阴道微生物组：种群特征与疾病干预治疗

高通量测序技术的发展显著加快了对人类微生物组的理解。将人体微生物组与疾病关联,力求阐明疾病的发生进程,是推进个性化精准医疗的重要研究方向。近年来,栖居于女性阴道的微生物菌群日益受到关注,发现其生态失调与疾病发生、演变密不可分。文中综述了阴道微生物组与生殖道疾病发生、进展和治疗的最新进展,同时对阴道微生物组培养组、益生菌工程化改造以及合成菌群在阴道微生物组学研究以及疾病干预与治疗方面的前景进行了展望。     

输卵管性不孕症孕妇女生殖道微生物群落研究

目的分析输卵管性不孕症妇女阴道微生物群落,研究阴道微生物菌落在输卵管性不孕症中的作用。方法选择2016年4月-2018年4月在本院就诊的女性246例,其中符合输卵管性不孕症诊断标准女性126例,设为不孕组;其余120例患者,设为对照组。对患者阴道微生物菌群分布,菌群密集度,菌群多样性,优势菌和炎症情况检测。结果两组间患者的年龄分组(P=0.916,0.564,0.734,0.423)、文化程度分布(P=0.937,0.488,0.522)、月经周期(P=0.839)、月经周期是否规律(P=0.943)、Nugent评分(P=0.614,0.089,0.108)等差异无统计学意义。对照组和不孕组患者阴道微生态各指标相比差异无统计学意义。不孕组患者解脲支原体阳性率明显高于对照组(P=0.000),沙眼衣原体阳性率明显高于对照组(P=0.005),阴道毛滴虫、线索细胞、假丝酵母菌等阳性率与对照组比较差异无统计学意义(Ps0.05)。结论阴道解脲支原体、沙眼衣原体感染与女性输卵管性不孕症有关,可能是引起女性不孕的主要因素之一。     

基于16S rRNA基因测序分析微生物群落多样性

微生物群落多样性的研究对于挖掘微生物资源,探索微生物群落功能,阐明微生物群落与生境间的关系具有重要意义。随着宏基因组概念的提出以及测序技术的快速发展,16S rRNA基因测序在微生物群落多样性的研究中已被广泛应用。文中系统地介绍了16S rRNA基因测序分析流程中的四个重要环节,包括测序平台与扩增区的选择、测序数据预处理以及多样性分析方法,就其面临的问题与挑战进行了探讨并对未来的研究方向进行了展望,以期为微生物群落多样性相关研究提供参考。     

基于高通量测序技术马肠道菌群研究进展

马肠道非常发达,其中定居着丰富又复杂的微生物菌群,这些微生物在宿主的生理、代谢、营养和免疫功能等方面有着重要作用.基于高通量测序的宏基因组学技术和分析手段的改进,对复杂环境中微生物的研究更加方便、透彻.本文就基于高通量测序的宏基因组技术在马肠道核心菌群、不同肠道段菌群结构、不同因素对肠道菌群结构的影响,以及马肠道微生物...     

女性生殖系统微生物群落及其与早产相关性研究进展

女性生殖系统微生物群落与女性生殖健康密切相关。阴道微生物的菌群变化伴随着多种阴道疾病的发生并可能通过宫颈感染子宫,孕期子宫内的炎症反应会导致胎儿早产(Preterm Birth,PTB),PTB被定义为妊娠满28周但不足37周所发生的分娩现象,PTB婴儿死亡率很高并面临着多种健康疾病,除了明确病原微生物的感染,PTB还伴随着生殖系统微生物群落失衡,将来有望通过根据女性生殖系统微生物群落的动态变化来预防PTB的发生。本综述将着重介绍女性生殖系统微生物群落与生殖健康,尤其是与PTB之间的联系。     

微生物分子生态学研究方法及其存在的缺陷

微生物群落多样性是微生物生态学和环境学研究的重点之一.分子生物学方法应用于微生物群落结构分析使得对环境样品中占大多数的不可培养微生物的研究成为了可能.然而,PCR过程中的偏差(bias)会引起结果并不能如实地再现原始的群落结构,随着多个宏基因组项目的研究,发现所谓的“通用引物”并不能覆盖全部的微生物类型,即使可以添加简并碱基,也无法与通过宏基因组得到的rRNA序列完全匹配,这导致了在诸多研究中会忽略环境中的微生物群落.即使是不经过PCR扩增的元基因组和元转录组学研究方法,对于分子微生物群落也存在着一定的问题.     

分子生物学方法在浮游病毒多样性研究中的应用

病毒是海洋及淡水微生物群落的重要组成部分,在调控微生物环路、驱动生物地球化学循环、维持浮游植物与细菌多样性等方面扮演着重要角色。然而,传统的病毒培养及定量技术难以对浮游病毒群落结构与多样性进行深入而全面的解析。微生物分子生态学技术的快速发展及广泛应用为此提供了新的途径。概述了克隆文库分析方法、凝胶脉冲场电泳(PFGE)技术、DNA指纹图谱、DNA微阵列、宏基因组技术等分子生物学方法的基本概念及其在研究浮游病毒的种群结构与遗传多样性及其与环境因素之间的相互关系等方面的应用状况。     

微生物宏基因组数据分析方法研究进展

随着测序技术的迅速发展,人们对宏基因组的研究逐渐深入。通过宏基因组学对微生物群落的测序和分析,以理解微生物组成与环境之间的相互作用。微生物宏基因组的分析摆脱了传统研究中微生物分离培养的技术限制,并获得了微生物群落的相对丰度和群落的功能等信息。用于微生物数据分析的工具和软件较多,对于研究者选择合适的分析方法具有一定困难。概述了微生物宏基因组分析方法的流程,总结了分析中常用的工具及软件,为研究者快速筛选分析方法,揭示数据背后的生物学意义提供参考。     

高危型HPV感染与阴道微生物组关系

目的 通过分析高危型人乳头瘤病毒(HPV)感染患者的阴道菌群,探讨HPV感染与阴道微生物组的关系。方法 选取于国际和平妇幼保健院体检中心体检的63例HPV阴性女性(对照组)及39例高危型HPV感染者(HPV感染组)为研究对象。对细菌16S rDNA的V1V2域扩增后,使用高通量测序法分析两组对象阴道微生物组的组成与结构差异。结果 在门水平上,两组对象均以厚壁菌门(Firmicutes)为主要优势门,其在感染组中占55.0%,在对照组中占88.4%。Alpha多样性指数分析表明感染组患者的阴道菌群多样性均高于对照组,差异有统计学意义(P0.05)。LEfSe结果显示加德纳菌属在感染组低级别鳞状上皮内病变(LSIL)型患者中为优势菌,乳杆菌属为对照组优势菌。结论 高危型HPV感染者阴道菌群多样性增加。加德纳菌属为LSIL与HPV阴性女性的差异菌,乳杆菌属为HPV阴性女性阴道特征菌。     

宏基因组方法揭示草地土壤微生物群落响应全球变化

草地在生物圈中发挥着重要的生态服务功能,而草地土壤微生物又是维持生态系统功能和稳定的关键要素之一。过去十几年间,宏基因组方法的进步为微生物群落分析提供了有力的工具。本文综述了宏基因组方法应用于草地土壤微生物群落响应全球变化的最新研究进展,特别是针对气候变化、大气组成变化、土地利用方式改变和外来物种入侵等条件下微生物群落的响应规律和反馈机制的研究。这些研究对于我们认识和了解微生物群落的生态功能十分重要,同时也对维持地球生态系统平衡具有积极的意义。最后,我们对未来应用宏基因组方法研究草地微生物群落进行了展望。     

The phylogenetic diversity of metagenomes

Phylogenetic diversity--patterns of phylogenetic relatedness among organisms in ecological communities--provides important insights into the mechanisms underlying community assembly. Studies that measure phylogenetic diversity in microbial communities have primarily been limited to a single marker gene approach, using the small subunit of the rRNA gene (SSU-rRNA) to quantify phylogenetic relationships among microbial taxa. In this study, we present an approach for inferring phylogenetic relationships among microorganisms based on the random metagenomic sequencing of DNA fragments. To overcome challenges caused by the fragmentary nature of metagenomic data, we leveraged fully sequenced bacterial genomes as a scaffold to enable inference of phylogenetic relationships among metagenomic sequences from multiple phylogenetic marker gene families. The resulting metagenomic phylogeny can be used to quantify the phylogenetic diversity of microbial communities based on metagenomic data sets. We applied this method to understand patterns of microbial phylogenetic diversity and community assembly along an oceanic depth gradient, and compared our findings to previous studies of this gradient using SSU-rRNA gene and metagenomic analyses. Bacterial phylogenetic diversity was highest at intermediate depths beneath the ocean surface, whereas taxonomic diversity (diversity measured by binning sequences into taxonomically similar groups) showed no relationship with depth. Phylogenetic diversity estimates based on the SSU-rRNA gene and the multi-gene metagenomic phylogeny were broadly concordant, suggesting that our approach will be applicable to other metagenomic data sets for which corresponding SSU-rRNA gene sequences are unavailable. Our approach opens up the possibility of using metagenomic data to study microbial diversity in a phylogenetic context.     

Robust estimation of microbial diversity in theory and in practice

Quantifying diversity is of central importance for the study of structure, function and evolution of microbial communities. The estimation of microbial diversity has received renewed attention with the advent of large-scale metagenomic studies. Here, we consider what the diversity observed in a sample tells us about the diversity of the community being sampled. First, we argue that one cannot reliably estimate the absolute and relative number of microbial species present in a community without making unsupported assumptions about species abundance distributions. The reason for this is that sample data do not contain information about the number of rare species in the tail of species abundance distributions. We illustrate the difficulty in comparing species richness estimates by applying Chao''s estimator of species richness to a set of in silico communities: they are ranked incorrectly in the presence of large numbers of rare species. Next, we extend our analysis to a general family of diversity metrics (‘Hill diversities''), and construct lower and upper estimates of diversity values consistent with the sample data. The theory generalizes Chao''s estimator, which we retrieve as the lower estimate of species richness. We show that Shannon and Simpson diversity can be robustly estimated for the in silico communities. We analyze nine metagenomic data sets from a wide range of environments, and show that our findings are relevant for empirically-sampled communities. Hence, we recommend the use of Shannon and Simpson diversity rather than species richness in efforts to quantify and compare microbial diversity.     

Microbial communities within field‐collected Culiseta melanura and Coquillettidia perturbans

Although mosquitoes are well‐known vectors of human and animal diseases, pathogens are only minor components of their total endogenous microbial communities. The midguts of many insects, including mosquitoes, contain diverse microbial communities. In this study, we used denaturing gradient gel electrophoresis to identify the diversity of bacteria in field‐collected adult female Culiseta melanura (Diptera: Culicidae) (Coquillett) and Coquillettidia perturbans (Diptera: Culicidae) (Walker). Few significant differences in bacterial fauna between the two mosquito species were found, but the results suggest that host life history may be a determinant of the endogenous bacterial communities in mosquitoes. In the present study, the dominant bacteria are frequently identified as major components of other mosquito species' microbial flora, suggesting the establishment of a stable association between the mosquitoes and the microbes after initial acquisition from the environment.     

Obstetric and Gynecological Diseases and Complications Resulting from Vaginal Dysbacteriosis

Accurate knowledge of the composition and ecology of vaginal microbial environment of a healthy woman is necessary for the understanding of normal flora and how to reduce the risk for diseases. Vagina and its microflora form a balanced ecosystem in which dominated bacteria are vaginal lactobacilli. There are dynamic changes in this ecosystem having structure and composition depending on many factors. The term dysbacteriosis defines any movement outside the normal range for the given biotope of obligate and/or facultative microflora. Such a change in the quantity and quality of the respective microbial balance is fraught with danger and requires correction and recovery. The purpose of this overview is to examine obstetric and gynecological diseases that can cause vaginal impaired microbial balance. Vaginal dysbacteriosis is a cause, predecessor, and often also consequence of vaginal infections. In essence, any vaginal infection can be seen as dysbacteriosis, developed to the most severe extent. Here, there is a dominant microorganism other than lactic acid bacteria in the vagina (clinically manifested or not, respectively), depletion of defense mechanisms of the vagina associated with the shift of lactobacilli from their dominant role in the vaginal balance, decrease in their number and species diversity, and a resulting change in the healthy status of the vagina. Vaginal dysbacteriosis can be found in pathogenetic mechanism, whereby many obstetric and gynecological diseases develop. Most of these diseases lead directly to increased maternal and infant morbidity and mortality, so it is important to understand the reasons for them and the arrangements for their prevention.     

A metagenomic approach to characterization of the vaginal microbiome signature in pregnancy

While current major national research efforts (i.e., the NIH Human Microbiome Project) will enable comprehensive metagenomic characterization of the adult human microbiota, how and when these diverse microbial communities take up residence in the host and during reproductive life are unexplored at a population level. Because microbial abundance and diversity might differ in pregnancy, we sought to generate comparative metagenomic signatures across gestational age strata. DNA was isolated from the vagina (introitus, posterior fornix, midvagina) and the V5V3 region of bacterial 16S rRNA genes were sequenced (454FLX Titanium platform). Sixty-eight samples from 24 healthy gravidae (18 to 40 confirmed weeks) were compared with 301 non-pregnant controls (60 subjects). Generated sequence data were quality filtered, taxonomically binned, normalized, and organized by phylogeny and into operational taxonomic units (OTU); principal coordinates analysis (PCoA) of the resultant beta diversity measures were used for visualization and analysis in association with sample clinical metadata. Altogether, 1.4 gigabytes of data containing >2.5 million reads (averaging 6,837 sequences/sample of 493 nt in length) were generated for computational analyses. Although gravidae were not excluded by virtue of a posterior fornix pH >4.5 at the time of screening, unique vaginal microbiome signature encompassing several specific OTUs and higher-level clades was nevertheless observed and confirmed using a combination of phylogenetic, non-phylogenetic, supervised, and unsupervised approaches. Both overall diversity and richness were reduced in pregnancy, with dominance of Lactobacillus species (L. iners crispatus, jensenii and johnsonii, and the orders Lactobacillales (and Lactobacillaceae family), Clostridiales, Bacteroidales, and Actinomycetales. This intergroup comparison using rigorous standardized sampling protocols and analytical methodologies provides robust initial evidence that the vaginal microbial 16S rRNA gene catalogue uniquely differs in pregnancy, with variance of taxa across vaginal subsite and gestational age.     

土壤宏基因组学技术及其应用

传统的基于培养的研究方法只能反映土壤中少数(0.1%～10 %)微生物的信息,而大部分微生物目前还不能培养,因而这部分微生物资源尚难以被有效地开发利用.宏基因组学是分子生物学技术应用于环境微生物生态学研究而形成的一个新概念,主要技术包括土壤DNA的提取、文库的构建和目标基因克隆的筛选.它可为揭示微生物生态功能及其分子基础提供更全面的遗传信息,并已在微生物新功能基因筛选、活性物质开发和微生物多样性研究等方面取得了显著成果.本文对土壤宏基因组学技术的方法和应用作了详细介绍.     

Signal Processing for Metagenomics: Extracting Information from the Soup

Traditionally, studies in microbial genomics have focused on single-genomes from cultured species, thereby limiting their focus to the small percentage of species that can be cultured outside their natural environment. Fortunately, recent advances in high-throughput sequencing and computational analyses have ushered in the new field of metagenomics, which aims to decode the genomes of microbes from natural communities without the need for cultivation. Although metagenomic studies have shed a great deal of insight into bacterial diversity and coding capacity, several computational challenges remain due to the massive size and complexity of metagenomic sequence data. Current tools and techniques are reviewed in this paper which address challenges in 1) genomic fragment annotation, 2) phylogenetic reconstruction, 3) functional classification of samples, and 4) interpreting complementary metaproteomics and metametabolomics data. Also surveyed are important applications of metagenomic studies, including microbial forensics and the roles of microbial communities in shaping human health and soil ecology.     

Metagenomics: Read Length Matters

Obtaining an unbiased view of the phylogenetic composition and functional diversity within a microbial community is one central objective of metagenomic analysis. New technologies, such as 454 pyrosequencing, have dramatically reduced sequencing costs, to a level where metagenomic analysis may become a viable alternative to more-focused assessments of the phylogenetic (e.g., 16S rRNA genes) and functional diversity of microbial communities. To determine whether the short (~100 to 200 bp) sequence reads obtained from pyrosequencing are appropriate for the phylogenetic and functional characterization of microbial communities, the results of BLAST and COG analyses were compared for long (~750 bp) and randomly derived short reads from each of two microbial and one virioplankton metagenome libraries. Overall, BLASTX searches against the GenBank nr database found far fewer homologs within the short-sequence libraries. This was especially pronounced for a Chesapeake Bay virioplankton metagenome library. Increasing the short-read sampling depth or the length of derived short reads (up to 400 bp) did not completely resolve the discrepancy in BLASTX homolog detection. Only in cases where the long-read sequence had a close homolog (low BLAST E-score) did the derived short-read sequence also find a significant homolog. Thus, more-distant homologs of microbial and viral genes are not detected by short-read sequences. Among COG hits, derived short reads sampled at a depth of two short reads per long read missed up to 72% of the COG hits found using long reads. Noting the current limitation in computational approaches for the analysis of short sequences, the use of short-read-length libraries does not appear to be an appropriate tool for the metagenomic characterization of microbial communities.     

阴道微生态与宫颈疾病发生发展的相关性研究

摘要：微生态学是近年新兴的学科,女性生殖道是一个重要的微生态区域。生殖道微生态体系是由正常的解剖结构、生殖道各种菌群、周期性内分泌变化、局部免疫系统四个部分组成。随着阴道微生态平衡理论的提出和发展,阴道微生态和HPV感染、宫颈上皮内瘤变、宫颈癌的内在关联引起了极大的重视。阴道微生态与宫颈疾病的发生发展相关,宫颈病变影响了阴道微生态环境,长期的阴道菌群失调也会反作用于宫颈,导致宫颈的病变加重甚至恶化,如此形成恶性循环。因此阴道微生态对宫颈疾病的诊断、治疗和预防将具有重要的临床意义。