Publications by authors named "Eun Bae Kim"

67 Publications

Strain diversity of plant-associated Lactiplantibacillus plantarum.

Microb Biotechnol 2021 Sep 25;14(5):1990-2008. Epub 2021 Jun 25.

Department of Food Science and Technology, University of California, Davis, Davis, CA, USA.

Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) is a lactic acid bacteria species found on plants that is essential for many plant food fermentations. In this study, we investigated the intraspecific phenotypic and genetic diversity of 13 L. plantarum strains isolated from different plant foods, including fermented olives and tomatoes, cactus fruit, teff injera, wheat boza and wheat sourdough starter. We found that strains from the same or similar plant food types frequently exhibited similar carbohydrate metabolism and stress tolerance responses. The isolates from acidic, brine-containing ferments (olives and tomatoes) were more resistant to MRS adjusted to pH 3.5 or containing 4% w/v NaCl, than those recovered from grain fermentations. Strains from fermented olives grew robustly on raffinose as the sole carbon source and were better able to grow in the presence of ethanol (8% v/v or sequential exposure of 8% (v/v) and then 12% (v/v) ethanol) than most isolates from other plant types and the reference strain NCIMB8826R. Cell free culture supernatants from the olive-associated strains were also more effective at inhibiting growth of an olive spoilage strain of Saccharomyces cerevisiae. Multi-locus sequence typing and comparative genomics indicated that isolates from the same source tended to be genetically related. However, despite these similarities, other traits were highly variable between strains from the same plant source, including the capacity for biofilm formation and survival at pH 2 or 50°C. Genomic comparisons were unable to resolve strain differences, with the exception of the most phenotypically impaired and robust isolates, highlighting the importance of utilizing phenotypic studies to investigate differences between strains of L. plantarum. The findings show that L. plantarum is adapted for growth on specific plants or plant food types, but that intraspecific variation may be important for ecological fitness and strain coexistence within individual habitats.
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http://dx.doi.org/10.1111/1751-7915.13871DOI Listing
September 2021

Cytoplasmic expression of a model antigen with M Cell-Targeting moiety in lactic acid bacteria and implication of the mechanism as a mucosal vaccine via oral route.

Vaccine 2021 07 12;39(30):4072-4081. Epub 2021 Jun 12.

Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang-gun 25354, Republic of Korea; Institute of Green-Bio Science & Technology, Seoul National University, Pyeongchang-gun 25354, Republic of Korea; Research Institute of Agriculture and Life Science, Seoul National University, Seoul 08826, Republic of Korea. Electronic address:

Lactic acid bacteria (LAB) have been widely studied as mucosal vaccine delivery carriers against many infectious diseases for heterologous expression of protein antigens. There are three antigen expression strategies for LAB: cytoplasmic expression (CE), cell surface display (SD), and extracellular secretion (ES). Despite the generally higher protein expression level and many observations of antigen-specific immunogenicity in CE, its application as a mucosal vaccine has been overlooked relative to SD and ES because of the antigens enclosed by the LAB cell wall. We hypothesized that the antigens in CE could be released from the LAB into the intestinal lumen before host bacterial access to gut-associated lymphoid tissue (GALT), which could contribute to antigen-specific immune responses after oral administration. To elucidate this hypothesis, three recombinant Lactobacillus plantarum (LP) strains were constructed to produce a model antigen, BmpB, with or without an M cell-targeting moiety, and their immunogenicities were analyzed comparatively as oral vaccines in mouse model. The data indicated that the recombinant LPs producing BmpBs with different conformations could induce mucosal immunity differentially. This suggests that the cytoplasmic antigens in LAB could be released into the intestinal lumen, subsequently translocated through M cells, and stimulate the GALT to generate antigen-specific immune responses. Therefore, the CE strategy has great potential, especially in the application of oral LAB vaccines as well as SD and ES strategies. This research provides a better understanding of the mechanism for recombinant oral LAB vaccines and gives insight to the future design of LAB vaccines and oral delivery applications for useful therapeutic proteins.
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http://dx.doi.org/10.1016/j.vaccine.2021.06.010DOI Listing
July 2021

Draft Genome of the Korean smelt Hypomesus nipponensis and its transcriptomic responses to heat stress in the liver and muscle.

G3 (Bethesda) 2021 May 4. Epub 2021 May 4.

Department of Applied Animal Science, College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Kangwon-do, Republic of Korea.

Pond smelt (Hypomesus nipponensis) is a cold-freshwater fish species as a winter economic resource of aquaculture in South Korea. Due to its high susceptibility to abnormal water temperature from global warming, a large number of smelt die in hot summer. Here, we present the first draft genome of H. nipponensis and transcriptomic changes in molecular mechanisms or intracellular responses under heat stress. We combined Illumina and PacBio sequencing technologies to generate the draft genome of H. nipponensis. Based on the reference genome, we conducted transcriptome analysis of liver and muscle tissues under normal (NT, 5 °C) versus warm (HT, 23 °C) conditions, to identify heat stress-induced genes and gene categories. We observed a total of 1,987 contigs, with N50 of 0.46 Mbp with a largest contig (3.03 Mbp) in the assembled genome. A total number of 20,644 protein coding genes were predicted, and 19,224 genes were functionally annotated: 15,955 genes for Gene Ontology (GO) terms; and 11,560 genes for KEGG Orthology (KO). We conducted the lost and gained genes analysis compared with three species that human, zebrafish and salmon. In the lost genes analysis, we detected smelt lost 4,461 (22.16%), 2,825 (10.62%), and 1,499 (3.09%) genes compare with above three species, respectively. In the gained genes analysis, we observed smelt gain 1,133 (5.49%), 1,670 (8.09%), and 229 (1.11%) genes compare with above species, respectively. From transcriptome analysis, a total of 297 and 331 differentially expressed genes (DEGs) with False discovery rate (FDR) < 0.05 were identified in the liver and muscle tissues, respectively. Gene enrichment analysis of DEGs indicates that up-regulated genes were significantly enriched for lipid biosynthetic process (GO : 0008610, P < 0.001) and regulation of apoptotic process (GO : 0042981, P < 0.01), and down-regulated genes by immune responses such as myeloid cell differentiation (GO : 0030099, P < 0.001) in the liver under heat stress. In muscle tissue, up-regulated genes were enriched for hypoxia (GO : 0001666, P < 0.05), transcription regulator activity (GO : 0140110, P < 0.001) and calcium-release channel activity (GO : 0015278, P < 0.01), and down-regulated genes for nicotinamide nucleotide biosynthetic process (GO : 0019359, P < 0.01). The results of KEGG pathway analysis were similar to that of gene enrichment analysis. The draft genome and transcriptomic of H. nipponensis will be used as a useful genetic resource for functional and evolutionary studies. Our findings will improve understanding of the molecular mechanisms and heat responses and will be useful for predicting survival of the smelt and its closely related species under global warming.
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http://dx.doi.org/10.1093/g3journal/jkab147DOI Listing
May 2021

Effects of Lactobacillus salivarius isolated from feces of fast-growing pigs on intestinal microbiota and morphology of suckling piglets.

Sci Rep 2021 03 24;11(1):6757. Epub 2021 Mar 24.

Department of Animal Resource Science, College of Animal Life Science, Kangwon National University, Chuncheon, 24341, Republic of Korea.

The study determined the effects of Lactobacillus salivarius (LS) administered early in the life of suckling piglets on their growth performance, gut morphology, and gut microbiota. Thirty litters of 3-day-old crossbreed piglets were randomly assigned to one of the three treatments, and treatments were commenced on day 3 after birth. During the whole period of the experiment, the piglets were kept with their mothers and left to suckle ad libitum while being supplemented with a milk formula with or without the bacterial probiotic supplemented. The control group (CON) was not treated with probiotics, the HLS group was treated with LS144 (HLS) screened from feces of fast-growing pigs with high body mass index (BMI) while the NLS group was supplemented with LS160 (NLS) screened from feces obtained from pigs of normal BMI. At the weaning time, a higher abundance of Actinobacteria, Lentisphaerae, and Elusimicrobia phyla were observed in NLS piglets, whereas the abundance of Fibrobacteres phylum was significantly reduced in NLS and HLS piglets compared with the CON. A greater abundance of Lactobacillus was detected in the HLS treatment compared with the CON. The abundance of Bacteroides and Fibrobacter was higher in the CON piglets compared with the HLS and NLS piglets. Compared with the CON group, the oral administration of LS significantly increased the number of Lactobacillus and villus height in the duodenum, jejunum, and ileum. Moreover, the villus height of the duodenum was significantly improved in the HLS treatment compared with the NLS treatment. Based on the findings in the neonatal piglet model, we suggest that oral supplementation of LS, particularly LS isolated from high BMI pigs, could be beneficial by improving the intestinal villus height.
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http://dx.doi.org/10.1038/s41598-021-85630-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990948PMC
March 2021

Insights into the Gut and Skin Microbiome of Freshwater Fish, Smelt (Hypomesus nipponensis).

Curr Microbiol 2021 May 18;78(5):1798-1806. Epub 2021 Mar 18.

Department of Applied Animal Science, College of Animal Life Sciences, Kangwon National University, Chuncheon, 24341, Kangwon-do, Republic of Korea.

Freshwater smelt (Hypomesus nipponensis) is a planktivorous fish found in the river of South Korea, Japan, China, and Russia. Because of its specific characteristics living in the cold temperature, this species is economically valuable in the various countries that held winter festival. The body size of the smelt is too small, so people consumed raw smelt during the winter festival sometimes. However, the microbial studies of smelt are nonexistent. Here, we characterized and compared the bacterial communities in the gut and skin of freshwater smelts. We amplified, sequenced, and analyzed the V4 regions of bacterial 16S rRNA genes from freshwater smelts. The microbial diversity in the skin (375 OTUs) was much greater than that in the gut (250 OTUs). At the phylum level, Proteobacteria (gut: 51.5%; skin: 52.9%), Firmicutes (gut: 30.6%; skin: 25.4%), Bacteroidetes (gut: 7.7%; skin: 14.7%), and Actinobacteria (gut: 5.2%; skin: 3.8%) were predominant in both organs. At the genus level, Sphingomonas (gut: 24.9%; skin: 4.4%, P < 0.01) was more abundant in the gut, whereas Acinetobacter (gut: 0.8%; skin: 11.8%, P = 0.02) and Pseudomonas (gut: 0.3%; skin: 2.1%, P = 0.01) were more abundant in the skin. Both beneficial (Lactobacillus) and harmful (Staphylococcus and Streptococcus) bacteria were detected in both organs, even under freshwater conditions. These results revealed that smelts have their own unique microbial communities in the gut and skin. Our findings broaden the understanding of planktivorous freshwater fish microbiomes and provide new insights into fish microbiomes for ensuring food safety.
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http://dx.doi.org/10.1007/s00284-021-02440-wDOI Listing
May 2021

Changes in Gut Microbial Community of Pig Feces in Response to Different Dietary Animal Protein Media.

J Microbiol Biotechnol 2020 Sep;30(9):1321-1334

Department of Applied Animal Science, College of Animal Life Science, Kangwon National University, Chuncheon, Kangwon-do, Republic of Korea.

Beef, pork, chicken and milk are considered representative protein sources in the human diet. Since the digestion of protein is important, the role of intestinal microflora is also important. Despite this, the pure effects of meat and milk intake on the microbiome are yet to be fully elucidated. To evaluate the effect of beef, pork, chicken and milk on intestinal microflora, we observed changes in the microbiome in response to different types of dietary animal proteins in vitro. Feces were collected from five 6-week-old pigs. The suspensions were pooled and inoculated into four different media containing beef, pork, chicken, or skim milk powder in distilled water. Changes in microbial communities were analyzed using 16S rRNA sequencing. The feces alone had the highest microbial alpha diversity. Among the treatment groups, beef showed the highest microbial diversity, followed by pork, chicken, and milk. The three dominant phyla were Proteobacteria, Firmicutes, and Bacteroidetes in all the groups. The most abundant genera in beef, pork, and chicken were , , and , whereas milk was enriched with , , and . Aerobic bacteria decreased while anaerobic and facultative anaerobic bacteria increased in protein-rich nutrients. Functional gene groups were found to be over-represented in protein-rich nutrients. Our results provide baseline information for understanding the roles of dietary animal proteins in reshaping the gut microbiome. Furthermore, growth-promotion by specific species/genus may be used as a cultivation tool for uncultured gut microorganisms.
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http://dx.doi.org/10.4014/jmb.2003.03021DOI Listing
September 2020

Changes in the Microbial Community of the Mottled Skate () During Alkaline Fermentation.

J Microbiol Biotechnol 2020 Aug;30(8):1195-1206

Department of Applied Animal Science, College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Republic of Korea.

, Cham-hong-eo in Korean, is a mottled skate which is belonging to the cartilaginous fish. Although this species is economically valuable in South Korea as an alkalinefermented food, there are few microbial studies on such fermentation. Here, we analyzed microbial changes and pH before, during, and after fermentation and examined the effect of inoculation by a skin microbiota mixture on the skate fermentation (control vs. treatment). To analyze microbial community, the V4 regions of bacterial 16S rRNA genes from the skates were amplified, sequenced and analyzed. During the skate fermentation, pH and total number of marine bacteria increased in both groups, while microbial diversity decreased after fermentation. , which was predominant in the initial skate, declined by fermentation (Day 0: 11.39 ± 5.52%; Day 20: 0.61 ± 0.9%), while the abundance of increased dramatically (Day 0: 1.42 ± 0.41%; Day 20: 64.92 ± 24.15%). From our co-occurrence analysis, the was positively correlated with Aerococcaceae (r = 0.638) and (r = 0.474), which also increased with fermentation, and negatively correlated with (r = -0.847) during fermentation. There are no critically significant differences between control and treatment. These results revealed that the alkaline fermentation of skates dramatically changed the microbiota, but the initial inoculation by a skin microbiota mixture didn't show critical changes in the final microbial community. Our results extended understanding of microbial interactions and provided the new insights of microbial changes during alkaline fermentation.
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http://dx.doi.org/10.4014/jmb.2003.03024DOI Listing
August 2020

Genome-based species-specific primers for rapid identification of six species of Lactobacillus acidophilus group using multiplex PCR.

PLoS One 2020 20;15(3):e0230550. Epub 2020 Mar 20.

Department of Animal Life Science, College of Animal Life Sciences, Kangwon National University, Chuncheon, Republic of Korea.

Many Lactobacillus species are frequently isolated from dairy products, animal guts, and the vaginas of healthy women. However, sequencing-based identification of isolated Lactobacillus strain is time/cost-consuming and lobor-intensive. In this study, we developed a multiplex PCR method to distinguish six closely related species in the Lactobacillus acidophilus group (L. gasseri, L. acidophilus, L. helveticus, L. jensenii, L. crispatus, and L. gallinarum), which is based on species-specific primer sets. Altogether, 86 genomes of 9 Lactobacillus species from the National Center of Biotechnology Information (NCBI) database were compared to detect species-specific genes and design six species-specific primer sets. The PCR conditions of the individual primer sets were optimized via gradient PCR methods. A final multiplex PCR condition was also optimized for a mixture of all six primer sets mixed. When identifying a single strain, the optimized multiplex PCR method can specifically detect one of the six species, but no band was amplified at least from the other Lactobacillus and Enterococcus species. These results indicated that species-specific primer sets designed from the genome comparison could identify one strain within the six Lactobacillus species by a single PCR reaction. Using the method described here, we will be able to save time, cost, and labor during species identification and screening of commercially important probiotic lactobacilli.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0230550PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7083307PMC
June 2020

Differences in microbiome and virome between cattle and horses in the same farm.

Asian-Australas J Anim Sci 2020 Jun 22;33(6):1042-1055. Epub 2019 Oct 22.

Department of Animal Life Science, College of Animal Life Science, Kangwon National University, Chuncheon 24341, Korea.

Objective: The ecosystem of an animal farm is composed of various elements, such as animals, farmers, plants, feed, soil, and microorganisms. A domesticated animal's health is largely connected with the reservoir of bacteria and viruses in animal farms. Although a few studies have focused on exploring the gut microbiome of animals, communities of microbiota and viruses in feedlots have not been thoroughly investigated.

Methods: Here, we collected feces and dust samples (4 groups: cattle feces, C_F; horse feces, H_F; cattle dust, C_D; and horse dust, H_D) from cattle and horse farms sharing the same housing and investigated their microbiome/virome communities by Illumina sequencing.

Results: Dust groups (C_D and H_D) showed higher microbial diversity than feces groups (C_F and H_F) regardless of animal species. From the microbial community analysis, all the samples from the four groups have major phyla such as Proteobacteria (min 37.1% to max 42.8%), Firmicutes (19.1% to 24.9%), Bacteroidetes (10.6% to 22.1%), and Actinobacteria (6.1% to 20.5%). The abundance of Streptococcus, which commonly recognized as equine pathogens, was significantly higher in the horse group (H_D and H_F). Over 99% among the classified virome reads were classified as Caudovirales, a group of tailed bacteriophages, in all four groups. Foot-and-mouth disease virus and equine adenovirus, which cause deadly diseases in cattle and horse, respectively, were not detected.

Conclusion: Our results will provide baseline information to understand different gut and environmental microbial ecology between two livestock species.
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http://dx.doi.org/10.5713/ajas.19.0267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206377PMC
June 2020

Draft Genome Sequence of Lactobacillus plantarum EBKLp545, Isolated from Piglet Feces.

Microbiol Resour Announc 2019 Apr 18;8(16). Epub 2019 Apr 18.

Laboratory of Microbial Genomics and Big Data, College of Animal Life Sciences, Kangwon National University, Chuncheon, Republic of Korea

strain EBKLp545 was isolated from piglet feces in South Korea and sequenced using an Illumina HiSeq system. This draft genome of strain EBKLp545 consists of 3,306,513 bp with 3,049 protein-coding genes in 138 contigs (≥500 bp), 54 noncoding RNA genes, and a 44.3% G+C content.
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http://dx.doi.org/10.1128/MRA.01739-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6473157PMC
April 2019

Draft Genome Sequence of Enterococcus plantarum Strain TRW2, Isolated from Lettuce.

Microbiol Resour Announc 2019 Feb 7;8(6). Epub 2019 Feb 7.

Department of Animal Life Science, Kangwon National University, Chuncheon, Kangwon-do, Republic of Korea.

We report the draft genome sequence for Enterococcus plantarum strain TRW2, isolated from the phyllosphere of romaine lettuce. The draft sequence consists of 3,383,441 bp, with a G+C content of 35.8% and 3,218 protein-coding genes. None of the 22,190 known antibiotic resistance genes were detected.
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http://dx.doi.org/10.1128/MRA.01428-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6368650PMC
February 2019

Effect of Ground Chopi () on Physicochemical Traits and Microbial Community of Chicken Summer Sausage during Manufacture.

Korean J Food Sci Anim Resour 2018 Oct 31;38(5):936-949. Epub 2018 Oct 31.

Department of Applied Animal Science, College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Korea.

Changes in microbial community and physicochemical traits of chicken summer sausage made from spent layer thigh added with different level (0%, 0.1%, 0.3%, and 0.5% w/w) of ground chopi () during manufacture were analyzed. The microbial community was profiled and analyzed by sequencing 16S rRNA gene using Illumina MiSeq. Samples were taken from raw sausage batter, after 15 h of fermentation, 8 h of cooking including cooling down, and 7 d of drying. The final pH of the sausage was reduced by the addition of ground chopi. However, no clear effect on water activity was observed. Ground chopi inhibited the development of red curing color after fermentation as it exhibited antimicrobial effect. However, the effect on species richness and microbial composition after cooking was unclear. Ground chopi delayed lipid oxidation during manufacture and the effect was dependent on the addition level. Fermentation reduced the species richness with a dominancy of lactic acid bacteria. The profile of microbiota in the raw batter was different from other stages, while the closest relationship was observed after cooking and drying. was predominant, followed by and in raw samples. became dominating after fermentation and so forth, whereas other predominant phylum decreased. At genus level, unclassified was the most abundant group found after fermentation and so forth. Therefore, the overall microbial composition aspects were mainly controlled during fermentation by the abundance of lactic acid bacteria, while bacterial counts and lipid oxidation were controlled by cooking and the addition of ground chopi.
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http://dx.doi.org/10.5851/kosfa.2018.e26DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6238041PMC
October 2018

Quality and Lactic Acid Bacteria Diversity of Pork Salami Containing Kimchi Powder.

Korean J Food Sci Anim Resour 2018 Oct 31;38(5):912-926. Epub 2018 Oct 31.

Department of Applied Animal Science, College of Animal Life Science, Kangwon National University, Chuncheon 24341, Korea.

We investigated the quality properties and bacteria diversity of pork salami containing homemade kimchi powder (KP). Pork salamis were manufactured with commercial starter culture (control), and 1% KP (KP1), 3% KP (KP3), and 5% KP (KP5). The salami was fermented and aged for 2 days and 56 days, respectively. The pH and A values of salamis with KP were significantly lower than that of the control (p<0.05). The 2-thiobarbituric acid reactive substance values of all salamis with KP increased but were below 0.2 mg MDA/kg. Salamis with KP, decreased the L* value, but increased the a* and b* values (p<0.05). The count was significantly higher in the salamis with KP than in the control by day 14 (p<0.05). The number of was higher in salamis with KP than in the control and was the highest in salamis in KP1. The KP1 protected lipid oxidation and showed low TBARS value of pork salami compared to the control. This study demonstrates that KP can improve pork salami properties and can serve as a potential natural compounds for fermented meat prodcuts like Metzgerei.
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http://dx.doi.org/10.5851/kosfa.2018.e24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6238042PMC
October 2018

Ruminal methane emissions, metabolic, and microbial profile of Holstein steers fed forage and concentrate, separately or as a total mixed ration.

PLoS One 2018 15;13(8):e0202446. Epub 2018 Aug 15.

Department of International Agricultural Technology, Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang, Gangwon, The Republic of Korea.

Few studies have examined the effects of feeding total mixed ration (TMR) versus roughage and concentrate separately (SF) on ruminant methane production. Therefore, this study compared differences in methane production, ruminal characteristics, total tract digestibility of nutrients, and rumen microbiome between the two feeding methods in Holstein steers. A total six Holstein steers of initial bodyweights 540 ± 34 kg were divided into two groups and assigned to a same experimental diet with two different feeding systems (TMR or SF) in a crossover design with 21 d periods. The experimental diet contained 73% concentrate and 27% forage and were fed twice a day. The total tract digestibility of crude protein, neutral detergent fibre, and organic matter were not affected by the two different feeding systems. Steers fed TMR emitted more methane (138.5 vs. 118.2 L/d; P < 0.05) and lost more gross energy as methane energy (4.0 vs. 3.5% gross energy intake; P = 0.005) compared to those fed SF. Steers fed TMR had greater (P < 0.05) total volatile fatty acid (VFA), ammonia-N concentrations and propionate proportion of total VFA at 1.5 h, whereas lower after that compared to steers fed SF. The greater (P < 0.05) acetate: propionate ratio at 4.5 h for steers fed TMR reflected the shift of H2 sink from propionate towards acetate synthesis. The lower (P < 0.05) isobutyrate and isovalerate proportions of total VFA observed in steers fed TMR implies decrease in net consumption of H2 for microbial protein synthesis compared to SF. There were no differences in both major bacterial and archaeal diversity between TMR and SF, unlike several minor bacterial abundances. The minor groups such as Coprococcus, Succiniclasticum, Butyrivibrio, and Succinivibrio were associated with the changes in ruminal VFA profiles or methanogenesis indirectly. Overall, these results indicate that SF reduces methane emissions from ruminants and increases propionate proportion of total VFA without affecting total tract digestion compared to TMR. There were no evidences that the response differed due to different major underlying microbial population.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0202446PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6093700PMC
February 2019

Pan-Genomics of Revealed Group-Specific Genomic Profiles without Habitat Association.

J Microbiol Biotechnol 2018 Aug;28(8):1352-1359

Laboratory of Microbial Genomics and Big Data, College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Republic of Korea.

is a lactic acid bacterium that promotes animal intestinal health as a probiotic and is found in a wide variety of habitats. Here, we investigated the genomic features of different clusters of strains via pan-genomic analysis. We compared the genomes of 108 strains that were available from the NCBI GenBank database. These genomes were 2.9-3.7 Mbp in size and 44-45% in G+C content. A total of 8,847 orthologs were collected, and 1,709 genes were identified to be shared as core genes by all the strains analyzed. On the basis of SNPs from the core genes, 108 strains were clustered into five major groups (G1-G5) that are different from previous reports and are not clearly associated with habitats. Analysis of group-specific enriched or depleted genes revealed that G1 and G2 were rich in genes for carbohydrate utilization (-arabinose, -rhamnose, and fructooligosaccharides) and that G3, G4, and G5 possessed more genes for the restriction-modification system and MazEF toxin-antitoxin. These results indicate that there are critical differences in gene content and survival strategies among genetically clustered strains, regardless of habitats.
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http://dx.doi.org/10.4014/jmb.1803.03029DOI Listing
August 2018

Regulation of CD4CD8CD25 and CD4CD8CD25 T cells by gut microbiota in chicken.

Sci Rep 2018 06 5;8(1):8627. Epub 2018 Jun 5.

Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Republic of Korea.

The gut microbiota in chicken has long been studied, mostly from the perspective of growth performance. However, there are some immunological studies regarding gut homeostasis in chicken. Although CD4CD25 T cells are reported to act as regulatory T cells (Tregs) in chicken, there have been no studies showing the relationship between gut microbiota and Tregs. Therefore, we established a model for 'antibiotics (ABX)-treated chickens' through administration of an antibiotic cocktail consisting of ampicillin, gentamycin, neomycin, metronidazole, and vancomycin in water for 7 days. CD4CD8CD25 and CD4CD8CD25 T cells in cecal tonsils were significantly decreased in this model. Gram-positive bacteria, especially Clostridia, was responsible for the changes in CD4CD8CD25 or CD4CD8CD25 T cells in cecal tonsils. Feeding ABX-treated chickens with acetate recovered CD4CD8CD25 and CD4CD8CD25 T cells in cecal tonsils. GPR43, a receptor for acetate, was highly expressed in CD4CD8CD25 T cells. In conclusion, our study demonstrated that the gut microbiota can regulate the population of CD4CD8CD25 and CD4CD8CD25 T cells, and that acetate is responsible for the induction of CD4CD8CD25 T cells in cecal tonsils via GPR43.
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http://dx.doi.org/10.1038/s41598-018-26763-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5988814PMC
June 2018

Tracing of the fecal microbiota of commercial pigs at five growth stages from birth to shipment.

Sci Rep 2018 04 16;8(1):6012. Epub 2018 Apr 16.

Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, Republic of Korea.

The intestinal microbiota affect various physiological traits of host animals such as brain development, obesity, age, and the immune system. In the swine industry, understanding the relationship between intestinal microbiota and growth stage is essential because growth stage is directly related to the feeding system of pigs, thus we studied the intestinal microbiota of 32 healthy pigs across five sows at 10, 21, 63, 93, and 147 d of ages. The intestinal microbiota were altered with growth of pigs and were separated into three distinct clusters. The relative abundance of several phyla and genera were significantly different between growth stages. We observed co-occurrence pattern of the intestinal microbiota at each growth stage. In addition, we predicted the functions of the intestinal microbiota and confirmed that several KEGG pathways were significantly different between growth stages. We also explored the relationship between the intestinal microbiota and innate factors such as the maternal effect and gender. When pigs were young, innate factors affected on construction of intestinal microbiota, however this tendency was disappeared with growth. Our findings broaden the understanding of microbial ecology, and the results will be used as a reference for investigating host-microbe interactions in the swine industry.
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http://dx.doi.org/10.1038/s41598-018-24508-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902624PMC
April 2018

Determination of Optimized Growth Medium and Cryoprotective Additives to Enhance the Growth and Survival of .

J Microbiol Biotechnol 2018 May;28(5):718-731

Research Institute of Eco-friendly Livestock Science, Institute of Green-Bio Science and Technology, Seoul National University, Pyeongchang 25354, Republic of Korea.

The beneficial effects of lactic acid bacteria (LAB) have been intensively investigated in recent decades with special focus on modulation of the host intestinal microbiota. Numerous discoveries of effective probiotics are driven by a significantly increasing demand for dietary supplements. Consequently, technological advances in the large-scale production and lyophilization are needed by probiotic-related industries for producing probiotic LAB for commercial use. Our study had a dual objective, to determine the optimum growth medium composition and to investigate appropriate cryoprotective additives (CPAs) for , and compare its responses with other species. The one-factor-at-a-time method and central composite design were applied to determine the optimal medium composition for cultivation. The following composition of the medium was established (per liter): 21.64 g maltose, 85 g yeast extract, 1.21 ml Tween 80, 6 g sodium acetate, 0.2 g MgSO₄∙7H₂O, 0.02 g MnSO₄∙H₂O, 1 g K₂HPO₄, 1.5 g KH₂PO₄, 0.01 g FeSO₄∙7H₂O, and 1 g sodium citrate. A cryoprotective additive combination comprising 10% (w/v) skim milk and 10% (w/v) sucrose supplemented with 2.5% (w/v) sodium glutamate was selected for , and its effectiveness was confirmed using culture-independent methods in the freeze-dried cells of the strains. In conclusion, the optimized medium enhanced the species-specific cultivation of . On the other hand, the cryoprotective effects of the selected CPA mixture may also be dependent on the bacterial strain. This study highlights the necessity for precise and advanced processing techniques for large-scale production of probiotics in the food and feed industries.
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http://dx.doi.org/10.4014/jmb.1801.01059DOI Listing
May 2018

Coelacanth-specific adaptive genes give insights into primitive evolution for water-to-land transition of tetrapods.

Mar Genomics 2018 Apr 21;38:89-95. Epub 2017 Dec 21.

Interdisciplinary Program in Bioinformatics, Seoul National University, Seoul, Republic of Korea; C&K genomics, Seoul National University Research Park, Seoul, Republic of Korea; Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea; Institute for Biomedical Sciences, Shinshu University, Nagano, Japan. Electronic address:

Coelacanth is a group of extant lobe-finned fishes in Sarcopterygii that provides evolutionary information for the missing link between ray-finned fish and tetrapod vertebrates. Its phenotypes, different from actinopterygian fishes, have been considered as primitive terrestrial traits such as cartilages in their fatty fins which are homologous with the humerus and femur. To investigate molecular evolution of coelacanth which led to its divergence into Sarcopterygii, we compared its protein coding sequences with 11 actinopterygian fishes. We identified 47 genes under positive selection specific to coelacanth, when compared to Holostei and Teleostei. Out of these, NCDN and 14 genes were associated with spatial learning and nitrogen metabolism, respectively. In homeobox gene superfamily, we identified coelacanth-specific amino acid substitutions, and also observed that one of replacements in SHOX was shared with extant tetrapods. Such molecular changes may cause primordial morphological change in the common ancestor of sarcopterygians. These results suggest that certain genes such as NCDN, MMS19, TRMT1, ALX1, DLX5 and SHOX might have played a role in the evolutionary transition between aquatic and terrestrial vertebrates.
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http://dx.doi.org/10.1016/j.margen.2017.12.004DOI Listing
April 2018

Selective pressure on the protein-coding genes of the pufferfish is correlated with phenotypic traits.

Mar Genomics 2018 Feb 19;37:182-186. Epub 2017 Dec 19.

Department of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Republic of Korea. Electronic address:

The pufferfish accumulates neurotoxic tetrodotoxin in its body and inflates by filling its stomach with water. These traits are unique to this species, and may be a result of adaptation post-divergence of Tetraodontidae. However, evolution of the protein-coding genes in the pufferfish has not yet been well elucidated. Detection of positive selection on these genes can help us understand the mechanisms associated with functional evolution. We downloaded well-annotated gene information of two pufferfish species, Takifugu rubripes and Tetraodon nigroviridis, from the public ENSEMBL database. In order to detect selective pressure on protein-coding sequences, we performed dN/dS estimation using codeml within the PAML software package. We selected one to one orthologous genes among seven fish species (Gasterosteus aculeatus, Oryzias latipes, Poecilia formosa, Takifugu rubripes, Tetraodon nigroviridis, and Xiphophorus maculatus). Results of dN/dS analysis on orthologous genes indicate that pufferfish showed high non-synonymous substitution rate for positively selected genes, and the evolutionary rate was faster during the diversification of two pufferfishes after divergence. Additionally, a candidate mechanism for regulation of neuro-toxicity of tetrodotoxin was identified from functional annotation of positively selected genes. These results support positive selection on protein-coding genes of the pufferfish with the acquisition of specific phenotypic traits.
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http://dx.doi.org/10.1016/j.margen.2017.11.015DOI Listing
February 2018

Tracing the spatio-temporal dynamics of endangered fin whales (Balaenoptera physalus) within baleen whale (Mysticeti) lineages: a mitogenomic perspective.

Genetica 2017 Dec 4;145(6):603-612. Epub 2017 Nov 4.

Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, 151-742, Republic of Korea.

To explore the spatio-temporal dynamics of endangered fin whales (Balaenoptera physalus) within the baleen whale (Mysticeti) lineages, we analyzed 148 published mitochondrial genome sequences of baleen whales. We used a Bayesian coalescent approach as well as Bayesian inferences and maximum likelihood methods. The results showed that the fin whales had a single maternal origin, and that there is a significant correlation between geographic location and evolution of global fin whales. The most recent common female ancestor of this species lived approximately 9.88 million years ago (Mya). Here, North Pacific fin whales first appeared about 7.48 Mya, followed by a subsequent divergence in Southern Hemisphere approximately 6.63 Mya and North Atlantic about 4.42 Mya. Relatively recently, approximately 1.76 and 1.42 Mya, there were two additional occurrences of North Pacific populations; one originated from the Southern Hemisphere and the other from an uncertain location. The evolutionary rate of this species was 1.002 × 10 substitutions/site/My. Our Bayesian skyline plot illustrates that the fin whale population has the rapid expansion event since ~ 2.5 Mya, during the Quaternary glaciation stage. Additionally, this study indicates that the fin whale has a sister group relationship with humpback whale (Meganoptera novaeangliae) within the baleen whale lineages. Of the 16 genomic regions, NADH5 showed the most powerful signal for baleen whale phylogenetics. Interestingly, fin whales have 16 species-specific amino acid residues in eight mitochondrial genes: NADH2, COX2, COX3, ATPase6, ATPase8, NADH4, NADH5, and Cytb.
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http://dx.doi.org/10.1007/s10709-017-9988-4DOI Listing
December 2017

Prohibition of antibiotic growth promoters has affected the genomic profiles of Lactobacillus salivarius inhabiting the swine intestine.

PLoS One 2017 23;12(10):e0186671. Epub 2017 Oct 23.

Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea.

After the introduction of a ban on the use of antibiotic growth promoters (AGPs) for livestock, the feeding environment, including the composition of animal intestinal microbiota, has changed rapidly. We hypothesized that the microbial genomes have also been affected by this legal prohibition, and investigated an important member of the swine gut microbiota, Lactobacillus salivarius, with a pan-genomic approach. Here, we isolated 21 L. salivarius strains composed of 6 strains isolated before the AGP prohibition (SBPs) and 15 strains isolated after the AGP prohibition (SAPs) at an interval of a decade, and the draft genomes were generated de novo. Several genomic differences between SBPs and SAPs were identified, although the number and function of antibiotic resistance genes were not different. SBPs showed larger genome size and a higher number of orthologs, as well as lower genetic diversity, than SAPs. SBPs had genes associated with the utilization of L-rhamnose and D-tagatose for energy production. Because these sugars are also used in exopolysaccharide (EPS) synthesis, we tried to identify differences in biofilm formation-associated genes. The genes for the production of EPSs and extracellular proteins were different in terms of amino acid sequences. Indeed, SAPs formed dense biofilm and survived better than SBPs in the swine intestinal environment. These results suggest that SAPs have evolved and adapted to protect themselves from new selection pressure of the swine intestinal microenvironment by forming dense biofilms, adopting a distinct antibiotic resistance strategy. This finding is particularly important to understand the evolutionary changes in host-microbe interaction and provide detailed insight for the development of effective probiotics for livestock.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186671PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5653324PMC
November 2017

Comparative genomics of Lactobacillus salivarius strains focusing on their host adaptation.

Microbiol Res 2017 Dec 24;205:48-58. Epub 2017 Aug 24.

Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea; Research Institute for Agriculture and Life Science, Seoul National University, Seoul, Republic of Korea. Electronic address:

Lactobacillus salivarius is an important member of the animal gut microflora and is a promising probiotic bacterium. However, there is a lack of research on the genomic diversity of L. salivarius species. In this study, we generated 21 L. salivarius draft genomes, and investigated the pan-genome of L. salivarius strains isolated from humans, pigs and chickens using all available genomes, focusing on host adaptation. Phylogenetic clustering showed a distinct categorization of L. salivarius strains depending on their hosts. In the pan-genome, 15 host-specific genes and 16 dual-host-shared genes that only one host isolate did not possess were identified. Comparison of 56 extracellular protein encoding genes and 124 orthologs related to exopolysaccharide production in the pan-genome revealed that extracellular components of the assayed bacteria have been globally acquired and mutated under the selection pressure for host adaptation. We also found the three host-specific genes that are responsible for energy production in L. salivarius. These results showed that L. salivarius has evolved to adapt to host habitats in two ways, by gaining the abilities for niche adhesion and efficient utilization of nutrients. Our study offers a deeper understanding of the probiotic species L. salivarius, and provides a basis for future studies on L. salivarius and other mutualistic bacteria.
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http://dx.doi.org/10.1016/j.micres.2017.08.008DOI Listing
December 2017

Evaluating the association between body weight and the intestinal microbiota of weaned piglets via 16S rRNA sequencing.

Appl Microbiol Biotechnol 2017 Jul 18;101(14):5903-5911. Epub 2017 May 18.

Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea.

Due to the ban on the use of antimicrobial growth promoters in livestock feeds, understanding the relationship between intestinal microbiota and the physiology of the host has become very important for improving livestock performance. In this study, we investigated the relationship between intestinal microbiota and body weights of weaned piglets. Lighter (n = 9) and heavier (n = 9) 9-week-old weaned piglets were selected from approximately one hundred individuals based on their body weights. Their fecal microbial communities were analyzed by sequencing the V4 region of the 16S rRNA gene. The microbial richness estimators of the heavier piglets were significantly higher than those of the lighter piglets. At the phylum level, the microbiota of the heavier group had significantly higher levels of Firmicutes and a higher Firmicutes-to-Bacteroidetes ratio than that of the lighter group. At the genus level, the levels of several genera, such as Anaerococcus and Lactococcus, were significantly different in the two groups. In particular, the lighter group had significantly higher levels of opportunistic pathogenic bacteria, such as Anaerotruncus and Bacteroides, compared with those of the heavier group. Moreover, the levels of bacteria expressing the components of several metabolic pathways were significantly different in the two groups. The microbiota of the heavier group had a significantly higher involvement in three KEGG pathways concerned with xenobiotic degradation than that of the lighter group. These results may provide insights into host-microbe interactions occurring in the piglet intestine and will be useful in establishing a strategy for improving growth performance in the swine industry.
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http://dx.doi.org/10.1007/s00253-017-8304-7DOI Listing
July 2017

Improved antimicrobial activity of Pediococcus acidilactici against Salmonella Gallinarum by UV mutagenesis and genome shuffling.

Appl Microbiol Biotechnol 2017 Jul 3;101(13):5353-5363. Epub 2017 May 3.

Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea.

Pediococcus acidilactici is a widely used probiotic, and Salmonella enterica serovar Gallinarum (SG) is a significant pathogen in the poultry industry. In this study, we improved the antimicrobial activity of P. acidilactici against SG using UV mutation and genome shuffling (GS). To improve antimicrobial activity against SG, UV mutagenesis was performed against wild-type P. acidilactici (WT), and five mutants showed improved antimicrobial activity. To further improve antimicrobial activity, GS was performed on five UV mutants. Following GS, four mutants showed improved antimicrobial activity compared with the UV mutants and WT. The antimicrobial activity of GS1 was highest among the mutants; however, the activity was reduced when the culture supernatant was treated with proteinase K, suggesting that the improved antimicrobial activity is due to a proteinous substance such as bacteriocin. To validate the activity of GS1 in vivo, we designed multi-species probiotics and performed broiler feeding experiments. Groups consisted of no treatment (NC), avilamycin-treated (PC), probiotic group 1 containing WT (T1), and probiotic group 2 containing GS1 (T2). In broiler feeding experiments, coliform bacteria were significantly reduced in T2 compared with NC, PC, and T1. The cecal microbiota was modulated and pathogenic bacteria were reduced by GS1 oral administration. In this study, GS1 showed improved antimicrobial activity against SG in vitro and reduced pathogenic bacteria in a broiler feeding experiment. These results suggest that GS1 can serve as an efficient probiotic, as an alternative to antibiotics in the poultry industry.
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http://dx.doi.org/10.1007/s00253-017-8293-6DOI Listing
July 2017

Pan-Genomic Approaches in Lactobacillus reuteri as a Porcine Probiotic: Investigation of Host Adaptation and Antipathogenic Activity.

Microb Ecol 2017 Oct 24;74(3):709-721. Epub 2017 Apr 24.

Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea.

After the introduction of a ban on the use of antibiotic growth promoters (AGPs) for livestock, reuterin-producing Lactobacillus reuteri is getting attention as an alternative to AGPs. In this study, we investigated genetic features of L. reuteri associated with host specificity and antipathogenic effect. We isolated 104 L. reuteri strains from porcine feces, and 16 strains, composed of eight strains exhibiting the higher antipathogenic effect (group HS) and eight strains exhibiting the lower effect (group LS), were selected for genomic comparison. We generated draft genomes of the 16 isolates and investigated their pan-genome together with the 26 National Center for Biotechnology Information-registered genomes. L. reuteri genomes organized six clades with multi-locus sequence analysis, and the clade IV includes the 16 isolates. First, we identified six L. reuteri clade IV-specific genes including three hypothetical protein-coding genes. The three annotated genes encode transposases and cell surface proteins, indicating that these genes are the result of adaptation to the host gastrointestinal epithelia and that these host-specific traits were acquired by horizontal gene transfer. We also identified differences between groups HS and LS in the pdu-cbi-cob-hem gene cluster, which is essential for reuterin and cobalamin synthesis, and six genes specific to group HS are revealed. While the strains of group HS possessed all genes of this cluster, LS strains have lost many genes of the cluster. This study provides a deeper understanding of the relationship between probiotic properties and genomic features of L. reuteri.
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http://dx.doi.org/10.1007/s00248-017-0977-zDOI Listing
October 2017

Age-associated molecular changes are deleterious and may modulate life span through diet.

Sci Adv 2017 Feb 17;3(2):e1601833. Epub 2017 Feb 17.

Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

Transition through life span is accompanied by numerous molecular changes, such as dysregulated gene expression, altered metabolite levels, and accumulated molecular damage. These changes are thought to be causal factors in aging; however, because they are numerous and are also influenced by genotype, environment, and other factors in addition to age, it is difficult to characterize the cumulative effect of these molecular changes on longevity. We reasoned that age-associated changes, such as molecular damage and tissue composition, may influence life span when used in the diet of organisms that are closely related to those that serve as a dietary source. To test this possibility, we used species-specific culture media and diets that incorporated molecular extracts of young and old organisms and compared the influence of these diets on the life span of yeast, fruitflies, and mice. In each case, the "old" diet or medium shortened the life span for one or both sexes. These findings suggest that age-associated molecular changes, such as cumulative damage and altered dietary composition, are deleterious and causally linked with aging and may affect life span through diet.
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http://dx.doi.org/10.1126/sciadv.1601833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5315447PMC
February 2017

Development of a Rapid Identification Method for the Differentiation of Enterococcus Species Using a Species-Specific Multiplex PCR Based on Comparative Genomics.

Curr Microbiol 2017 Apr 22;74(4):476-483. Epub 2017 Feb 22.

Department of Animal Life Science, College of Animal Life Sciences, Kangwon National University, Chuncheon, Kangwon-do, Republic of Korea.

Enterococci are lactic acid bacteria that are commonly found in food and in animal gut. Since 16 S ribosomal RNA (rRNA) sequences, genetic markers for bacterial identification, are similar among several Enterococcus species, it is very difficult to determine the correct species based on only 16 S rRNA sequences. Therefore, we developed a rapid method for the identification of different Enterococcus species using comparative genomics. We compared 38 genomes of 13 Enterococcus species retrieved from the National Center of Biotechnology Information database and identified 25,623 orthologs. Among the orthologs, four genes were specific to four Enterococcus species (Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, and Enterococcus durans). We designed species-specific primer sets targeting the genes and developed a multiplex PCR using primer sets that could distinguish the four Enterococcus species among the nine strains of Enterococcus species that were available locally. The multiplex PCR method also distinguished the four species isolated from various environments, such as feces of chicken and cow, meat of chicken, cow, and pigs, and fermented soybeans (Cheonggukjang and Doenjang). These results indicated that our novel multiplex PCR using species-specific primers could identify the four Enterococcus species in a rapid and easy way. This method will be useful to distinguish Enterococcus species in food, feed, and clinical settings.
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http://dx.doi.org/10.1007/s00284-017-1210-5DOI Listing
April 2017

WITHDRAWN: Evaluating the association between body weight and the intestinal microbiota of weaned piglets via 16S rRNA sequencing.

Vet Microbiol 2016 11 15;196:55-62. Epub 2016 Oct 15.

Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea; Research Institute for Agriculture and Life Science, Seoul National University, Seoul, Republic of Korea. Electronic address:

This article has been removed: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). The publisher regrets that an error occurred due to a technical issue which led to the premature publication of this paper. This error bears no reflection on the article or its authors. The publisher apologizes to the authors and the readers for this unfortunate error.
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http://dx.doi.org/10.1016/j.vetmic.2016.10.020DOI Listing
November 2016
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