• 제목/요약/키워드: 16S rRNA gene-based sequencing

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Polyphasic Analysis of the Bacterial Community in the Rhizosphere and Roots of Cyperus rotundus L. Grown in a Petroleum-Contaminated Soil

  • Jurelevicius, Diogo;Korenblum, Elisa;Casella, Renata;Vital, Ronalt Leite;Seldin, Lucy
    • Journal of Microbiology and Biotechnology
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    • 제20권5호
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    • pp.862-870
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    • 2010
  • Cyperus rotundus L. is a perennial herb that was found to be dominating an area in northeast Brazil previously contaminated with petroleum. In order to increase our knowledge of microorganism-plant interactions in phytoremediation, the bacterial community present in the rhizosphere and roots of C. rotundus was evaluated by culture-dependent and molecular approaches. PCR-DGGE analysis based on the 16S rRNA gene showed that the bacterial community in bulk soil, rhizosphere, and root samples had a high degree of similarity. A complex population of alkane-utilizing bacteria and a variable nitrogen-fixing population were observed via PCR-DGGE analysis of alkB and nifH genes, respectively. In addition, two clone libraries were generated from alkB fragments obtained by PCR of bulk and rhizosphere soil DNA samples. Statistical analyses of these libraries showed that the compositions of their respective populations were different in terms of alkB gene sequences. Using culturedependent techniques, 209 bacterial strains were isolated from the rhizosphere and rhizoplane/roots of C. rotundus. Dot-blot analysis showed that 17 strains contained both alkB and nifH gene sequences. Partial 16S rRNA gene sequencing revealed that these strains are affiliated with the genera Bosea, Cupriavidus, Enterobacter, Gordonia, Mycoplana, Pandoraea, Pseudomonas, Rhizobium, and Rhodococcus. These isolates can be considered to have great potential for the phytoremediation of soil with C. rotundus in this tropical soil area.

국내 액상 발효유용 유산균 스타터 미생물의 동정 및 생리적 특성 (Identification and Characterization of Lactic Acid Bacteria Starters Isolated from the Commercial Drink-Yogurt Products)

  • 전상록;송태석;김지연;신원철;허송욱;윤성식
    • 한국축산식품학회지
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    • 제27권4호
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    • pp.509-516
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    • 2007
  • Starters of lactic acid bacteria(LAB) were isolated from the commercial yoghurt products and the four isolates have been studied on their identification and some physiological characteristics. For the purpose of identification, microscopic examination, API test, and 16s rRNA gene sequencing were conducted. Isolate A from a yogurt product of local dairy company A was shown to be Gram-positive rod-shaped bacterium. All strains isolated were turned out to be as Lactobacillus paracasei by using a API 50 CHL kit. In contrast, isolate A was identified as a strain of Lactobacillus helveticus based on the 16S rRNA sequencing data, and L. casei ssp. casei for both B and D and L. paracasei for C. All the isolates survived the simulated gastric juice, pH 2.0 within 3 hours and sharply decreased in viability so that no viable cell was observed after 4.5 hours incubation. In addition, the four isolated strains were almost identical in antibiotic susceptibility to six different kinds of antibiotics including erythromycin ($15\;{\mu}g$), ampicillin ($10\;{\mu}g$), gentamycin ($10\;{\mu}g$), neomycin ($30\;{\mu}g$), but rather resistant to colistin ($10\;{\mu}g$) and streptomycin ($10\;{\mu}g$). It was noteworthy that four isolates were confirmed to produce antibacterial substance against foodborne pathogens of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli 0157:H7 as test organisms based on the inhibitory zones on an MRS soft agar medium. At presence, the inhibitory factor is unknown so that further studies are required to ascertain the active factor responsible for the inhibitory activities.

Microbial Community Diversity in Anaerobic Reactors Digesting Turkey, Chicken, and Swine Wastes

  • Ziganshina, Elvira E.;Belostotskiy, Dmitry E.;Shushlyaev, Roman V.;Miluykov, Vasili A.;Vankov, Petr Y.;Ziganshin, Ayrat M.
    • Journal of Microbiology and Biotechnology
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    • 제24권11호
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    • pp.1464-1472
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    • 2014
  • The microbial community structures of two continuous stirred tank reactors digesting turkey manure with pine wood shavings as well as chicken and swine manure were investigated. The reactor fed with chicken/swine wastes displayed the highest organic acids concentration (up to 15.2 g/l) and ammonia concentration (up to 3.7 g/l ammonium nitrogen) and generated a higher biogas yield (up to $366ml/g_{VS}$) compared with the reactor supplied with turkey wastes (1.5-1.8 g/l of organic acids and 1.6-1.7 g/l of ammonium levels; biogas yield was up to $195ml/g_{VS}$). The microbial community diversity was assessed using both sequencing and profiling terminal restriction fragment length polymorphisms of 16S rRNA genes. Additionally, methanogens were analyzed using methyl coenzyme M reductase alpha subunit (mcrA) genes. The bacterial community was dominated by members of unclassified Clostridiales with the prevalence of specific clostridial phylotypes in each reactor, indicating the effect of the substrate type on the community structure. Of the methanogenic archaea, methanogens of the genus Methanosarcina were found in high proportions in both reactors with specific methanosarcinas in each reactor, whereas the strict hydrogenotrophic methanogens of Methanoculleus sp. were found at significant levels only in the reactor fed with chicken/swine manure (based on the analyses of 16S rRNA gene). This suggests that among methanogenic archaea, Methanosarcina species which have different metabolic capabilities, including aceticlastic and hydrogenotrophic methanogenesis, were mainly involved in anaerobic digestion of turkey wastes.

유아의 분변으로부터 항리스테리아 활성의 Bifidobacterium 속 균주의 분리 및 동정 (Isolation and Identification of the Antilisterial Bifidobacterium Isolates from the Infants Fecal Samples)

  • 김송이;김기환;윤순용;윤성식
    • Journal of Dairy Science and Biotechnology
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    • 제24권1호
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    • pp.19-28
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    • 2006
  • 유아 분변으로부터 분리한 52 균주는 그람 양성균인 Listeria monocytogenes KCCM 40307$^T$에 대하여 항균활성이 있었고 그 중에서도 선별 균주 A24의 항균활성이 45% 이상으로 가장 높았다. Bifidobacterium longum A24의 생육 및 항균 물질 생산을 검토하였을 때 균체의 생육은 28시간 배양 시 최고에 도달하였고 항균 활성은 36시 간 배양 시 최고를 나타내었다. 선별균주 A24는 16S rRNA-based molecular typing 결과Bifidobacterium 속 균주임을 확인할 수 있었고 형태학적 ${\cdot}$ 생화학적인 방법으로 검토하여 보았을 때 Bifidobacterium longum으로 판단되었으며, 16s rDNA sequencing 결과 최종적으로 Bifidobacterium longum로 동정 되었으며 이것을 Bifidobacterium longum A24로 명명하였다. Bifidobacterium longum A24의 항균 활성 물질은 균체의 생육이 가장 좋은 28시간 배양에서가 아니라 그보다 늦은 36시간 배양에서 최고를 나타내었고 그 이후로는 활성이 감소하는 경향을 보였다. 이것은 Bifidobacterium longum A24이 생성하는 항균 활성 물질이 bacteriocin과 같은 2차 대사 산물임을 암시하는 결과로 해석된다.

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Analysis of Microbiota in Bellflower Root, Platycodon grandiflorum, Obtained from South Korea

  • Kim, Daeho;Hong, Sanghyun;Na, Hongjun;Chun, Jihwan;Guevarra, Robin B.;Kim, You-Tae;Ryu, Sangryeol;Kim, Hyeun Bum;Lee, Ju-Hoon
    • Journal of Microbiology and Biotechnology
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    • 제28권4호
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    • pp.551-560
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    • 2018
  • Bellflower root (Platycodon grandiflorum), which belongs to the Campanulaceae family, is a perennial grass that grows naturally in Korea, northeastern China, and Japan. Bellflower is widely consumed as both food and medicine owing to its high nutritional value and potential therapeutic effects. Since foodborne disease outbreaks often come from vegetables, understanding the public health risk of microorganisms on fresh vegetables is pivotal to predict and prevent foodborne disease outbreaks. We investigated the microbial communities on the bellflower root (n = 10). 16S rRNA gene amplicon sequencing targeting the V6-V9 regions of 16S rRNA genes was conducted via the 454-Titanium platform. The sequence quality was checked and phylogenetic assessments were performed using the RDP classifier implemented in QIIME with a bootstrap cutoff of 80%. Principal coordinate analysis was performed using the weighted Fast UniFrac distance. The average number of sequence reads generated per sample was 67,192 sequences. At the phylum level, bacterial communities from the bellflower root were composed primarily of Proteobacteria, Firmicutes, and Actinobacteria in March and September samples. Genera Serratia, Pseudomonas, and Pantoea comprised more than 54% of the total bellflower root bacteria. Principal coordinate analysis plots demonstrated that the microbial community of bellflower root in March samples was different from those in September samples. Potential pathogenic genera, such as Pantoea, were detected in bellflower root samples. Even though further studies will be required to determine if these species are associated with foodborne illness, our results indicate that the 16S rRNA gene-based sequencing approach can be used to detect pathogenic bacteria on fresh vegetables.

Microbiological Characteristics of Gouda Cheese Manufactured with Pasteurized and Raw Milk during Ripening Using Next Generation Sequencing

  • Park, Wonseo;Yoo, Jayeon;Oh, Sangnam;Ham, Jun-sang;Jeong, Seok-geun;Kim, Younghoon
    • 한국축산식품학회지
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    • 제39권4호
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    • pp.585-600
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    • 2019
  • Gouda cheese, one of most popular cheeses in the Korea, has been produced from only pasteurized milk in Korean dairy farms. Recently, it has become legally possible to produce ripened cheese manufactured with raw milk in Korea. In the present study, we investigated the physico-chemical and microbiological characteristics of Gouda cheese manufactured with raw (R-GC) or pasteurized milk (P-GC) during manufacturing and ripening. Particularly, this study characterized the bacterial community structure of two cheese types, which are produced without pasteurization during ripening based on next generation sequencing of 16S rRNA gene amplicons. During ripening, protein and fat content increased slightly, whereas moisture content decreased in both P-GC and R-GC. At the 6 wk of ripening, R-GC became softer and smoother and hence, the values of hardness and gumminess, chewiness in R-GC was lower than that of P-GC. Metagenomic analysis revealed that the bacterial genera used a starter cultures, namely Lactococcus and Leuconostoc were predominant in both P-GC and R-GC. Moreover, in R-GC, the proportion of coliform bacteria such as Escherichia, Leclercia, Raoultella, and Pseudomonas were detected initially but not during ripening. Taken together, our finding indicates the potential of manufacturing with Gouda cheese from raw milk and the benefits of next generation sequencing for microbial community composition during cheese ripening.

Molecular Identification of Vaginal Lactobacillus spp. Isolated from Korean Women

  • CHANG, CHUNG EUN;SYLVIA I. PAVLOVA;LIN TAO;EUN-KI KIM;SEUNG CHUL KIM;HYUN SHIK YUN;JAE-SEONG SO
    • Journal of Microbiology and Biotechnology
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    • 제12권2호
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    • pp.312-317
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    • 2002
  • Indigenous lactobacilli were isolated from vaginas of Korean women for possible use in ecological treatment of bacterial vaginosis. Vaginal swab samples were obtained from a gynecological clinic and streaked on Rogosa SL agar plates to select the most predominant lactobacilli in each sample. The preliminary identification of the isolates as lactobacilli was based on microscopic observation of Gram-positive rod-shaped cell morphology. The initial characterization was performed on 108 isolates in terms of their cell surface hydrophobicity (CSH), antimicrobial activity, and hydrogen peroxide (H₂O₂) production capability, and 10 isolates were then selected for further molecular identification. For a rapid procedure to identify lactobacilli, polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses of the l6S rRNA genes were applied. The 10 selected lactobacilli and 9 different reference strains of Lactobacillus spp. were characterized by PCR-RFLP where the amplified l6S rDNA was digested with 7 different restriction endonucleases prior to analysis. DNA sequencing of the 16S rRNA gene of one particular isolate, KLB 46, that had been identified as L. crispatus by the PCR-RFLP analysis, further confirmed its identity as L. crispatus.

Biochemical and Molecular Characterization of High Population Density Bacteria Isolated from Sunflower

  • Goes, Kelly Campos Guerra Pinheiro De;Fisher, Maria Luisa De Castro;Cattelan, Alexandre Jose;Nogueira, Marco Antonio;Carvalho, Claudio Guilherme Portela De;Oliveira, Andre Luiz Martinez De
    • Journal of Microbiology and Biotechnology
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    • 제22권4호
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    • pp.437-447
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    • 2012
  • Natural and beneficial associations between plants and bacteria have demonstrated potential commercial application for several agricultural crops. The sunflower has acquired increasing importance in Brazilian agribusiness owing to its agronomic characteristics such as the tolerance to edaphoclimatic variations, resistance to pests and diseases, and adaptation to the implements commonly used for maize and soybean, as well as the versatility of the products and by-products obtained from its cultivation. A study of the cultivable bacteria associated with two sunflower cultivars, using classical microbiological methods, successfully obtained isolates from different plant tissues (roots, stems, florets, and rhizosphere). Out of 57 plant-growth-promoting isolates obtained, 45 were identified at the genus level and phylogenetically positioned based on 16S rRNA gene sequencing: 42 Bacillus (B. subtilis, B. cereus, B. thuringiensis, B. pumilus, B. megaterium, and Bacillus sp.) and 3 Methylobacterium komagatae. Random amplified polymorphic DNA (RAPD) analysis showed a broad diversity among the Bacillus isolates, which clustered into 2 groups with 75% similarity and 13 subgroups with 85% similarity, suggesting that the genetic distance correlated with the source of isolation. The isolates were also analyzed for certain growth-promoting activities. Auxin synthesis was widely distributed among the isolates, with values ranging from 93.34 to 1653.37 ${\mu}M$ auxin per ${\mu}g$ of protein. The phosphate solubilization index ranged from 1.25 to 3.89, and siderophore index varied from 1.15 to 5.25. From a total of 57 isolates, 3 showed an ability to biologically fix atmospheric nitrogen, and 7 showed antagonism against the pathogen Sclerotinia sclerotiorum. The results of biochemical characterization allowed identification of potential candidates for the development of biofertilizers targeted to the sunflower crop.

A comprehensive longitudinal study of gut microbiota dynamic changes in laying hens at four growth stages prior to egg production

  • Seojin Choi;Eun Bae Kim
    • Animal Bioscience
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    • 제36권11호
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    • pp.1727-1737
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    • 2023
  • Objective: The poultry industry is a primary source of animal protein worldwide. The gut microbiota of poultry birds, such as chickens and ducks, is critical in maintaining their health, growth, and productivity. This study aimed to identify longitudinal changes in the gut microbiota of laying hens from birth to the pre-laying stage. Methods: From a total of 80 Hy-Line Brown laying hens, birds were selected based on weight at equal intervals to collect feces (n = 20 per growth) and ileal contents (n = 10 per growth) for each growth stage (days 10, 21, 58, and 101). The V4 regions of the 16S rRNA gene were amplified after extracting DNA from feces and ileal contents. Amplicon sequencing was performed using Illumina, followed by analysis. Results: Microbial diversity increased with growth stages, regardless of sampling sites. Microbial community analysis indicated that Firmicutes, Proteobacteria, and Bacteroidetes were the dominant phyla in the feces and ileal. The abundance of Lactobacillus was highest on day 10, and that of Escherichia-shigella was higher on day 21 than those at the other stages at the genus level (for the feces and ileal contents; p<0.05). Furthermore, Turicibacter was the most abundant genus after changing feed (for the feces and ileal contents; p<0.05). The fecal Ruminococcus torques and ileal Lysinibacillus were negatively correlated with the body weights of chickens (p<0.05). Conclusion: The gut microbiota of laying hens changes during the four growth stages, and interactions between microbiota and feed may be present. Our findings provide valuable data for understanding the gut microbiota of laying hens at various growth stages and future applied studies.

Effects of Sampling Techniques and Sites on Rumen Microbiome and Fermentation Parameters in Hanwoo Steers

  • Song, Jaeyong;Choi, Hyuck;Jeong, Jin Young;Lee, Seul;Lee, Hyun Jung;Baek, Youlchang;Ji, Sang Yun;Kim, Minseok
    • Journal of Microbiology and Biotechnology
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    • 제28권10호
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    • pp.1700-1705
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    • 2018
  • We evaluated the influence of sampling technique (cannulation vs. stomach tube) and site (dorsal sac vs. ventral sac) on the rumen microbiome and fermentation parameters in Hanwoo steers. Rumen samples were collected from three cannulated Hanwoo steers via both a stomach tube and cannulation, and 16S rRNA gene amplicons were sequenced on the MiSeq platform to investigate the rumen microbiome composition among samples obtained via 1) the stomach tube, 2) dorsal sac via rumen cannulation, and 3) ventral sac via rumen cannulation. A total of 722,001 high-quality 16S rRNA gene sequences were obtained from the three groups and subjected to phylogenetic analysis. There was no significant difference in the composition of the major taxa or alpha diversity among the three groups (p>0.05). Bacteroidetes and Firmicutes represented the first and second most dominant phyla, respectively, and their abundances did not differ among the three groups (p>0.05). Beta diversity principal coordinate analysis also did not separate the rumen microbiome based on the three sample groups. Moreover, there was no effect of sampling site or method on fermentation parameters, including pH and volatile fatty acids (p>0.05). Overall, this study demonstrates that the rumen microbiome and fermentation parameters are not affected by different sampling techniques and sampling sites. Therefore, a stomach tube can be a feasible alternative method to collect representative rumen samples rather than the standard and more invasive method of rumen cannulation in Hanwoo steers.