• Journal of Animal Environmental Science
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• v.21 no.3
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• pp.93-98
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• 2015

The objective of this study was to review application of next-generation sequencing (NGS) to investigate microbiome in the livestock sector. Since the 16S rRNA gene is used as a phylogenetic marker, unculturable members of microbiome in nature or managed environments have been investigated using the NGS technique based on 16S rRNA genes. However, few NGS studies have been conducted to investigate microbiome in the livestock sector. The 16S rRNA gene sequences obtained from NGS are classified to microbial taxa against the 16S rRNA gene reference database such as RDP, Greengenes and Silva databases. The sequences also are clustered into species-level OTUs at 97% sequence similarity. Microbiome similarity among treatment groups is visualized using principal coordinates analysis, while microbiome shared among treatment groups is visualized using a venn diagram. The use of the NGS technique will contribute to elucidating roles of microbiome in the livestock sector.

• Korean Journal of Veterinary Service
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• v.37 no.3
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• pp.157-164
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• 2014

Clostridium chauvoei is the etiologic agent of blackleg, a high mortality rated disease infection mainly cattle. In the present study, the partial sequences of 16S rRNA and flagellin gene of C. chauvoei isolated in Jeonbuk, Korea were determined and compared with those of reference strain. Oligonucleotide primers were designed to amplify a 811 bp fragment of 16S rRNA gene and 1229 bp fragment of flagellin gene. Sequencing analysis of 16S rRNA gene showed high homology to the reference strains ranging 82.3% to 100%, while flagellin gene were different from published foreign clostridia, showing 98.7% to 72.0% nucleotide sequence homology. Phylogenetic analysis based on 16S rRNA gene revealed the close phylogenetic relationship of C. chauvoei and C. septicum in cluster I, which includes C. carnis, C. tertium, C. quinii, C. celatum, C. perfringens, C. absonum, C. botulinum B. Phylogentic analysis also revealed that flagellin gene formed a single cluster with C. chauvoei, C. septicum, C. novyi A, C. novyi B, C. tyrobutylicum, C. acetobutylicum. The genetic informations obtained from this study could be useful for the molecular study of C. chauvoei.

• Korean Journal of Food Science and Technology
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• v.53 no.3
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• pp.334-343
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• 2021

Various vegetables, including cucumbers, have a high probability of foodborne illness because they are usually eaten raw. In this study, we analyzed the 16S rRNA gene sequences of the cucumber (Cucumis sativus L.) microbiota. The diversity indices of cucumber cultivated in May were higher than in cucumber cultivated in November. At the phylum level, Proteobacteria, Firmicutes, and Actinobacteria were predominant. The classes generally comprised Gammaproteobacteria, Bacilli, Alphaproteobacteria, and Actinobacteria. At the genus level, the proportions of Aureimonas, Escherichia, and Microbacterium in samples from May were relatively high, whereas Enterococcus, Pseudomonas, and Rhizobium accounted for a higher proportion in samples from November. Moreover, it is noteworthy that potential pathogenic genera such as Acinetobacter, Aerococcus, Aureimonas, Enterobacter, Enterococcus, Escherichia, Pantoea, Pseudomonas, and Staphylococcus were detected. Although further studies on the characteristics of potential pathogens are required, our results can be used to improve the food safety of vegetables.

• Korean Journal of Veterinary Service
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• v.39 no.1
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• pp.1-6
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• 2016

In this study, Bacillus licheniformis which has been used as probiotics was isolated from Korean traditional fermented soybean. A total of 69 strains were presumptively identified as B. licheniformis by phenotypic methods. Based on PCR amplification and 16S rRNA gene sequencing, the multilocus sequence typing of gyrA and rpoB, followed by phylogenetic analysis was performed. The isolates were distinctly differentiated and found to be closely related to B. amyloliquefaciens, B. subtilis, and B. aerius. The partial 16S rRNA gene sequences of those strains matched those of B. sonorensis (97%) and B. aerius (98%) in the phylogenetic tree. In contrast, multilocus phylogenetic analysis (MLPA) showed that only 61 (86.9%) out of 69 strains were B. licheniformis. The rest of those strains were found to be B. subtilis (5.8%), B. amyloliquefaciens (2.9%), and B. sonorensis (2.9%), respectively. Therefore, our results suggested that since the 16S rRNA gene sequencing alone was not sufficient to compare and discriminate closely related lineages of Bacillus spp., it was required to analyze the MLPA simultaneously to avoid any misleading phenotype-based grouping of these closely related species.

• Microbiology and Biotechnology Letters
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• v.39 no.2
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• pp.97-103
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• 2011

Two Gram positive bacterial strains, designated strain GC-1 and GC-4, were isolated from coastal seawater near Jeju Island in the Republic of Korea. The two strains were identified as members of the genus Bacillus, based on 16S rRNA gene sequencing and data for physiological characteristics analyses. A subtle difference in physiological and genotypical characteristics has led us to designate the strains GC-1 and GC-4. The strain GC-1 showed a 99.91% similarity in 16S rRNA gene sequencing with B. tequiliensis and B. subtilis subsp. inaquosorum and the strain GC-4 showed a 100% similarity in 16S rRNA gene sequencing with those of B. altitudinis, B. stratosphericus, and B. aerophilus. However, both strains exhibited different physiological and genotypical characteristics in many aspects from those of their phylogenetically closest neighbors listed above, which implies that genus Bacillus has diversified into various species during its evolutionary process.

• Journal of Microbiology and Biotechnology
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• v.27 no.7
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• pp.1288-1299
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• 2017

Composting is widely used to transform waste into valuable agricultural organic fertilizer. Anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the global nitrogen cycle, but their role in composting remains poorly understood. In the present study, the community structure, diversity, and abundance of anammox bacteria were analyzed using cloning and sequencing methods by targeting the 16S rRNA gene and the hydrazine oxidase gene (hzo) in samples isolated from compost produced from cow manure and rice straw. A total of 25 operational taxonomic units were classified based on 16S rRNA gene clone libraries, and 14 operational taxonomic units were classified based on hzo gene clone libraries. The phylogenetic tree analysis of the 16S rRNA gene and deduced HZO protein sequences from the corresponding encoding genes indicated that the majority of the obtained clones were related to the known anammox bacteria Candidatus "Brocadia," Candidatus "Kuenenia," and Candidatus "Scalindua." The abundances of anammox bacteria were determined by quantitative PCR, and between $2.13{\times}10^5$ and $1.15{\times}10^6$ 16S rRNA gene copies per gram of compost were found. This study provides the first demonstration of the existence of anammox bacteria with limited diversity in cow manure composting.

• Microbiology and Biotechnology Letters
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• v.47 no.3
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• pp.401-411
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• 2019

All Idiomarina species are isolated from saline environments; microorganisms in such extreme habitats develop metabolic adaptations and can produce compounds such as proteases with an industrial potential. ARDRA and 16S rRNA gene sequencing are established methods for performing phylogenetic analysis and taxonomic identification. However, 16S-23S ITS is more variable than the 16S rRNA gene within a genus, and is therefore, used as a marker to achieve a more precise identification. In this study, ten protease producing Idiomarina strains isolated from the Peruvian salterns were characterized using biochemical and molecular methods to determine their bacterial diversity and industrial potential. In addition, comparison between the length and nucleotide sequences of a 16S-23S ITS region allowed us to assess the inter and intraspecies variability. Based on the 16S rRNA gene, two species of Idiomarina were identified (I. zobellii and I. fontislapidosi). However, biochemical tests revealed that there were differences between the strains of the same species. Moreover, it was found that the ITS contains two tRNA genes, $tRNA^{Ile(GAT)}$ and $tRNA^{Ala(TGC)}$, which are separated by an ISR of a variable size between strains of I. zobellii. In one strain of I. zobellii (PM21), we found nonconserved nucleotides that were previously not reported in the $tRNA^{Ala}$ gene sequences of Idiomarina spp. Thus, based on the biochemical and molecular characteristics, we can conclude that protease producing Idiomarina strains have industrial potential; only two I. zobellii strains (PM48 and PM72) exhibited the same properties. The differences between the other strains could be explained by the presence of subspecies.

• Korean Journal of Microbiology
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• v.55 no.3
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• pp.220-225
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• 2019

Studies based on microbial community analyses have increased in the recent decade since the development of next generation sequencing technology. Associations of gut microbiota with host's health are one of the major outcomes of microbial ecology filed. The major approach for microbial community analysis includes the sequencing of variable regions of 16S rRNA genes, which does not provide the information of bacterial activities. Here, we conducted RNA-based microbial community analysis and compared results obtained from DNA- and its cDNA-based microbial community analyses. Our results indicated that these two approaches differed in the ratio of Firmicutes and Bacteroidetes, known as an obesity indicator, as well as abundance of some key bacteria in gut metabolisms such as butyrate producers and probiotics strains. Therefore, cDNA-based microbial community may provide different insights regarding roles of gut microbiota compared to the previous studies where DNA-based microbial community analyses were performed.

• Journal of Species Research
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• v.9 no.4
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• pp.339-345
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• 2020

The Earth contains billions of microbial species, although the vast majority cannot be cultured in laboratories and are thus considered unidentified and uncharacterized. Extremophiles are microorganisms that thrive in extreme conditions, including temperature, salinity, and pH. Extremophilic microorganisms have provided important insights for biological, metabolic, and evolutionary studies. Between 2017 and 2019, as part of a comprehensive investigation to identify bacterial species in Korea, eight bacterial strains were isolated from marine and non-marine environments in Jeju Island. These strains were cultured under extreme salinity or pH conditions. Phylogenetic analysis using 16S ribosomal RNA(rRNA) gene sequencing indicated that all eight strains belonged to the phyla Gammaproteobacteria, Bacilli, and Alphaproteobacteria. Based on their high 16S rRNA gene sequence similarities(>98.7%) and the formation of strong monophyletic clades with their closest related species, all isolated strains were considered as an unrecorded strain, previously unidentified species. Gram stain reaction, culture conditions, colony and cell morphology, biochemical characteristics, isolation source, and National Institute of Biological Resources(NIBR) IDs are described in this article. The characterization of these unrecorded strains provides information on microorganisms living in Korea.

• Korean Journal of Veterinary Research
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• v.58 no.1
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• pp.51-55
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• 2018

Bovine mastitis (BM) has resulted in enormous economic loss in the dairy industry and coagulase-negative staphylococci (CNS) have caused subclinical BM. Although VITEK 2 GP ID card (VITEK 2) has been used for CNS identification, the probability of identification varies. The rpoB sequence typing (RSTing) method has been used for molecular diagnosis and epidemiology of bacterial infections. In this study, we undertook RSTing of CNS and compared the results with those of VITEK2 and 16S rRNA gene sequencing. As compared VITEK2, the molecular-based methods were more reliable for species identification; moreover, RSTing provided more molecular epidemiological information than that from 16S rRNA gene sequencing.