16S amplicon sequencing

16S 扩增子测序
  • 文章类型: Journal Article
    背景:龋齿的状况与口腔微生物组的变化密切相关。在这项研究中,我们比较了儿童严重早期龋齿(S-ECC)患儿在全身麻醉和门诊治疗前后牙菌斑微生物组的多样性和结构.
    方法:选择40名在全身麻醉(C1)或门诊(C2)下完成全口牙科治疗的3至5岁S-ECC患儿,每组20人。记录患儿的基本信息和口腔健康状况,以及治疗前牙菌斑的微生物群落结构和多样性(C1,C2),治疗后的第二天(C2_0D),治疗后7天(C1_7D,C2_7D),治疗后1个月(C1_1M,C2_1M),和治疗后3个月(C1_3M,C2_3M)通过16SrRNA高通量测序技术进行分析。
    结果:(1)α多样性检验表明,多重组菌群丰富度在治疗后明显大于治疗前(P<0.05),其余α多样性指数在两组间差异不显著(P>0.05)。β多样性分析表明,C1_7D组和C2_3M组的植物区系结构与各组内其他时间点的差异有统计学意义(P<0.05)。(2)治疗前和治疗后两组均存在核心菌群。根据两组儿童的龋齿状况,它们的植物丰度比例可能会发生变化。单约会组和多约会组治疗后7天,Leptotrichia丰度显着降低(P<0.05)。C1_1M和C1_3M组比C1和C1_7D组显著丰富(P<0.05)。链球菌,C1_7D组的嗜血杆菌和副流感嗜血杆菌明显高于其他组(P<0.05)。
    结论:全身麻醉下的单次治疗可在治疗后7天内引起微生物群落结构和组成的巨大变化,而多次预约治疗可能会导致口腔菌群多样性的缓慢变化。
    BACKGROUND: The status of dental caries is closely related to changes in the oral microbiome. In this study, we compared the diversity and structure of the dental plaque microbiome in children with severe early childhood caries (S-ECC) before and after general anaesthesia and outpatient treatment.
    METHODS: Forty children aged 3 to 5 years with S-ECC who had completed whole-mouth dental treatment under general anaesthesia (C1) or in outpatient settings (C2) were selected, 20 in each group. The basic information and oral health status of the children were recorded, and the microbial community structure and diversity of dental plaque before treatment (C1, C2), the day after treatment(C2_0D), 7 days after treatment (C1_7D, C2_7D), 1 month after treatment (C1_1M, C2_1M), and 3 months after treatment (C1_3M, C2_3M) were analysed via 16 S rRNA high-throughput sequencing technology.
    RESULTS: (1) The alpha diversity test showed that the flora richness in the multiappointment group was significantly greater at posttreatment than at pretreatment (P < 0.05), and the remaining alpha diversity index did not significantly differ between the 2 groups (P > 0.05). The beta diversity analysis revealed that the flora structures of the C1_7D group and the C2_3M group were significantly different from those of the other time points within the respective groups (P < 0.05). (2) The core flora existed in both the pre- and posttreatment groups, and the proportion of their flora abundance could be altered depending on the caries status of the children in both groups. Leptotrichia abundance was significantly (P < 0.05) lower at 7 days posttreatment in both the single- and multiappointment groups. Corynebacterium and Corynebacterium_matruchotii were significantly more abundant in the C1_1M and C1_3M groups than in the C1 and C1_7D groups (P < 0.05). Streptococcus, Haemophilus and Haemophilus_parainfluenzae were significantly more abundant in the C1_7D group than in the other groups (P < 0.05).
    CONCLUSIONS: A single session of treatment under general anaesthesia can cause dramatic changes in the microbial community structure and composition within 7 days after treatment, whereas treatment over multiple appointments may cause slow changes in oral flora diversity.
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  • 文章类型: Journal Article
    肠道微生物组通过对T细胞的影响影响炎症环境,其影响刺激小鼠破骨细胞生成和骨丢失的免疫介质和炎性细胞因子的产生。然而,很少有关于肠道微生物组和骨骼健康的大型人类研究。我们在两个大型队列中调查了人体肠道微生物组和桡骨和胫骨的高分辨率外周定量计算机断层扫描(HR-pQCT)扫描之间的关联;弗雷明汉心脏研究(FHS[n=1227,年龄范围:32-89]),和男性骨质疏松症研究(MrOS[n=836,年龄范围:78-98])。来自研究参与者的粪便样品进行16SrRNA基因的V4高变区的扩增和测序。对每个队列分别处理所得16SrRNA测序数据,将DADA2管道纳入16SbioBakery工作流程。使用SILVA参考数据库对所得扩增子序列变体进行分类。控制多个协变量,我们使用在微生物组多变量关联线性模型(MaAslin2)中实施的一般线性模型,测试了微生物类群丰度与HR-pQCT测量值之间的关联.FHS中37个微生物属的丰度,和MrOS的4个属,与各种骨骼测量(错误发现率[FDR]≤0.1)相关,包括DTU089与骨骼测量的关联,在两个队列中独立复制。分类群-骨关联的荟萃分析进一步显示(FDR≤0.25),属的丰度更高;Akkermansia和DTU089与较低半径的总vBMD相关,和胫骨皮质vBMD。相反,属的丰度较高;天花科NK4A136组,和粪杆菌与较大的胫骨皮质vBMD有关。我们还通过测试预测的(基于16SrRNA扩增子序列数据)代谢途径丰度和每个队列中的骨表型之间的关联来研究微生物类群的功能能力。虽然在两个队列中都没有观察到一致的功能关联,荟萃分析揭示了8个途径,包括组氨酸的超途径,嘌呤,嘧啶生物合成,与胫骨皮质室的骨测量相关。总之,我们的研究结果表明,肠道微生物组和骨骼代谢之间存在联系。
    The gut microbiome affects the inflammatory environment through effects on T-cells, which influence the production of immune mediators and inflammatory cytokines that stimulate osteoclastogenesis and bone loss in mice. However, there are few large human studies of the gut microbiome and skeletal health. We investigated the association between the human gut microbiome and high resolution peripheral quantitative computed tomography (HR-pQCT) scans of the radius and tibia in two large cohorts; Framingham Heart Study (FHS [n=1227, age range: 32 - 89]), and the Osteoporosis in Men Study (MrOS [n=836, age range: 78 - 98]). Stool samples from study participants underwent amplification and sequencing of the V4 hypervariable region of the 16S rRNA gene. The resulting 16S rRNA sequencing data were processed separately for each cohort, with the DADA2 pipeline incorporated in the16S bioBakery workflow. Resulting amplicon sequence variants were assigned taxonomies using the SILVA reference database. Controlling for multiple covariates, we tested for associations between microbial taxa abundances and HR-pQCT measures using general linear models as implemented in microbiome multivariable association with linear model (MaAslin2). Abundance of 37 microbial genera in FHS, and 4 genera in MrOS, were associated with various skeletal measures (false discovery rate [FDR] ≤ 0.1) including the association of DTU089 with bone measures, which was independently replicated in the two cohorts. A meta-analysis of the taxa-bone associations further revealed (FDR ≤ 0.25) that greater abundances of the genera; Akkermansia and DTU089, were associated with lower radius total vBMD, and tibia cortical vBMD respectively. Conversely, higher abundances of the genera; Lachnospiraceae NK4A136 group, and Faecalibacterium were associated with greater tibia cortical vBMD. We also investigated functional capabilities of microbial taxa by testing for associations between predicted (based on 16S rRNA amplicon sequence data) metabolic pathways abundance and bone phenotypes in each cohort. While there were no concordant functional associations observed in both cohorts, a meta-analysis revealed 8 pathways including the super-pathway of histidine, purine, and pyrimidine biosynthesis, associated with bone measures of the tibia cortical compartment. In conclusion, our findings suggest that there is a link between the gut microbiome and skeletal metabolism.
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  • 文章类型: Journal Article
    Diet has a major influence on the composition and metabolic output of the gut microbiome. Higher-protein diets are often recommended for older consumers; however, the effect of high-protein diets on the gut microbiota and faecal volatile organic compounds (VOC) of elderly participants is unknown. The purpose of the study was to establish if the faecal microbiota composition and VOC in older men are different after a diet containing the recommended dietary intake (RDA) of protein compared with a diet containing twice the RDA (2RDA). Healthy males (74⋅2 (sd 3⋅6) years; n 28) were randomised to consume the RDA of protein (0⋅8 g protein/kg body weight per d) or 2RDA, for 10 weeks. Dietary protein was provided via whole foods rather than supplementation or fortification. The diets were matched for dietary fibre from fruit and vegetables. Faecal samples were collected pre- and post-intervention for microbiota profiling by 16S ribosomal RNA amplicon sequencing and VOC analysis by head space/solid-phase microextraction/GC-MS. After correcting for multiple comparisons, no significant differences in the abundance of faecal microbiota or VOC associated with protein fermentation were evident between the RDA and 2RDA diets. Therefore, in the present study, a twofold difference in dietary protein intake did not alter gut microbiota or VOC indicative of altered protein fermentation.
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  • 文章类型: Journal Article
    这项研究旨在阐明有机土壤改良剂对添加和不添加矿物肥料的散装土壤中细菌共存的长期影响。以往的研究大多忽略了细菌共生结构,主要集中在参数物种多样性和物种丰度变化上。在这里,我们对两种常用的土壤改良剂进行了系统的比较,粪肥和稻草,关于它们对Speyer长期现场试验中细菌共现的影响,德国。该方法涉及16S扩增子测序与细菌网络分析相结合,比较不同的肥料制度。结果表明,与对照处理相比,用粪肥施肥的地块中细菌多样性的增加以及芽孢杆菌的细菌积累。在秸秆改良的地块中,既没有发现多样性的增加,也没有发现指示性物种。此外,网络分析显示,矿物肥料添加对细菌共生结构有明显的影响。最重要的是,无论矿物施肥制度如何,这两种有机修正案都增加了网络的复杂性。同时,粪肥和秸秆的影响表现出差异,这可以通过其营养/化学含量的差异来解释。结论是,细菌相互作用是评估有关土壤健康和可持续性的修正效果的关键参数。
    This study intended to elucidate the long-term effects of organic soil amendments on bacterial co-occurrence in bulk soil with and without addition of mineral fertiliser. Previous research mostly neglected the bacterial co-occurrence structure and focussed mainly on the parameters species diversity and abundance changes of species. Here we present a systematic comparison of two frequently used soil amendments, manure and straw, with regard to their impact on bacterial co-occurrence in a long-term field trial in Speyer, Germany. The approach involved 16S amplicon sequencing in combination with a bacterial network analysis, comparing the different fertiliser regimes. The results show an increase of bacterial diversity as well as an accumulation of bacteria of the order Bacillales in plots fertilised with manure compared to a control treatment. In the straw-amended plots neither an increase in diversity was found nor were indicative species detectable. Furthermore, network analysis revealed a clear impact of mineral fertiliser addition on bacterial co-occurrence structure. Most importantly, both organic amendments increased network complexity irrespective of mineral fertilisation regime. At the same time, the effects of manure and straw exhibited differences that might be explained by differences in their nutritional/chemical contents. It is concluded that bacterial interactions are a crucial parameter for the assessment of amendment effects regarding soil health and sustainability.
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