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ISSN : 1226-9999(Print)
ISSN : 2287-7851(Online)
Korean J. Environ. Biol. Vol.38 No.2 pp.289-298
DOI : https://doi.org/10.11626/KJEB.2020.38.2.289

Description of 17 unrecorded bacterial species isolated from freshwater showing antibiotic resistance in Korea

Kiwoon Baek, Eui-Jin Kim1, Ji-Hye Han1, Ahyoung Choi2,*
Bioresources Industrialization Support Department, Nakdonggang National Institute of Biological Resources (NNIBR), Sangju 37242, Republic of Korea
1Microbial Research Department, Nakdonggang National Institute of Biological Resources (NNIBR), Sangju 37242, Republic of Korea
2Strategic Planning Department, Nakdonggang National Institute of Biological Resources (NNIBR), Sangju 37242, Republic of Korea
*Corresponding author Ahyoung Choi Tel. 054-530-0712 E-mail. aychoi@nnibr.re.kr
15/05/2020 11/06/2020 12/06/2020

Abstract


As part of the research program “2018 Rapid screening and identification of freshwater microorganisms using MALDI-TOF/MS library” freshwater samples were collected from a branch of the Nakdong River. Almost 300 antibiotic-resistant bacterial strains were isolated from freshwater samples and subsequently identified by 16S rRNA gene sequencing. Seventeen strains among the isolates shared high 16S rRNA gene sequence similarity (>99.0%) with known species that were not previously recorded in Korea, and each of the isolates also formed a robust phylogenetic clade with the closest species. These species were phylogenetically diverse, belonging to four phyla, seven classes, 10 orders, and 13 genera. At the genus and class level, the previously unrecorded species belonged to Rhodovarius, Xanthobacter, and Shinella of the class Alphaproteobacteria; Ottowia, Simplicispira, and Zoogloea of Betaproteobacteria; Pseudomonas, Acinetobacter, and Shewanella of Gammaproteobacteria; Arcobacter of Epsilonproteobacteria; Sphingobacterium of Sphingobacteriia; Trichococcus of Bacilli; and Leucobacter of Actinobacteria. The previously unrecorded species were further characterized by examining their gram-staining, colony and cell morphology, biochemical properties, and phylogenetic position.



초록


    Ministry of Environment
    NNIBR202001106

    INTRODUCTION

    Freshwater streams may have an adverse effect on ecosystems and human water resources, resulting in an increase in antibiotic-resistant bacteria owing to the inflow of wastewater and an increase in residual antibiotics (Kümmerer 2009). Antibiotic-resistant bacteria are bacteria that are not controlled or killed by antibiotics (Engelberg-Kulka et al. 2004). They can survive and reproduce even in the presence of antibiotics. In particular, the livestock field is known to be highly dependent on antibiotics. Antibiotic resistance in animals is increasing owing to the misuse of antibiotics rather than regulated medications, mainly via feed containing antibiotics. Bacteria that are resistant to many antibiotics are known as multi-resistant organisms (MRO) (Woodford et al. 2011). Livestock products with increased resistance pose a risk because antibiotic-resistant bacteria can multiply and spread to people taking antibiotics (Ji et al. 2012). Antibiotic resistance is a serious public health problem; thus, identifying and securing resistant bacteria is highly important.

    As part of the research program “2018 Rapid screening and identification of freshwater microorganisms using MALDI-TOF/MS library”, freshwater samples were collected from a branch of the Nakdong River in 2018. We selected the Gwangpyeong Stream, where wastewater is discharged from the national industrial complex in Gumi-si, Gyeongsangbuk-do, and conducted on-site surveys of possible areas with industrial wastewater contamination. Using phylogenetic analyses based on 16S rRNA gene sequencing, 17 bacterial species were identified that had not been previously recorded in Korea. Here, we report the phylogenetic information and phenotypic characteristics of these bacterial species.

    MATERIALS AND METHODS

    During a screening of bacteria with antibacterial activity, almost 300 bacterial strains were isolated from freshwater in the brand of Nakdong River in 2018, using standard dilution plating method on Reasoner’s 2A (R2A), Nutrient agar (NA) and Muller Hinton agar (HMA) media added to antibiotics and subsequently incubated at 20-25°C for 3-7 days. Antibiotics used a total of seven antibiotics, including synergistic antibiotics such as 1% penicillin-streptomycin solution (Sigma-Aldrich, St. Louis, MO), 5% penicillin- streptomycin-neomycin solution (Sigma-Aldrich), 5% ampicillin-gentamicin solution (Sigma-Aldrich), 5% carbenicillin-gentamicin solution (Sigma-Aldrich), and antibiotics with different mechanisms of action such as 1% tetracycline solution (Sigma-Aldrich), 10% lincomycin solution (Sigma-Aldrich) and 5% novobiocin (Sigma-Aldrich) (Standiford et al. 1970). The concentration of antibiotics was increased by 10% based on Minimum Inhibitory Concentration value for accurate isolation of resistant bacteria. All strains were purified as single colonies after serial dilution spreading and the pure cultures were stored as 20% glycerol suspension at -80°C and as lyophilized ampoules. The designation of strains, culture medium, and incubation conditions are summarized in Table 1.

    Colony morphology of bacterial strains was observed on agar plates with a magnifying glass after the cells were cultivated to their stationary phase. Cellular morphology and cell size were examined by transmission electron microscopy. Gram staining was performed using a Gramstaining kit (bioMérieux, Marcy I’Etoile, France). Biochemical characteristics were tested by using API 20NE galleries (bioMérieux) according to the manufacturer’s instructions.

    Bacterial DNA extraction, PCR amplification, and 16S rRNA gene sequencing were performed using standard procedures. For the phylogenetic analysis, amplification of the 16S rRNA gene from strains were performed using a standard PCR method with a PCR pre-mix and two universal bacteria-specific primers (Bac 8F (5′-AGAGTTTGATCCTGGCTCAG- 3′) and Bac 1492R (5′-GGYTACCTTGTTACGACTT- 3′) (Lane 1991). The 16S rRNA gene sequences were compared with other bacterial strains with validly published names using the EzBioCloud database (Yoon et al. 2017). The cutoff value of 98.7% sequence similarity was applied for identification. Bacterial strains showing 98.7% or higher sequence similarity with known bacterial species never reported in Korea were selected as unreported bacterial species. For phylogenetic analyses, multiple sequence alignments between the 16S rRNA gene sequences of the isolates and those of the reference type strains were carried out using ClustalW and manually checked with EzEditor (Jeon et al. 2014). Based on the sequences aligned, phylogenetic trees were generated by using a neighbor-joining method (Saitou and Nei 1987) with the Kimura 2-parameter model (Kimura 1980) implemented in MEGA 7.0 software (Kumar et al. 2016). The robustness of the inferred phylogenetic trees was evaluated by bootstrap analyses based on 1,000 random re-samplings (Felsenstein 1985).

    RESULTS AND DISCUSSION

    A total of 334 bacterial strains with antibacterial activity were isolated from freshwater during a screening of bacteria. Of these strains, the 17 unrecorded species belonging to four phyla, seven classes, ten orders, and thirteen genera were found in Korea. The taxonomic composition and identification results of these species are summarized in Table 1. At the genus level, these unreported species belonged to the genera Rhodovarius, Xanthobacter, and Shinella (two species) of the Alphaproteobacteria, Ottowia, Simplicispira, and Zoogloea of the class Betaproteobacteria, Pseudomonas (two species), Acinetobacter (three species) and Shewanella of the class Gammaproteobacteria, Arcobacter of the class Epsilonproteobacteria, Sphingobacterium of the class Sphingobacteriia, Trichococcus of the class Bacilli and Leucobacter of the Actinobacteria. Phylogenetic tree of bacterial strains assigned to the classes Alphaproteobac teria and Betaproteobacteria is shown in Fig. 1, to the class Gammaproteobacteria is shown in Fig. 2 and to the four of classes Epsilonproteobacteria, Sphingobacteriia, Bacilli and Actinobacteria is shown in Fig. 3, respectively. As expected from the high 16S rRNA gene sequence similarities of the ten strains with their closest relatives, each strain formed a robust phylogenetic clade with the most closely related species in the trees. The 17 unrecorded antibiotic resistant bacterial species were Gram-stain-negative or positive, rod or short-rod or oval-shaped bacteria (Fig. 4). Other detailed morphological and physiological characteristics are given in the strain descriptions.

    In summary, in this study, 17 bacterial species were identified as unrecorded bacterial species that have not previously been reported from Korean ecosystems. Although 17 unrecorded antibiotic resistant bacterial species had some physiological characteristics different from the previously known species, but their morphology and phylogenetic analysis showed that they belonged to those species. Therefore, here we describe the characteristics of these unreported bacterial species.

    1. Description of Rhodovarius lipocyclicus KPS-102

    Cells are Gram-stain-negative, non-flagellated and short rod-shaped. Colonies grown on R2A are circular, convex and pink colored after incubation for 3 days at 25°C. Positive for urease in API 20NE, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, esculin hydrolysis, gelatinase and β-galactosidase. 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. Strain KPS-102 displays the highest 16S rRNA gene sequence similarity with Rhodovarius lipocyclicus CCUG 44693T (100.0%). Strain KPS- 102 (=NNIBRBA2018141BA29) was isolated from freshwater. The GenBank accession number of 16S rRNA gene sequence is MK402055.

    2. Description of Xanthobacter agilis KPS-34

    Cells are Gram-stain-positive, non-flagellated and rodshaped. Colonies grown on MHA are circular, convex and ivory colored after incubation for 3 days at 25°C. Positive for nitrate reduction and urease in API 20NE, but negative for indole production, glucose fermentation, arginine dihydrolase, esculin hydrolysis, gelatinase and β-galactosidase. Adipic acid and malic acid are utilized. Does not utilize D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, D-maltose, potassium gluconate, capric acid, trisodium citrate and phenylacetic acid. Strain KPS-34 displays the highest 16S rRNA gene sequence similarity with Xanthobacter agilis SA35T (99.0%). Strain KPS-34 (=NNIBRBA2018141BA32) was isolated from freshwater. The GenBank accession number of 16S rRNA gene sequence is MK402058.

    3. Description of Shinella curvata KPS-58

    Cells are Gram-stain-negative, non-flagellated and rodshaped. Colonies grown on NA are circular, convex and white colored after incubation for 2 days at 25°C. Positive for esculin hydrolysis, gelatinase and β-galactosidase in API 20NE, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase and urease. D-Glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose and malic acid are uti-lized. Does not utilize potassium gluconate, capric acid, adipic acid, trisodium citrate and phenylacetic acid. Strain KPS-58 displays the highest 16S rRNA gene sequence similarity with Shinella curvata C3T (99.4%). Strain KPS- 58 (=NNIBRBA2018141BA34) was isolated from freshwater. The GenBank accession number of 16S rRNA gene sequence is MK402060.

    4. Description of Shinella yambaruensis KPS-69

    Cells are Gram-stain-negative, non-flagellated and rodshaped. Colonies grown on NA are circular, convex and white colored after incubation for 3 days at 25°C. Positive for gelatinase and β-galactosidase in API 20NE, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease and esculin hydrolysis. D-Glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, malic acid and phenylacetic acid are utilized. Does not utilize potassium gluconate, capric acid, adipic acid and trisodium citrate. Strain KPS-69 displays the highest 16S rRNA gene sequence similarity with Shinella yambaruensis MS4T (100.0%). Strain KPS-69 (=NNIBRBA2018141BA35) was isolated from freshwater. The GenBank accession number of 16S rRNA gene sequence is MK402061.

    5. Description of Ottowia beijingensis KPS-14

    Cells are Gram-stain-negative, non-flagellated and rodshaped. Colonies grown on MHA are circular, convex and ivory colored after incubation for 3 days at 25°C. Negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, esculin hydrolysis, gelatinase and β-galactosidase in API 20NE. Adipic acid is utilized. Does not utilize D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, malic acid, trisodium citrate and phenylacetic acid. Strain KPS-14 displays the highest 16S rRNA gene sequence similarity with Ottowia beijingensis GCS-AN-3T (99.9%). Strain KPS-14 (=NNIBR BA2018141BA30) was isolated from freshwater. The Gen- Bank accession number of 16S rRNA gene sequence is MK402056.

    6. Description of Simplicispira metamorpha KPS-73

    Cells are Gram-stain-negative, non-flagellated and rodshaped. Colonies grown on NA are circular, convex with entire edge and ivory colored after incubation for 3 days at 25°C. Positive for gelatinase in API 20NE, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, esculin hydrolysis and β-galactosidase. Phenylacetic acid is utilized. Does not utilize D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl- glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid and trisodium citrate. Strain KPS-73 displays the highest 16S rRNA gene sequence similarity with Simplicispira metamorpha DSM 1837T (99.3%). Strain KPS-73 (=NNIBRBA2018141BA36) was isolated from freshwater. The GenBank accession number of 16S rRNA gene sequence is MK402062.

    7. Description of Zoogloea oleivorans KPW-80

    Cells are Gram-stain-negative, non-flagellated and short rod-shaped. Colonies grown on R2A are circular, convex with entire edge and white colored after incubation for 3 days at 25°C. Positive for urease in API 20NE, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, esculin hydrolysis, gelatinase and β-galactosidase. 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. Strain KPW-80 displays the highest 16S rRNA gene sequence similarity with Zoogloea oleivorans CCUG 44693T (100.0 %). Strain KPW-80 (=NNIBRBA2018141BA41) was isolated from freshwater. The GenBank accession number of 16S rRNA gene sequence is MK402067.

    8. Description of Pseudomonas litoralis 4AnSW4-16

    Cells are Gram-stain-positive, non-flagellated and rodshaped. Colonies grown on MHA are circular, convex, smooth and ivory colored after incubation for 2 days at 25°C. Positive for glucose fermentation in API 20NE, but negative for nitrate reduction, indole production, arginine dihydrolase, urease, esculin hydrolysis, gelatinase and β-galactosidase. Adipic acid and malic acid are utilized. Does not utilize D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, trisodium citrate and phenylacetic acid. Strain 4AnSW4-16 displays the highest 16S rRNA gene sequence similarity with Pseudomonas litoralis 2SM5T (99.6%). Strain 4AnSW4-16 (=NNIBRBA2018141 BA25) was isolated from freshwater. The GenBank accession number of 16S rRNA gene sequence is MK402051.

    9. Description of Pseudomonas protegens KPS-50

    Cells are Gram-stain-negative, non-flagellated and short rod-shaped. Colonies grown on NA are circular, convex, smooth and ivory colored after incubation for 3 days at 25°C. Positive for nitrate reduction, glucose fermentation, arginine dihydrolase and gelatinase in API 20NE, but negative for indole production, urease, esculin hydrolysis and β-galactosidase. D-Glucose, D-mannose, D-mannitol, N-acetyl-glucosamine, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid are utilized. Dose not utilize L-arabinose and D-maltose. Strain KPS-50 displays the highest 16S rRNA gene sequence similarity with Pseudomonas protegens CHA0T (100.0%). Strain KPS-50 (=NNIBRBA2018141BA33) was isolated from freshwater. The GenBank accession number of 16S rRNA gene sequence is MK402059.

    10. Description of Acinetobacter celticus KPW-39

    Cells are Gram-stain-positive, non-flagellated and ovalshaped. Colonies grown on MHA are circular, convex and ivory colored after incubation for 3 days at 25°C. Negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, esculin hydrolysis, gelatinase and β-galactosidase in API 20NE. Capric acid and malic acid are utilized. Does not utilize D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, adipic acid, trisodium citrate and phenylacetic acid. Strain KPW-39 displays the highest 16S rRNA gene sequence similarity with Acinetobacter celticus ANC 4603T (99.9%). Strain KPW-39 (=NNIBRBA2018141BA38) was isolated from freshwater. The GenBank accession number of 16S rRNA gene sequence is MK402064.

    11. Description of Acinetobacter bohemicus KPW-4

    Cells are Gram-stain-positive, non-flagellated and ovalshaped. Colonies grown on MHA are circular, convex, slightly and ivory colored after incubation for 3 days at 25°C. Negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, esculin hydrolysis, gelatinase and β-galactosidase in API 20NE. Capric acid and malic acid are utilized. Does not utilize D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl- glucosamine, D-maltose, potassium gluconate, adipic acid, trisodium citrate and phenylacetic acid. Strain KPW- 39 displays the highest 16S rRNA gene sequence similarity with Acinetobacter bohemicus ANC 3994T (99.7%). Strain KPW-4 (=NNIBRBA2018141BA39) was isolated from freshwater. The GenBank accession number of 16S rRNA gene sequence is MK402065.

    12. Description of Acinetobacter harbinensis KPW-8

    Cells are Gram-stain-positive, non-flagellated and ovalshaped. Colonies grown on MHA are circular, convex, slightly and ivory colored after incubation for 3 days at 25°C. Negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, esculin hydrolysis, gelatinase and β-galactosidase in API 20NE. Capric acid is utilized. Does not utilize D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, adipic acid, malic acid, trisodium citrate and phenylacetic acid. Strain KPW-8 displays the highest 16S rRNA gene sequence similarity with Acinetobacter harbinensis HITLi 7T (99.9%). Strain KPW-8 (=NNIBRBA2018141BA40) was isolated from freshwater. The GenBank accession number of 16S rRNA gene sequence is MK402066.

    13. Description of Shewanella glacialipiscicola KPS-21

    Cells are Gram-stain-negative, non-flagellated and rodshaped. Colonies grown on MHA are circular, convex with entire edge and ivory colored after incubation for 2 days at 25°C. Positive for nitrate reduction, esculin hydrolysis and gelatinase in API 20NE, but negative for indole production, glucose fermentation, arginine dihydrolase, urease and β-galactosidase. N-Acetyl-glucosamine, potassium gluconate and malic acid are utilized. Does not utilize D-glucose, L-arabinose, D-mannose, D-mannitol, D-maltose, capric acid, adipic acid, trisodium citrate and phenylacetic acid. Strain KPS-21 displays the highest 16S rRNA gene sequence similarity with Shewanella glacialipiscicola T147T (100.0%). Strain KPS-21 (=NNIBRBA2018141BA31) was isolated from freshwater. The GenBank accession number of 16S rRNA gene sequence is MK402057.

    14. Description of Arcobacter cibarius 4KPW53

    Cells are Gram-stain-negative, non-flagellated and rodshaped. Colonies grown on R2A are circular, convex and pink colored after incubation for 4 days at 25°C. Negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, esculin hydrolysis, gelatinase and β-galactosidase in API 20NE. 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. Strain 4KPW53 displays the highest 16S rRNA gene sequence similarity with Arcobacter cibarius LMG 21996T (99.3%). Strain 4KPW53 (=NNIBRBA2018141 BA28) was isolated from freshwater. The GenBank accession number of 16S rRNA gene sequence is MK402054.

    15. Description of Sphingobacterium kitahiroshimense KPW-32

    Cells are Gram-stain-negative, non-flagellated and rodshaped. Colonies grown on MHA are circular, convex with entire edge and ivory colored after incubation for 3 days at 25°C. Positive for esculin hydrolysis and β-galactosidase in API 20NE, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease and gelatinase. D-Glucose, L-arabinose, D-mannose, N-acetyl-glucosamine and D-maltose are utilized. Does not utilize D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. Strain KPW-32 displays the highest 16S rRNA gene sequence similarity with Sphingobacterium kitahiroshimense 10CT (99.7%). Strain KPW-32 (=NNIBRBA2018141 BA37) was isolated from freshwater. The GenBank accession number of 16S rRNA gene sequence is MK402063.

    16. Description of Trichococcus flocculiformis 4KPW16

    Cells are Gram-stain-positive, non-flagellated and rodshaped. Colonies grown on MHA are circular, convex and white colored after incubation for 3 days at 25°C. Positive for nitrate reduction, esculin hydrolysis and β-galactosidase in API 20NE, but negative for indole production, glucose fermentation, arginine dihydrolase, urease and gelatinase. 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. Strain 4KPW16 displays the highest 16S rRNA gene sequence similarity with Trichococcus flocculiformis DSM 2094T (99.9%). Strain 4KPW16 (=NNIBRBA2018141BA26) was isolated from freshwater. The GenBank accession number of 16S rRNA gene sequence is MK402052.

    17. Description of Leucobacter komagatae 4KPW17

    Cells are Gram-stain-positive, non-flagellated and rodshaped. Colonies grown on MHA are circular, convex, smooth and ivory colored after incubation for 2 days at 25°C. Positive for gelatinase in API 20NE, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, esculin hydrolysis and β-galactosidase. 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. Strain 4KPW17 displays the highest 16S rRNA gene sequence similarity with Leucobacter komagatae IFO 15245T (99.3%). Strain 4KPW17 (=NNIBRBA2018141BA27) was isolated from freshwater. The GenBank accession number of 16S rRNA gene sequence is MK402053.

    ACKNOWLEDGEMENTS

    This study was supported by a grant from the Nakdonggang National Institute of Biological Resources (NNIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NNIBR202001106).

    Figure

    KJEB-38-2-289_F1.gif

    Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationship between the strains isolated in this study and their relatives in the classes Alphaproteobacteria and Betaproteobacteria. Bootstrap values (>50%) are shown at the nodes. Bar, 0.02 substitutions per nucleotide position.

    KJEB-38-2-289_F2.gif

    Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationship between the strains isolated in this study and their relatives in the class Gammaproteobacteria. Bootstrap values (>50%) are shown at the nodes. Bar, 0.02 substitutions per nucleotide position.

    KJEB-38-2-289_F3.gif

    Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationship between the strains isolated in this study and their relatives in the classes Epsilonproteobacteria, Sphingobacteriia, Bacilli, and Actinobacteria. Bootstrap values (>50%) are shown at the nodes. Bar, 0.05 substitutions per nucleotide position.

    KJEB-38-2-289_F4.gif

    Transmission electron micrographs of cells of the strains isolated in this study. Strains: 1, KPS -102; 2, KPS-34; 3, KPS-58; 4, KPS- 69; 5, KPS-14; 6, KPS-73; 7, KPW-80; 8, 4AnSW4-16; 9, KPS-50; 10, KPW-39; 11, KPW-4; 12, KPW-8; 13, KPS-21; 14, 4KPW53; 15, KPW-32; 16, 4KPW16; 17, 4KPW17.

    Table

    Summary of antibiotic -resistant bacterial strains isolated from freshwater and their taxonomic affiliations

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