Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

AIM:To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome.METHODS:A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral,esophageal,and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species.Candidate primers evaluated were from the European rRNA database.To assess the effect of sequence length on accuracy of classification,16S rRNA genes of various lengths were created by trimming the full length sequences.Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs.The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP).The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes,represented by 36 phyla.RESULTS:Truncation to 100 nucleotides(nt)downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87(39.7%)of the 219 sequences,compared with misclassification of only 29(13.2%)sequences with truncation to 350 nt.Among 350-nt sequence reads within various regions of the 16S rRNA gene,the reverse read of an amplicon generated using the 343F/798R primers had the least(8.2%)effect on classification.In comparison,truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0%of the 219 sequences.The 343F/798R amplicon accurately assigned 91.8%of the 219 sequences at the species level.Weighted by abundance of the species in the esophageal dataset,the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage(92%).Modification of the 343F/798R primers to 347F/803R increased their universality among foregut species.Assuming that a typicalpolymerase chain reaction can tolerate 2 mismatches between a primer and a template,the modified 347F and 803R primers should be able to anneal 98%and 99.6%of all 16S rRNA genes in the RDP database.CONCLUSION:347F/803R is the most suitable pair of primers for classification of foregut 16S rRNA genes but also possess universality suitable for analyses of other complex microbiomes.

Alterations in the human oral microbiome in cholangiocarcinoma

Dear Editor,Alterations in the human microbiome are closely related to various hepatobiliary diseases.Gut microbial dysbiosis has been found in patients with cholangiocarcinoma(CCA)[1].However,the characteristics of oral microbiome in patients with CCA have not been studied.

A New Technique for Use in Culturing Prokaryotes Comprising the Mouse Intestinal Microbiome

The microbiome has a profound impact on host fitness. pH, oxygen, nutrients, or other factors such as food or pharmaceuticals, subject the microbiome to variations in the gastrointestinal tract. This variation is a cause for concern given dysbiosis of the microbiome is correlated with various disease states. Currently, much research relies on model organisms to study microbial communities since intact microbiomes are challenging to utilize. The objective of this study is to culture an explanted colon microbiome of 4 Balb/c mice to develop an in vitro tool for future microbiome studies. We cultured homogenates of the distal colons of 4 mice in trans-well culture dishes. These dishes were incubated for 24 hours in two different oxygen concentration levels and the pH was compared before and after incubation of the cultures. To analyze the integrity of the microbiome, we utilized massively paralleled DNA sequencing with 16S metagenomics to characterize fecal and colon samples to speculate whether future studies may utilize feces in constructing an in vitro microbial community to spare animal lives. We found that pH and familial relationships had a profound impact on community structure while oxygen did not have a significant influence. The feces and the colon were similar in community profiles, which lends credence to utilizing feces in future studies. The gut microbiome is of great interest and great importance for studies in a variety of different diseases. Many laboratories do not have access to germ-free mice, which is one optimal way to study mammalian microbiomes, but this technique allowed for the in vitro culturing of a majority of the prokaryotes isolated from the colons of mice. This may allow an alternative to study the interactions of this very diverse population of microorganisms without the need for germ-free conditions.

Recent advances and perspectives in next generation sequencing application to the genetic research of type 2 diabetes

Type 2 diabetes(T2D)mellitus is a common complex disease that currently affects more than 400 million people worldwide and has become a global health problem.High-throughput sequencing technologies such as whole-genome and whole-exome sequencing approaches have provided numerous new insights into the molecular bases of T2D.Recent advances in the application of sequencing technologies to T2D research include,but are not limited to:(1)Fine mapping of causal rare and common genetic variants;(2)Identification of confident genelevel associations;(3)Identification of novel candidate genes by specific scoring approaches;(4)Interrogation of disease-relevant genes and pathways by transcriptional profiling and epigenome mapping techniques;and(5)Investigation of microbial community alterations in patients with T2D.In this work we review these advances in application of next-generation sequencing methods for elucidation of T2D pathogenesis,as well as progress and challenges in implementation of this new knowledge about T2D genetics in diagnosis,prevention,and treatment of the disease.

Marginal bone loss around non-submerged implants is associated with salivary microbiome during bone healing

Marginal bone loss during bone healing exists around non-submerged dental implants. The aim of this study was to identify the relationship between different degrees of marginal bone loss during bone healing and the salivary microbiome. One hundred patients were recruited, and marginal bone loss around their implants was measured using cone beam computed tomography during a 3-month healing period. The patients were divided into three groups according to the severity of marginal bone loss.Saliva samples were collected from all subjected and were analysed using 16 SMiSeq sequencing. Although the overall structure of the microbial community was not dramatically altered, the relative abundance of several taxonomic groups noticeably changed. The abundance of species in the phyla Spirochaeta and Synergistetes increased significantly as the bone loss became more severe. Species within the genus Treponema also exhibited increased abundance, whereas Veillonella, Haemophilus and Leptotrichia exhibited reduced abundances, in groups with more bone loss. Porphyromonasgingivalis, Treponemadenticola and Streptococcus intermedius were significantly more abundant in the moderate group and/or severe group. The severity of marginal bone loss around the non-submerged implant was associated with dissimilar taxonomic compositions. An increased severity of marginal bone loss was related to increased proportions of periodontal pathogenic species. These data suggest a potential role of microbes in the progression of marginal bone loss during bone healing.

Gut microbiome in allogeneic hematopoietic stem cell transplantation and specific changes associated with acute graft vs host disease

Allogeneic hematopoietic stem cell transplantation(aHSCT)is a standard validated therapy for patients suffering from malignant and nonmalignant hematological diseases.However,aHSCT procedures are limited by potentially life-threatening complications,and one of the most serious complications is acute graft-versus-host disease(GVHD).During the last decades,DNA sequencing technologies were used to investigate relationship between composition or function of the gut microbiome and disease states.Even if it remains unclear whether these microbiome alterations are causative or secondary to the presence of the disease,they may be useful for diagnosis,prevention and therapy in aHSCT recipients.Here,we summarized the most recent findings of the association between human gut microbiome changes and acute GVHD in patients receiving aHSCT.

Influence of group B streptococcus and vaginal cleanliness on the vaginal microbiome of pregnant women

BACKGROUND The vaginal microbiome plays a critical role in the health of pregnant women and their newborns.Group B Streptococcus(GBS)and vaginal cleanliness significantly affect the vaginal microecosystem and are closely associated with vaginal diseases.AIM To explore the effects of GBS status and vaginal cleanliness on vaginal microecosystems.METHODS We collected 160 vaginal swabs from pregnant women and divided them into the following four groups based on GBS status and vaginal cleanliness:GBS-positive+vaginal cleanliness I–II degree,GBS-negative+vaginal cleanliness I–II degree,GBS-positive+vaginal cleanliness III–IV degree,and GBS-negative+vaginal cleanliness III–IV degree.Samples were subjected to 16S rRNA gene amplicon sequencing.RESULTS Alpha diversity analysis showed that the Shannon index did not significantly differ between the four groups.We identified significant variation in taxa abundance between the GBS-positive and GBS-negative groups and between the vaginal cleanliness I–II degree and III–IV degree groups.Principal coordinate analysis and non-metric multidimensional scaling analysis further confirmed the microbial diversity of the four groups.Moreover,the linear discriminant analysis demonstrated that Lactobacillus jensenii and Actinobacteria were strongly associated with GBS-positive status,and Lactobacillus iners,Lactobacillaceae,Lactobacillus,Lactobacillales,Bacilli and Firmicutes were closely correlated with GBS-negative status.CONCLUSION GBS status and vaginal cleanliness significantly affect vaginal microbiome differences in pregnant women.Our findings provide instructional information for clinical antibiotic treatment in pregnant women with different GBS statuses and vaginal cleanliness degrees.

Fecal microbiome of growing pigs fed a cereal based diet including chicory(Cichorium intybus L.) or ribwort(Plantago lanceolata L.) forage

Background： The purpose of this study was to investigate how inclusion of chicory forage or ribwort forage in a cereal-based diet influenced the fecal microbial community（microbiome） in newly weaned（35 days of age） piglets.The piglets were fed a cereal-based diet without（B） and with inclusion（80 and 160 g/kg air-dry forage） of vegetative shoots of chicory（C） and leaves of ribwort（R） forage in a 35-day growth trial. Fecal samples were collected at the start（D0）, 17（D17） and 35（D35） days after weaning and profiles of the microbial consortia were generated using terminal restriction fragment length polymorphism（T-RFLP）. 454-FLX pyrosequencing of 16 S r RNA gene amplicons was used to analyze the microbial composition in a subset of the samples already analyzed with T-RFLP.Results： The microbial clustering pattern was primarily dependent on age of the pigs, but diet effects could also be observed. Lactobacilli and enterobacteria were more abundant at D0, whereas the genera Streptococcus, Treponema,Clostridium, Clostridiaceae1 and Coprococcus were present in higher abundances at D35. Pigs fed ribwort had an increased abundance of sequences classified as Treponema and a reduction in lactobacilli. However, the abundance of Prevotellaceae increased with age in on both the chicory and the ribwort diet. Moreover, there were significant correlations between the abundance of Bacteroides and the digested amount of galactose, uronic acids and total non-starch polysaccharides, and between the abundance of Bacteroidales and the digested amount of xylose.Conclusion： This study demonstrated that both chicory and ribwort inclusion in the diet of newly weaned pigs influenced the composition of the fecal microbiota and that digestion of specific dietary components was correlated with species composition of the microbiota. Moreover, this study showed that the gut will be exposed to a dramatic shift in the microbial community structure several weeks after weaning.

Analysis of the Human Oral Microbiome of Smokers and Non-Smokers Using PCR-RFLP and Ribotyping

Recent advances in the field of microbial and medical ecology emphasize the critical role played by oral bacteria in the delicate dynamic equilibrium of human health and disease, creating the need to define the bacterial communities associated with healthy and non-healthy conditions and to capture shifts in community structure germane to diagnosis. Employing PCR-RFLP of the 16S rDNA gene from metagenomes and plate-wash (cultured) bacteria of oral wash from 10 volunteers, this study evaluated the stability of oral bacteria in healthy subjects and documented community shifts in smokers. Sequence analysis of selected 16S gene amplicons cloned with the Gene Hunter PCR-Trap vector and pCR 4-TOPO cloning kits was conducted to determine the bacteria identity and diversity indices of the two groups. Ribopatterns generated by the restriction enzymes HaeIII and Sau3AI were significantly (p AluI using the GelCompare II software cluster analysis. A stable core of bacteria DNA fingerprint was detected in all healthy subjects, and remained unchanged over the study period of 3 months. Signature bands (1500 bp with HaeIII) in smokers and in non-smokers (800 bp and 700 bp with Sau3A1) were evidently suggesting the presence of potential biomarkers of healthy and non-healthy states. There was no significant difference in the DNA fingerprints of cultured and metagenomic extracts. The genera Xanthomonas, Streptococcus and phylum Candidatus occurred in large numbers in both groups, however, a major shift in composition with the dominance of gram-negative bacteria in smokers compared to healthy subjects was quite remarkable. Taxonomic diversity in smokers was quite high, including members of the genera Rothia, Synechococcus, Neisseria, Thiomargarita and Pyrobaculum. These data highlight the presence of a stable core microbiome amidst a wide diversity, identify a distinct smokers’ cluster and open the way for the search for potential biomarkers for specific diseases.

Comparison of in Clinic-Based Fecal Microbiome Collection Techniques for Increase in Study Participation and Utilization of Microbiome Analysis

We compared the collection techniques of fecal specimens for DNA extraction and fecal microbiome analysis by utilizing the glove from a standard-of-care digital rectal exam (DRE) and the rectal swab from a pre-prostate biopsy bacterial rectal culture collected in clinical care settings. DNA yield from the swab technique compared to the glove technique yielded similar amounts of DNA (18.1 vs. 13.1 ng/μL, p = 0.06), slightly favoring the swab technique. However, utilizing DNA yield cutoffs of 15 ng/μL (37% vs. 29%, p = 0.18) and 30 ng/μL (15% and 9%, p = 0.16), we identified no differences in yield between the swab versus glove technique, respectively. Absorbance values for overall DNA quality were significantly different in favor of the glove technique (mean 1.6 vs. 2.0, p < 0.001). Using an absorbance value of 1.5 as an indication of DNA quality, only 26% (19/91) met the cutoff value using the swab group compared to 47.3% (53/112) if the glove technique was used (p < 0.001). Similar results occurred for the RNA quality with an absorbance value cutoff of 2.0 (2.2% vs. 30.4%, p < 0.001). To increase sampling feasibility and improve population sampling, gloves used from a DRE may be utilized as a consistent and efficient fecal DNA collection technique for fecal microbiome analysis. DNA yield and quality from the glove technique are comparable to—if not better than—rectal swab collection.

Metagenomic Shotgun Sequencing Provides Prevalence Data for Pathogens, and Source-Tracking Indices Useful in Public Health Risk Assessment of Environmental Waters

State-approved membrane filtration (MF) techniques for water quality assessments were contrasted with metagenomic shotgun sequencing (MSS) protocols to evaluate their efficacy in providing precise health-risk indices for surface waters. Samples from a freshwater receiving pond (ABI-1002) and two upstream storm water ditches (ABI-1003) and (ABI-1004) yielded alarmingly high Fecal coliform MF densities of 220, >2000 and >2000 CFU/100ml respectively. The indicator, Enterococcus bacteria exceeded allowable limits in all but the equipment control (ABI-1001). Using MSS, the relative numerical abundance of pathogenic bacteria, virulence and antibiotic resistance genes revealed the status and potential pollution sources of each ditch. High levels of Shigella sp. (0 (ABI-1001), 4945 (ABI-1002), 55,008 (ABI-1003), and 2221 (ABI-1004) genomic reads/100ml) correlated with virulence genes and antibiotic resistance genes found in fecal samples for ABI1003 and not ABI1004. Traditional culture methods (TCM) showed possible fecal contamination in two of the four samples, and no contamination in the others. MSS clearly distinguished between fecal and environmental bacteria contamination sources, and pinpointed actual risks from pathogens. Our data underscore the potential utility of MSS in precision risk assessment for public and biodiversity health and tracking of environmental microbiomes shifts by field managers and policy makers.

Application of artificial intelligence in microbiome study promotes precision medicine for gastric cancer

The microbiome has been identified as a causing factor for many cancers.Helicobacter pylori contributes to the development of gastric cancer(GC)and impacts disease treatments.The rapid development of sequencing technology is increasingly producing large-scale and complex big data.However,there are many obstacles in the analysis of these data by humans,which limit clinicians from making rapid decisions.Recently,the emergence of artificial intelligence(AI),including machine learning and deep learning,has greatly assisted clinicians in processing and interpreting large microbiome data.This paper reviews the application of AI in the study of the microbiome and discusses its potential in the diagnosis and therapy of GC.We also exemplify strategies for implementing microbiome-based precision medicines for patients with GC.

圈养与野外近人环境猕猴肠道菌群的比较分析

通过对来自北京动物园10只圈养猕猴(Macaca mulatta)和来自河南济源五龙口风景区10只处于野外近人环境中的猕猴的粪便微生物进行鸟枪法测序,分析两种不同环境下猕猴的肠道菌群组成和差异。结果表明:20份样品共检测出9个门、18个纲、22个目、31个科、46个属和226个种。通过分析发现,圈养和野外近人环境群体共享了绝大部分细菌(200种),但是近人环境群体相比于圈养群体,独有更多种类的微生物。圈养猕猴肠道微生物主要由厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)和绿弯菌门(Chloroflexi)组成。处于野外近人环境中的猕猴肠道微生物主要由厚壁菌门、变形菌门(Proteobacteria)和拟杆菌门组成。圈养猕猴和近人猕猴属水平上的肠道微生物结构存在显著差异(p=0.001)。圈养猕猴和野外近人环境猕猴肠道菌群组成的多样性与差异,可为猕猴的圈养以及景区野生动物管理提供指导和理论基础。

基于下一代测序的精液菌群研究进展

精液菌群在男性生殖健康中发挥着重要作用,其构成可能与精子质量、少弱精子症的发生、辅助生殖技术妊娠结局相关。同时,由于精液菌群可能向伴侣及后代传递,因此对女性生殖健康及后代的健康都可能产生影响。常规细菌培养方法因耗时、条件限制等难以发现完整的精液菌群结构。下一代测序技术(NGS)可以揭示完整的精液菌群组成,有助于阐明人类生理学和病理生理学基础上复杂的微生物-宿主相互作用。本文就基于NGS研究的精液菌群构成、与男性不育症的关系、损伤精子质量的作用机制等内容进行综述,总结使用NGS来描述不同男性生殖道解剖部位的细菌定植模式的研究结果。目前的研究结果揭示了男性精液呈现独特的多样性菌群结构,精液菌群结构异常与精子质量和生育状态密切相关,精液可能具有支持健康功能的原生菌群。但迄今为止,精液菌群的来源、构成、及其对生育力和妊娠结局的影响尚不明确,精液菌群多样性影响精子的具体机制还需要具有大样本量和适当的对照标本进一步研究。

基于高通量测序的眼-鼻区系微生物菌群研究进展

近年来,微生物在眼部的作用日益受到重视。传统的眼部微生物鉴定主要集中于涂片、细菌培养等检查手段,但受到培养条件等各种因素限制导致阳性率低。随着人类宏基因组计划的启动实施,高通量测序技术因其通量高、灵敏度及特异性高、信息量丰富、成本低的特点可将微生物群落定义到属、种水平,有助于更好的理解眼部疾病的病因、诊断、治疗及预防,推动眼科实现精准化医疗进程。由于眼部与鼻部在解剖结构上相通,本文旨在探讨眼部和鼻部微生物菌群策略的研究进展及眼-鼻区系微生物可能的传播途径,为眼鼻相关疾病的研究提供基础。

人类呼吸道微生物组及检测技术进展

人类呼吸道微生物组在健康和疾病状态下具有巨大的多样性和异质性,快速准确的微生物检测是区分呼吸道感染和非感染的关键。近年来,以宏基因组技术为代表的新型检测技术在病原诊断领域迅速发展,在提高病原微生物检出率的同时,也在分析人类宿主反应以及预测耐药性方面有了很大突破。本文阐述分子技术在呼吸道微生物组中的研究进展和成果,启发读者今后对新技术开发和临床转化进行思考,旨在探索基于呼吸道微生物组对呼吸道疾病的治疗策略。

High-throughput 16S rDNA sequencing of the pulmonary microbiome of rats with allergic asthma

A decrease in microbial infection in adolescents is implicated with an increase in the incidence of asthma and allergic diseases in adulthood,indicating that the microbiome plays a critical role in asthma.However,the microbial composition of the lower respiratory tract remains unclear,hindering the further exploration of the pathogenesis of asthma.This study aims to explore the microbial distribution and composition in the lungs of normal rats and rats with allergic asthma via 16S rDNA sequencing.The DNA of the pulmonary microbiome was extracted from the left lungs collected from normal control group(NC),saline control group(SC),and allergic asthma group(AA)under aseptic conditions.After the 16s rDNA V4eV5 region was amplified,the products were sequenced using Illumina high-throughput technology and subjected to operational taxonomic unit(OTU)cluster and taxonomy analysis.The OTU values of AA increased significantly compared with those of NC and SC.Microbiome structure analysis showed that the dominant phylum of the pulmonary microbiome changed from Proteobacteria in NC to Firmicutes in AA.Linear discriminant analysis indicated that the key microbiomes involved in the three groups varied.

烟草青枯病根际土壤细菌群落动态变化分析

为了解山东烟草种植区烟草青枯病病害发生过程中的土壤微生物组群落动态变化,通过高通量测序,系统分析了山东沂水县地区烟草根际微生物组。扩增子测序分析表明,与移栽前相比,青枯病发病初期根际土壤微生物多样性显著降低,但是随着病害的发展,后续根际土壤微生物多样性又有恢复趋势;进一步对比移栽前和青枯病病发初期烟草根际微生物组成的差异,探究了整个病害发生的动态过程与根际细菌群落变化之间的规律,并通过构建相关性网络发现了小粒胞菌属(Granulicella)、无色杆菌属(Achromobacter)、出芽单胞菌属(Gemmatimonas)、居土杆菌属(Humibacter)、弯曲杆状菌属(Flexivirga)、束缚菌属(Conexibacter)与青枯病的发生存在潜在的作用关系。综上所述,本研究通过时间维度分析烟草发病动态过程根际细菌组成的差异,发现了潜在的烟草青枯病防治菌属以及预警菌属,为后续山东烟草土传病害预警和防控奠定了基础。

青少年正畸固定矫治中釉质健康与脱矿状态下龈上菌斑真菌微生物组的比较研究

目的牙釉质脱矿是固定矫治常见的并发症。本研究比较了固定矫治中釉质脱矿与健康状态下龈上菌斑真菌组的差异,探索口腔真菌与正畸釉质脱矿的关系。方法选取54名固定矫治患者,釉质脱矿组31人,健康对照组23人。收集龈上菌斑,通过转录间隔子测序比较两组真菌组差异,使用RT-qPCR分析白色念珠菌的丰度变化。结果两组真菌微生物多样性和优势物种丰度均存在显著差异。脱矿组真菌微生物多样性降低,念珠菌属及白色念珠菌在脱矿组中富集,且白色念珠菌在脱矿组中丰度较高(P<0.05)。结论青少年固定矫治脱矿状态下龈上菌斑真菌组与健康状态存在显著差异,白色念珠菌更易富集在脱矿菌斑中,可能与釉质脱矿的发生发展密切相关。

基于16S rDNA测序的慢性牙髓炎及根尖周炎感染根管内菌群多样性研究

目的采用16S rDNA测序技术比较慢性牙髓炎和慢性根尖周炎患牙根管内微生物群落的构成差异,了解微生物与牙髓疾病的关系。方法临床采集需要进行根管治疗的慢性牙髓炎患牙和慢性根尖周炎患牙根管内微生物群落样本。提取样本中的细菌总DNA,PCR扩增其16S rDNA片段上的V3-V4高变区基因片段,经NovaSeq测序后进行统计分析和生物信息学分析,包括种系发育分析、多样性分析及组间差异分析。结果总计收集到慢性牙髓炎患牙6颗,慢性根尖周炎7颗,经高通量测序后共得到8510个操作分类单元(OTU),菌群多样性分析显示慢性牙髓炎和慢性根尖周炎在菌群构成的差异有统计学意义(P<0.05)。其中,变形菌门、酸杆菌门及放线菌门在慢性牙髓炎患牙根内相对丰度显著高于慢性根尖周炎。拟杆菌门和互养菌门的相对丰度在慢性根尖周炎患牙根管中显著增加。结论牙髓疾病患牙根管中感染微生物存在复杂多样性。慢性牙髓炎和慢性根尖周炎感染根管内的微生物群落构成上具有一定差异,根管内微生物组成的变化可能与牙髓疾病发生发展相关。