figure 2. These 23 unrecorded species belong to 15 genera: Empedobacter and Flavobacterium of the class Flavobacteriia; Aquamicrobium, Paracoccus, and Sphingomonas of the class Alphaproteobacteria; Achromobacter, Delftia, and Deefgea of the class Betaproteobacteria; and Aeromonas, Providencia, Yersinia (three species), Marinomonas, Acinetobacter, Pseudomonas (six species), and Proteus of the class Gammaproteobacteria.

These 42 unrecorded species displayed diverse cellular morphologies, including rods, short rods, and cocci (Fig. 3), with or without flagella. The colony colors were white, cream, ivory, yellow, or pink. All species grew aerobically, and some strains produced spores. The enzymatic and physiological properties examined using API 20NE kits varied depending on the species. Detailed cultural, morphological, and biochemical characteristics are shown in the species description section. Some physiological characteristics of the 42 unrecorded species differed from those of previously identified species; however, the morphologies and phylogenetic analyses proved that they were of the same species. The presence of these 42 species has been reported overseas, but until now, were unrecorded in Korea. Their phenotypic properties are described here.

Description of Brachybacterium huguangmaarense FEB3-24

Cells are Gram-stain-positive, aerobic, non-flagellated, and coccoid to short rod-shaped. Colonies grown on R2A are circular, convex, smooth, and ivory colored after incubation for 2-3 days at 30°C. Esculin is hydrolyzed, but not gelatin. Nitrate is not reduced. Glucose is not fermented and indole is not produced. Enzyme activity of β-galactosidase is observed, but not oxidase, arginine dihydrolase, and urease. D-glucose, D-mannose (weakly), D-mannito l (weakly), N-acetyl-glucosamine (weakly), D-maltose, and potassium gluconate are utilized as sole carbon sources, but not L-arabinose, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. Strain FEB3-24 (=NNIBR2017301BA49) was isolated from intestine of Anquilla japonica collected from fishery, Gochang, Jeollabuk- do, Korea. The GenBank accession number for the 16S rRNA gene sequence is MG780346.

Description of Empedobacter falsenii FEB3-06

Cells are Gram-stain-negative, aerobic, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, convex, smooth, and ivory colored after incubation for 2-3 days at 30°C. Esculin and gelatin are hydrolyzed. Nitrate is not reduced and glucose is not fermented. Indole is produced. Enzyme activities of oxidase and β-galactosidase are observed, but not arginine dihydrolase and urease. D-glucose, N-acetyl-glucosamine (weakly), D-maltose (weakly), trisodium citrate (weakly), and phenylacetic acid (weakly) are utilized as sole carbon sources, but not L-arabinose, D-mannose, potassium gluconate, capric acid, adipic acid, and malic acid. Strain FEB3-06 (=NNIBR2017BA48) was isolated from intestine of Anquilla japonica collected from from fishery, Gochang, Jeollabuk-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MG780344.

Description of Delftia lacutris FEB3-05

Cells are Gram-stain-negative, aerobic, flagellated, and rod-shaped. Colonies grown on NA are circular, convex, smooth, and ivory colored after incubation for 2-3 days at 25°C. Esculin and gelatin are not hydrolyzed and nitrate is reduced to nitrite. Glucose is not fermented and indole is not produced. Enzyme activities of oxidase, arginine dihydrolase, and urease are observed, but not β-galactosidase. D-mannitol, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid are utilized as sole carbon sources, but not D-glucose, L-arabinose, D-mannose, N-acetyl-glucosamine, and D-maltose. Strain FEB3-05 (=NNIBR2017BA47) was isolated from intestine of Anquilla japonica collected from fishery, Gochang, Jeollabuk-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MG780343.

Description of Sphingomonas olei 19SCL-58

Cells are Gram-stain-negative, aerobic, flagellated, and rod-shaped. Colonies grown on R2A are circular, convex, smooth, and yellow colored after incubation for 2-3 days at 25°C. Esculin is hydrolyzed, but not gelatin. Nitrate is not reduced. Glucose is not fermented and indole is not produced. Enzyme activities of oxidase, arginine dihydrolase, urease, and β-galactosidase are not observed. D-glucose and capric acid are utilized as sole carbon sources, but not L-arabinose, D-mannose, D-mannitol, N-acetyl- glucosamine, D-maltose, potassium gluconate, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. Strain 19SCL-58 (=NNIBR2019642BA185) was isolated from Corbicula leana collected from the Yeongok stream, Gangneung, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559812.

Description of Proteus terrae 19SCL-27

Cells are Gram-stain-negative, aerobic, flagellated, and rod-shaped. Colonies grown on R2A are circular, convex, smooths and cream white colored after incubation for 2-3 days at 25°C. Esculin and gelatin are hydrolyzed and nitrate is reduced to nitrite. Glucose is fermented and indole is produced. Enzyme activity of urease is observed, but not oxidase, arginine dihydrolase, and β-galactosidase. D-glucose, N-acetyl-glucosamine, D-maltose, and adipic acid are utilized as sole carbon sources, but not L-arabinose, D-mannose, D-mannitol, potassium gluconate, capric acid, malic acid, trisodium citrate, and phenylacetic acid. Strain 19SCL-27 (=NNIBR2019642BA166) was isolated from Corbicula leana collected from the Yeongok stream, Gangneung, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559811.

Description of Williamsia serinedens S8-55

Cells are Gram-stain-positive, aerobic, non-flagellated, and rod-shaped. Colonies grown on TSA are circular, convex, smooth, and pinkish ivory colored after incubation for 2-3 days at 25°C. Esculin is hydrolyzed, but not gelatin. Nitrate is reduced to nitrogen. Glucose is not fermented and indole is not produced. Enzyme activities of urease and β-galactosidase are observed, but not oxidase and arginine dihydrolase. D-mannitol, malic acid and trisodium citrate are utilized as sole carbon sources, but not D-glucose, L-arabinose, D-mannose, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, and phenylacetic acid. Strain S8-55 (=NNIBR2017BA51) was isolated from intestine of Coreoperca herzi collected from the Nakdong River, Sangju, Gyeonsangjuk-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MG780347.

Description of Gordonia polyisoprenivorans CFH1-11

Cells are Gram-stain-negative, aerobic, non-flagellated, and rod-shaped. Colonies grown on R2A are circular with entire margin, brittle, opaque, and pinkish white colored after incubation for 2-3 days at 25°C. Esculin is hydrolyzed, but not gelatin. Nitrate is not reduced and indole is not produced. Glucose is fermented. Enzyme activities of urease and β-galactosidase are observed, but not oxidase and arginine dihydrolase. D-glucose, L-arabinose, D-mannose, D-mannitol, potassium gluconate, malic acid, trisodium citrate and phenylacetic acid are utilized as sole carbon sources, but not N-acetyl-glucosamine, D-maltose, capric acid, and adipic acid. Strain CFH1-11 (=NNIBR2019642BA20) was isolated from intestine of Hypomesus nipponensis collected from the Soyang Lake, Chuncheon, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559440.

Description of Arthrobacter stackebrandtii IFH1-28

Cells are Gram-stain-positive, aerobic, non-flagellated, and coccoid to short rods shaped. Colonies grown on NA are circular entire margin, smooth, and ivory to yellow colored after incubation for 2-3 days at 25°C. Esculin is hydrolyzed, but not gelatin. Nitrate is reduced and glucose is fermented. Indole is not produced. Enzyme activities of oxidase (weakly) and β-galactosidase are observed, but not arginine dihydrolase and urease. D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, malic acid, and trisodium citrate are utilized as sole carbon sources, but not adipic acid and phenylacetic acid. Strain IFH1-28 (=NNIBR2019642BA78) was isolated from intestine of Hypomesus nipponensis collected from the Soyang Lake, Inje, Gangwon- do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559455.

Description of Flavobacterium frigidimaris CFH1-34

Cells are Gram-stain-negative, aerobic, non-flagellated, and rod-shaped. Colonies grown on Reasoner’s 2A agar (R2A) are circular, smooth, undulate margin, opaque, and yellow colored after incubation for 2-3 days at 25°C. Esculin is hydrolyzed, but not gelatin. Nitrate is reduced to nitrogen. Glucose is not fermented and indole is not produced. Enzyme activities of urease and β-galactosidase are observed, but not oxidase and arginine dihydrolase. The strain does not utilize D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid as sole carbon sources. Strain CFH1-34 (=NNIBR2019642BA36) was isolated from intestine of Hypomesus nipponensis collected from the Soyang Lake, Chuncheon, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559444.

Description of Fictibacillus enclensis FH1-17

Cells are Gram-stain-positive, aerobic, flagellated, and rod-shaped. Colonies grown on TSA are circular, smooth, convex, opaque, and cream colored after incubation for 2-3 days at 30°C. Esculin is hydrolyzed, but not gelatin. Nitrate is not reduced and indole is not produced. Glucose is fermented. Enzyme activity of β-galactosidase is observed, but not oxidase, urease, and arginine dihydrolase. D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, adipic acid, malic acid, trisodium citrate, and phenylacetic acid are utilized as sole carbon sources, but not capric acid. Strain FH1-17 (=NNIBR2018142BA16) was isolated from intestine of Hypomesus nipponensis collected from the Soyang Lake, Chuncheon, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MK396594.

Description of Psychrobacillus soli FH1-09

Cells are Gram-stain-positive, aerobic, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, raised, moist, and ivory colored after incubation for 2-3 days at 30°C. Esculin and gelatin are hydrolyzed. Nitrate is reduced to nitrogen. Glucose is not fermented and indole is not produced. Enzyme activity of urease is observed, but not oxidase, arginine dihydrolase, and β-galactosidase. D-glucose, potassium gluconate, and adipic acid are utilized as sole carbon sources, but not L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, capric acid, malic acid, trisodium citrate, and phenylacetic acid. Strain FH1-09 (=NNIBR2018142BA8) was isolated from intestine of Hypomesus nipponensis collected from the Soyang Lake, Chuncheon, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MK396593.

Description of Paenibacillus mendelii IFH1-14

Cells are Gram-stain-positive, aerobic, flagellated, and rod-shaped. Colonies grown on R2A are circular with entire margin, smooth, and ivory colored after incubation for 2-3 days at 25°C. Esculin is hydrolyzed, but not gelatin. Nitrate is not reduced and indole is not produced. Glucose is fermented. Enzyme activities of oxidase and arginine dihydrolase are observed, but not urease and β-galactosidase. D-glucose, L-arabinose, D-mannose, D-mannitol, potassium gluconate, capric acid, malic acid, and trisodium citrate are utilized as sole carbon sources, but not N-acetyl- glucosamine, D-maltose, adipic acid, and phenylacetic acid. Strain IFH1-14 (=NNIBR2019642BA74) was isolated from intestine of Hypomesus nipponensis collected from the Soyang lake, Inje, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559453.

Description of Paenibacillus silvae FH1-05

Cells are Gram-stain-positive, aerobic, flagellated, and rod-shaped. Colonies grown on TSA are circular, raised, and ivory colored after incubation for 2-3 days at 30°C. Esculin and gelatin are hydrolyzed. Nitrate is not reduced and indole is not produced. Glucose is fermented. Enzyme activities of oxidase and β-galactosidase are observed, but not arginine dihydrolase and urease. D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, and potassium gluconate are utilized as sole carbon sources, but not capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. Strain FH1-05 (=NNIBR2018142BA4) was isolated from intestine of Hypomesus nipponensis collected from the Soyang Lake, Chuncheon, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MK396592.

Description of Rummeliibacillus pycnus FH4-10

Cells are Gram-stain-positive, aerobic, flagellated, and rod-shaped. Colonies grown on TSA are circular, smooth, convex, translucent, and yellowish cream colored after incubation for 2-3 days at 30°C. Esculin and gelatin are not hydrolyzed. Nitrate is not reduced and indole is not produced. Glucose is not fermented. Enzyme activity of oxidase is observed, but not arginine dihydrolase, urease, and β-galactosidase. Potassium gluconate, adipic acid, malic acid, trisodium citrate and phenylacetic acid are utilized as sole carbon sources, but not D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose and capric acid. Strain FH4-10 (=NNIBR2018142BA84) was isolated from intestine of Hypomesus nipponensis collected from the Pangok Reservoir, Sangju, Gyeongsangbuk-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MK396595.

Description of Enterococcus faecalis CFHS-18

Cells are Gram-stain-positive, facultative anaerobic, non-flagellated, and coccoid-shaped. Colonies grown on 1/10 diluted MA are circular with entire margin, convex, smooth, and ivory colored after incubation for 2-3 days at 25°C. Gelatin is hydrolyzed, but not esculin. Nitrate is not reduced and indole is not produced. Glucose is not fermented. Enzyme activities of arginine dihydrolase, urease, and oxidase (weakly) are observed, but not β-galactosidase. D-glucose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, adipic acid, and trisodium citrate are utilized as sole carbon sources, but not L-arabinose, D-mannose, capric acid, malic acid, and phenylacetic acid. Strain CFHS-18 (=NNIBR2019642BA52) was isolated from intestine of Hypomesus nipponensis collected from the Soyang Lake, Chuncheon, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559450.

Description of Vagococcus fluvialis FH5-03

Cells are Gram-stain-positive, aerobic, flagellated, and coccoid-shaped. Colonies grown on TSA are circular, smooth, convex, opaque, and cream colored after incubation for 2-3 days at 37°C. Esculin is hydrolyzed, but not gelatin. Nitrate is not reduced and indole is not produced. Glucose is fermented. Enzyme activity of β-galactosidase is observed, but not arginine dihydrolase, urease, and oxidase. D-glucose and trisodium citrate are utilized as sole carbon sources, but not L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid and phenylacetic acid. Strain FH5-03 (=NNIBR2018142BA119) was isolated from intestine of Hypomesus nipponensis collected from the Songnim Reservoir, Gyeongsan, Gyeongsangbuk-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MK396597.

Description of Paracoccus sphaerophysae IFH1-29

Cells are Gram-stain-negative, aerobic, non-flagellated, and coccoid-shaped. Colonies grown on R2A are circular with entire margin, smooth, and ivory to yellow colored after incubation for 2-3 days at 25°C. Esculin is hydrolyzed weakly, but not gelatin. Nitrate is not reduced. Indole is not produced and glucose is not fermented. Enzyme activity of oxidase is observed, but not arginine dihydrolase, urease, and β-galactosidase. D-glucose, L-arabinose, potassium gluconate and malic acid are utilized as sole carbon sources, but not D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, capric acid, adipic acid, trisodium citrate, and phenylacetic acid. Strain IFH1-29 (=NNIBR2019642BA79) was isolated from intestine of Hypomesus nipponensis collected from the Soyang Lake, Inje, Gangwon- do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559456.

Description of Deefgea rivuli IFH1-02

Cells are Gram-stain-negative, aerobic, flagellated, and rod-shaped. Colonies grown on R2A are circular entire margin, flat, smooth, and ivory colored after incubation for 2-3 days at 25°C. Esculin is hydrolyzed weakly, but not gelatin. Nitrate is not reduced to nitrogen and indole is not produced. Glucose is fermented. Enzyme activity of oxidase is observed, but not arginine dihydrolase, urease, and β-galactosidase. D-mannose, N-acetyl-glucosamine, and potassium gluconate are utilized as sole carbon sources, but not D-glucose, L-arabinose, D-mannitol, D-maltose, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. Strain IFH1-02 (=NNIBR2019642BA68) was isolated from intestine of Hypomesus nipponensis collected from the Soyang Lake, Inje, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559452.

Description of Aeromonas sobria CFH1-09

Cells are Gram-stain-negative, aerobic, flagellated, and short rod-shaped. Colonies grown on NA are circular with entire margin, smooth, translucent, and cream white colored after incubation for 2-3 days at 25°C. Esculin and gelatin are hydrolyzed and nitrate is reduced. Indole is produced and glucose is fermented. Enzyme activities of oxidase, arginine dihydrolase, and β-galactosidase are observed, but not urease. D-glucose, L-arabinose (weakly), D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, malic acid and trisodium citrate are utilized as sole carbon sources, but not adipic acid and phenylacetic acid. Strain CFH1- 09 (=NNIBR2019642BA19) was isolated from intestine of Hypomesus nipponensis collected from the Soyang Lake, Chuncheon, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559439.

Description of Providencia heimbachae FH5-02

Cells are Gram-stain-negative, aerobic, flagellated, and rod-shaped. Colonies grown on NA are circular, smooth, convex, opaque, and cream colored after incubation for 2-3 days at 25°C. Esculin is hydrolyzed, but not gelatin. Nitrate is reduced and glucose is fermented. Indole is not produced. Enzyme activity of arginine dihydrolase is observed, but not urease, β-galactosidase, and oxidase. D-glucose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, malic acid, trisodium citrate, and phenylacetic acid are utilized as sole carbon sources, but not L-arabinose, capric acid, and adipic acid. Strain FH5-02 (=NNIBR2018142BA118) was isolated from Hypomesus nipponensis collected from the Songnim Reservoir, Gyeongsan, Gyeongsangbuk-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MK396596.

Description of Yersinia entomophaga CFH1-25

Cells are Gram-stain-negative, aerobic, flagellated, and rod-shaped. Colonies grown on R2A are circular with entire margin, convex, smooth, and cream white colored after incubation for 2-3 days at 25°C. Esculin and gelatin are hydrolyzed. Nitrate is reduced and indole is produced. Glucose is not fermented. Enzyme activities of arginine dihydrolase and β-galactosidase are observed, but not oxidase and urease. D-glucose, D-mannose, D-mannitol, N-acetyl- glucosamine, D-maltose, potassium gluconate, adipic acid, malic acid, and trisodium citrate are utilized as sole carbon sources, but not L-arabinose, capric acid, and phenylacetic acid. Strain CFH1-25 (=NNIBR2019642BA29) was isolated from intestine of Hypomesus nipponensis collected from the Soyang Lake, Chuncheon, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559442.

Description of Yersinia enterocolitica subsp. palearctica IFH1-18

Cells are Gram-stain-negative, aerobic, non-flagellated, and rod-shaped. Colonies grown on R2A are circular entire margin, smooth, and ivory colored after incubation for 2-3 days at 25°C. Esculin is hydrolyzed, but not gelatin. Nitrate is not reduced and indole is not produced. Glucose is fermented. Enzyme activities of arginine dihydrolase, urease, β-galactosidase, and oxidase (weakly) are observed. D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, malic acid, and trisodium citrate are utilized as sole carbon sources, but not capric acid, adipic acid and phenylacetic acid. Strain IFH1-18 (=NNIBR2019642BA75) was isolated from intestine of Hypomesus nipponensis collected from the Soyang Lake, Inje, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559454.

Description of Pseudomonas aeruginosa CFHS-01

Cells are Gram-stain-negative, aerobic, flagellated, and rod-shaped. Colonies grown on R2A are circular with entire margin, smooth, and ivory colored after incubation for 2-3 days at 25°C. Esculin is hydrolyze, but not gelatin. Nitrate is reduced to nitrogen. Indole is not produced and glucose is not fermented. Enzyme activities of arginine dihydrolase, urease, and oxidase are observed, but not β-galactosidase. D-glucose, D-mannitol, N-acetyl-glucosamine, potassium gluconate, capric acid, adipic acid, malic acid, and trisodium citrate are utilized as sole carbon sources, but not L-arabinose, D-mannose, D-maltose and phenylacetic acid. Strain CFHS-01 (=NNIBR2019642BA40) was isolated from intestine of Hypomesus nipponensis collected from the Soyang Lake, Chuncheon, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559445.

Description of Pseudomonas lundensis CFHS-10

Cells are Gram-stain-negative, aerobic, flagellated, and rod-shaped. Colonies grown on R2A are circular with entire margin, smooth, and ivory colored after incubation for 2-3 days at 25°C. Gelatin is hydrolyzed, but not esculin. Nitrate is not reduced and indole is not produced. Glucose is fermented. Enzyme activities of arginine dihydrolase, urease, and oxidase (weakly) are observed, but not β-galactosidase. D-glucose, L-arabinose, D-mannose, potassium gluconate, capric acid, malic acid, and trisodium citrate are utilized as sole carbon sources, but not D-mannitol, N-acetyl- glucosamine, D-maltose, adipic acid, and phenylacetic acid. Strain CFHS-10 (=NNIBR2019642BA45) was isolated from intestine of Hypomesus nipponensis collected from the Soyang Lake, Chuncheon, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559447.

Description of Pseudomonas paralactis CFH1-01

Cells are Gram-stain-negative, aerobic, flagellated, and rod-shaped. Colonies grown on TSA are circular, flat, smooth, opaque, and ivory colored after incubation for 2-3 days at 25°C. Gelatin is hydrolyzed, but not esculin. Nitrate is not reduced and indole is not produced. Glucose is not fermented. Enzyme activities of arginine dihydrolase, urease, and oxidase are observed, but not β-galactosidase. D-glucose L-arabinose, D-mannose, D-mannitol, N-acetyl- glucosamine, D-maltose, potassium gluconate, capric acid, malic acid, and trisodium citrate are utilized as sole carbon sources, but not adipic acid and phenylacetic acid. Strain CFH1-01 (=NNIBR2019642BA11) was isolated from intestine of Hypomesus nipponensis collected from the Soyang Lake, Chuncheon, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559438.

Description of Pseudomonas synxantha CFHS-02

Cells are Gram-stain-negative, aerobic, flagellated, and rod-shaped. Colonies grown on R2A are circular with entire margin, smooth, and ivory colored after incubation for 2-3 days at 25°C. Gelatin and esculin are hydrolyzed and nitrate is reduced. Indole is not produced and glucose is not fermented. Enzyme activities of arginine dihydrolase and oxidase are observed, but not urease and β-galactosidase. D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, potassium gluconate, capric acid, malic acid, and trisodium citrate are utilized as sole carbon sources, but not D-maltose, adipic acid, and phenylacetic acid. Strain CFHS-02 (=NNIBR2019642BA41) was isolated from intestine of Hypomesus nipponensis collected from the Soyang Lake, Chuncheon, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559446.

Description of Pseudomonas versuta CFHS-14

Cells are Gram-stain-negative, aerobic, flagellated, and rod-shaped. Colonies grown on R2A are circular with entire margin, smooth, and ivory colored after incubation for 2-3 days at 25°C. Esculin is hydrolyzed, but not gelatin. Nitrate is not reduced and indole is not produced. Glucose is fermented. Enzyme activities of arginine dihydrolase and oxidase are observed, but not urease and β-galactosidase. D-glucose, L-arabinose, D-mannose, D-mannitol, potassium gluconate, capric acid, malic acid, and trisodium citrate are utilized as sole carbon sources, but not N-acetyl-glucosamine, D-maltose, adipic acid and phenylacetic acid. Strain CFHS-14 (=NNIBR2019642BA49) was isolated from intestine of Hypomesus nipponensis collected from the Soyang Lake, Chuncheon, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559449.

Description of Pseudomonas weihenstephanensis CFHS-12

Cells are Gram-stain-negative, aerobic, flagellated, and rod-shaped. Colonies grown on R2A are circular with entire margin, smooth, and ivory colored after incubation for 2-3 days at 25°C. Gelatin is hydrolyzed, but not esculin. Nitrate is not reduced and indole is not produced. Glucose is fermented. Enzyme activities of arginine dihydrolase, urease, and oxidase (weakly) are observed, but not β-galactosidase. D-glucose, D-mannose (weakly), potassium gluconate, capric acid, malic acid, and trisodium citrate are utilized as sole carbon sources, but not L-arabinose, D-mannitol, N-acetyl-glucosamine, D-maltose, adipic acid, and phenylacetic acid. Strain CFHS-12 (=NNIBR2019642BA47) was isolated from intestine of Hypomesus nipponensis collected from the Soyang Lake, Chuncheon, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559448.

Description of Yersinia ruckeri FI2-07

Cells are Gram-stain-negative, aerobic, flagellated, and rod-shaped. Colonies grown on NA are circular, convex, smooth, and cream colored after incubation for 2-3 days at 25°C. Esculin and gelatin are hydrolyzed. Nitrate is reduced and glucose is fermented. Indole is not produced. Enzyme activities of arginine dihydrolase and β-galactosidase are observed, but not oxidase and urease. D-glucose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, malic acid, and trisodium citrate are utilized as sole carbon sources, but not L-arabinose, capric acid, and phenylacetic acid. Strain FI2-07 (=NNIBR2018142BA242) was isolated from intestine of Iksookimia koreensis collected from the Geum River, Geumsan, Chungcheongnam-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MK396587.

Description of Acinetobacter pittii FI1-01

Cells are Gram-stain-negative, aerobic, non-flagellated, and diplococoid or short rods-shaped. Colonies grown on TSA are circular, convex, smooth, opaque, and ivory colored after incubation for 2-3 days at 37°C. Esculin and gelatin are hydrolyzed weakly. Nitrate is not reduced and indole is not produced. Glucose is fermented. Enzyme activities of oxidase, urease, arginine dihyrolase, and β-galactosidase are not observed. L-arabinose, capric acid, adipic acid, malic acid, and trisodium citrate are utilized as sole carbon sources, but not D-glucose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, and phenylacetic acid. Strain FI1-01 (=NNIBR2018142BA228) was isolated from intestine of Iksookimia koreensis collected from the Geum River, Geumsan, Chungcheongnam-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MK396586.

Description of Flavobacterium frigidarium FEB2-04

Cells are Gram-stain-negative, aerobic, non-flagellated, and rod-shaped. Colonies grown on MA are circular, convex, smooth, and yellow colored after incubation for 3-4 days at 15°C. Esculin and gelatin are hydrolyzed. Nitrate is not reduced and indole is not produced. Glucose is not fermented. Enzyme activity of β-galactosidase is observed, but not arginine dihydrolase, urease, and oxidase. D-glucose, D-mannose, D-mannitol, and D-maltose are utilized as sole carbon sources, but not L-arabinose, N-acetyl- glucosamine, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. Strain FEB2-04 (=NNIBR2017BA46) was isolated from intestine of Leucopsarion petersii collected from the Jangji Stream, Goseong, Gyeongsangnam-do, Korea. The Gen- Bank accession number of 16S rRNA gene sequence is MG780342.

Description of Corynebacterium stationis FMD-28

Cells are Gram-stain-positive, aerobic, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, smooth, convex, and ivory colored after incubation for 2-3 days at 30°C. Esculin is hydrolyzed weakly, but not gelatin. Nitrate is reduced and glucose is fermented. Indole is not produced. Enzyme activities of oxidase, urease, arginine dihydrolase, and β-galactosidase is not observed. D-glucose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, malic acid, trisodium citrate, and phenylacetic acid are utilized as sole carbon sources, but not L-arabinose, potassium gluconate, capric acid, and adipic acid. Strain FMD- 28 (=NNIBR2018142BA300) was isolated from intestine of Micropterus salmoides collected from the Nakdong River, Sangju, Gyeongsangbuk-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MK396590.

Description of Mycobacteroides saopaulense FMS-15

Cells are Gram-stain-positive, aerobic, non-flagellated, and rod-shaped. Colonies grown on R2A are irregular, dry, brittle, opaque, and white colored after incubation for 2-3 days at 37°C. Esculin is hydrolyzed, but not gelatin. Nitrate is reduced. Indole is not produced and glucose is not fermented. Enzyme activities of arginine dihydrolase, urease, β-galactosidase, and oxidase are not observed. The strain does not utilize D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid as sole carbon sources. Strain FMS-15 (=NNIBR2018142BA318) was isolated from intestine of Micropterus salmoides collected from the Nakdong River, Sangju, Gyengsangbuk-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MK396591.

Description of Mycolicibacterium sarraceniae FMD-25

Cells are Gram-stain-positive, aerobic, non-flagellated, and rod-shaped. Colonies grown on NA are circular, smooth, convex, and yellowish ivory colored after incubation for 2-3 days at 30°C. Esculin and gelatin are not hydrolyzed. Nitrate is reduced. Indole is not produced and glucose in not fermented. Enzyme activity of urease is observed, but not arginine dihydrolase, β-galactosidase, and oxidase. D-glucose, D-mannitol, potassium gluconate, and malic acid are utilized as sole carbon sources, but not L-arabinose, D-mannose, N-acetyl-glucosamine, D-maltose, capric acid, adipic acid, trisodium citrate, and phenylacetic acid. Strain FMD-25 (=NNIBR2018142BA298) was isolated from intestine of Micropterus salmoides collected from the Nakdong River, Sangju, Gyeongsangbuk-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MK396589.

Description of Modestobacter versicolor FMS-37

Cells are Gram-stain-positive, aerobic, flagellated, and rod-shaped. Colonies grown on NA are circular, crateriform, and pinkish ivory colored after incubation for 2-3 days at 30°C. Esculin and gelatin are hydrolyzed. Nitrate is not reduced and indole is not produced. Glucose is not fermented. Enzyme activity of β-galactosidase is observed, but not arginine dihydrolase, urease, and oxidase. D-glucose, D-maltose, potassium gluconate, adipic acid (weakly), malic acid (weakly), and phenylacetic acid are utilized as sole carbon sources, but not L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, capric acid, and trisodium citrate. Strain FMS-37 (=NNIBR2018142BA338) was isolated from intestine of Micropterus salmoides collected from the Nakdong River, Sangju, Gyeongsangbuk-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MK396600.

Description of Sanguibacter inulinus 19FMS-10

Cells are Gram-stain-positive, aerobic, flagellated, and rod-shaped. Colonies grown on R2A are circular, convex, smooth, and lemon colored after incubation for 2-3 days at 25°C. Esculin is hydrolyzed, but not gelatin. Nitrate is not reduced and indole is not produced. Glucose is fermented. Enzyme activities of β-galactosidase and oxidase (weakly) is observed, but not arginine dihydrolase and urease. D-glucose, L-arabinose, D-mannose, N-acetyl-glucosamine, D-maltose, and potassium gluconate are utilized as sole carbon sources, but not D-mannitol, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. Strain 19FMS-10 (=NNIBR2019642BA87) was isolated from intestine of Micropterus salmoides collected from the Yeongok stream, Gangneung, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559813.

Description of Cohnella damuensis FMD-08

Cells are Gram-stain-positive, aerobic, flagellated, and rod-shaped. Colonies grown on NA are circular, flat, opaque, and cream white colored after incubation for 2-3 days at 30°C. Esculin is hydrolyzed weakly, but not gelatin. Nitrate is reduced to nitrogen. Indole is not produced and glucose is not fermented. Enzyme activities of urease and oxidase are observed, but not arginine dihydrolase and β-galactosidase. D-mannitol, N-acetyl-glucosamine, potassium gluconate, capric acid, adipic acid, and malic acid are utilized as sole carbon sources, but not D-glucose, L-arabinose, D-mannose, D-maltose, trisodium citrate, and phenylacetic acid. Strain FMD-08 (=NNIBR2018142BA285) was isolated from intestine of Micropterus salmoides collected from the Nakdong River, Sangju, Gyeongsangbuk-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MK396588.

Description of Mycolicibacterium bacteremicum 19FPP-03

Cells are Gram-stain-positive, aerobic, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, convex, smooth, and yellowish ivory colored after incubation for 2-3 days at 25°C. Esculin is hydrolyzed weakly, but not gelatin. Nitrate is reduced. Indole is not produced and glucose is not fermented. Enzyme activities of urease and β-galactosidase are observed, but not arginine dihydrolase and oxidase. D-mannitol, potassium gluconate, and malic acid are utilized as sole carbon sources, but not D-glucose, L-arabinose, D-mannose, N-acetyl-glucosamine, D-maltose, capric acid, adipic acid, trisodium citrate, and phenylacetic acid. Strain 19FPP-03 (=NNIBR2019642BA105) was isolated from intestine of Pseudorasbora parva collected from the Yeongok stream, Gangneung, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559459.

Description of Fictibacillus aquaticus 19FPP-20

Cells are Gram-stain-positive, aerobic, flagellated, and rod-shaped. Colonies grown on R2A are circular with entire margin, flat, smooth, and cream white colored after incubation for 2-3 days at 25°C. Esculin and gelatin are hydrolyzed. Nitrate is not reduced and indole is not produced. Glucose is not fermented. Enzyme activity of β-galactosidase is observed, but not arginine dihydrolase, urease, and oxidase. D-glucose and D-maltose are utilized as sole carbon sources, but not L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. Strain 19FPP-20 (=NNIBR2019642BA114) was isolated from intestine of Pseudorasbora parva collected from the Yeongok stream, Gangneung, Gangwon-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MN559460.

Description of Aquamicrobium lusatiense P4N-04

Cells are Gram-stain-negative, aerobic, flagellated, and rod-shaped. Colonies grown on NA are irregular with undulate margins, umbonate, and cream colored after incubation for 2-3 days at 30°C. Esculin is hydrolyzed, but not gelatin. Nitrate is not reduced and indole is not produced. Glucose is not fermented. Enzyme activity of β-galactosidase is observed, but not arginine dihydrolase, urease, and oxidase. D-glucose and trisodium citrate are utilized as sole carbon sources, but not L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, and phenylacetic acid. Strain P4N-04 (=NNIBR2018142BA270) was isolated from intestine of Silurus asotus collected from fishery, Jeongeup, Jeollabuk-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MK396598.

Description of Achromobacter pulmonis PI3-03

Cells are Gram-stain-negative, aerobic, flagellated, and rod-shaped. Colonies grown on TSA are circular, smooth, convex, opaque, and cream colored after incubation for 2-3 days at 30°C. Esculin and gelatin are not hydrolyzed. Nitrate is reduced. Indole is not produced and glucose is not fermented. Enzyme activities of urease and oxidase, but not arginine dihydrolase and β-galactosidase. N-acetyl- glucosamine (weakly), potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and pheD-glucose, L-arabinose, D-mannose, D-mannitol and D-maltose. Strain PI3-03 (=NNIBR2018142BA279) was isolated from intestine of Silurus asotus collected from fishery, Jeongeup, Jeollabuk-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MK396599.

Description of Marinomonas pontica FEB1-13

Cells are Gram-stain-negative, aerobic, flagellated, and rod-shaped. Colonies grown on MA are irregular, convex, smooth, and ivory colored after incubation for 2-3 days at 20°C. Esculin is hydrolyzed, but not gelatin. Nitrate is not reduced and indole is not produced. Glucose is fermented. Enzyme activities of urease (weakly) and β-galactosidase are observed, but not arginine dihydrolase and oxidase. D-glucose, D-mannose, D-mannitol, D-maltose, potassium gluconate, malic acid, and trisodium citrate are utilized as sole carbon sources, but not L-arabinose, N-acetyl-glucosamine, capric acid, adipic acid and phenylacetic acid. Strain FEB1-13 (=NNIBR2017BA45) was isolated from intestine of Takifugu niphobles collected from the Jangji Stream, Goseong, Gyeongsangbuk-do, Korea. The GenBank accession number of 16S rRNA gene sequence is MG780341.