基于16S rRNA高通量测序技术的妊娠期和非妊娠期妇女口腔菌群差异研究

目的使用16S rRNA基因高通量测序技术分析妊娠期和非妊娠期妇女口腔菌群的差异。方法选择2019年11月—2020年11月定期在新疆医科大学第一附属医院产科就诊的10例25~35岁妊娠晚期(孕29~39周)妇女(妊娠期组)和10名非妊娠期女性健康志愿者(非妊娠期组)为研究对象,采集所有研究对象的牙菌斑样本,提取牙菌斑菌群总DNA,进行扩增,并采用16S rRNA基因高通量测序技术分析菌群多样性和菌群丰度。结果妊娠期组的Chao1指数、Observed-species指数、PD-whole-tree指数、Shannon指数分别为(5.50±0.36)、(5.70±0.48)、(5.50±0.36)、(12.35±3.78);非妊娠期组的Chao1指数、Observed-species指数、PD-whole-tree指数、Shannon指数分别为(15.50±4.63)、(15.30±4.42)、(15.50±4.63)、(8.65±1.57)。妊娠期组的Chao1指数、Observed-species指数、PD-whole-tree指数均低于非妊娠期组,差异均有统计学意义(均P<0.05);妊娠期组的Shannon指数高于非妊娠期组,但差异无统计学意义(P>0.05)。不同分组的口腔菌群组成在操作分类单元(OTU)水平有明显差异。梭杆菌门、拟杆菌门、厚壁菌门、变形菌门及放线菌门是两组口腔菌群共有的优势菌门。链球菌属、奈瑟菌属及卟啉单胞菌属在非妊娠期组表现出丰度高于妊娠期组的趋势,而妊娠期组普雷沃菌属的相对丰度高于非妊娠期组,罗氏菌属及放线菌属在两样本中表现出较低的丰度值。结论妊娠晚期妇女口腔菌群物种丰度和物种均匀度呈现降低的趋势,可为后期深入研究与妊娠相关的口腔微生态失调的病因学提供依据,并为预测妊娠期并发症的危险因素提供一种潜在的非侵入性指标。     

呼和浩特地区鼠类群落多样性研究

目的研究呼和浩特地区1984-2007年鼠类群落多样性变化。方法1984-2007年每年4-10月中旬利用直线夹日法调查,利用香浓—威纳指数计算多样性指数,用皮洛公式计算均匀性指数。结果1984-2007年布放364 697夹日,捕获鼠10 851只,共8种鼠。黑线仓鼠和长爪沙鼠为呼和浩特地区的优势种。结论各年度多样性指数和均匀性指数变化较大,多样性指数随着群落中鼠的种数以及黑线仓鼠和长爪沙鼠的优势度、群落捕获率和2个优势鼠种的捕获率而变化。     

16S rRNA基因测序在医院环境微生物群落分布特征分析中的研究进展

16S rRNA基因测序技术可以有效识别医院内物体表面、水体、医疗设备等各种环境中微生物的群落结构,帮助追踪医院环境中微生物分布及传播关系。本文主要介绍16S rRNA基因测序在医院环境微生物群落分布特征分析中的研究进展,以期为院内感染传播研究和控制策略提供一些思路。     

基于高通量测序的家用冰箱菌群分析

目的 了解家用冰箱不同环境中的微生物菌群情况,发现潜在致病菌,为人们日常使用冰箱提供一定参考意见。方法 采集7个家庭的家用冰箱4 ℃、-20 ℃环境内部及冷凝水共19份样本,通过高通量测序方法检测16s rRNA,分析其菌群情况;并进一步和致病菌数据库进行比对分析,发现潜在致病菌。结果 共检出30个不同的细菌门,其中变形菌门(74.59%)、厚壁菌门(15.73%)及拟杆菌门(7.80%)含量最高。三种环境中,冷凝水及4 ℃环境菌群特征比较接近,且G^-占比高于-20 ℃环境。4 ℃和冷凝水中,假单胞菌属和不动杆菌属分别含量最多,其次为大肠杆菌;而-20 ℃中,不动杆菌属和巨型球菌数丰度最高。共发现20种潜在致病菌,其中条件致病菌不动杆菌属、假单胞菌属及芽孢杆菌属丰度最高,同时还发现多种食源性致病菌,包括李斯特菌属、沙门菌属及大肠菌属等。结论 冰箱环境中存在G^-为主的多种菌群及潜在致病菌,日常清理冰箱及其重要。     

深度测序分析早产儿坏死性小肠结肠炎肠道菌群多样性变化

目的利用深度测序方法分析早产儿坏死性小肠结肠炎(NEC)肠道菌群多样性变化。方法采集NEC组和匹配对照组各16例早产儿的粪便标本,采用高通量测序技术分析肠道菌群16S rDNA PCR产物,分析早产儿NEC的肠道菌群多样性及其变化。结果 NEC组的Chao指数值为(57.25±60.5),对照组为(58.19±92.1),两组比较差异无统计学意义(P0.05)。两组样本Shannon指数值比较发现,NEC组的Shannon指数值为(0.87±0.65),对照组为(1.31±0.56),NEC组明显低于对照组,差异有统计学意义(P0.05)。两组肠道菌群门主要为厚壁菌门和变形菌门两大类。NEC组的Firmicutes占比为(0.40±0.41),对照组为(0.42±0.33),两组比较差异无统计学意义(P0.05)。NEC组的Proteobacteria占比为(0.52±0.43),对照组为(0.54±0.35),两组比较差异无统计学意义(P0.05)。结论肠道细菌多样性下降可能与NEC的发生密切相关。     

16S rRNA、16S-23S rRNA基因测序分析检测主要血流感染病原菌比较

目的比较细菌16S rRNA、16S-23S rRNA 基因测序分析在血流感染病原菌检测中的作用。方法提取临床上血流感染常见的金黄色葡萄菌、表皮葡萄球菌、大肠埃希菌、粪肠球菌、肺炎链球菌、铜绿假单胞菌、阴沟肠杆菌、鲍曼不动杆菌、洛菲不动杆菌、肺炎克雷伯杆菌、化脓性链球菌、奇异变形杆菌、潘尼变形杆菌、屎肠球菌、粘质沙雷菌、宋内志贺菌、产气肠杆菌、小肠结肠炎耶尔森菌、腐生葡萄球菌基因组 DNA,运用 16S rRNA、16S-23S rRNA 基因进行 PCR 扩增。扩增产物经测序后在美国国家生物技术中心(NCBI)上进行比对分析,确定菌种。结果在所分析的 19 种临床血流感染常见细菌中,16S rRNA 基因测序分析可将除粘质沙雷菌外的细菌鉴定到种的水平,但无法完全区分近缘种属;16S-23SrRNA 成功鉴定 17 种细菌,除大肠埃希菌、宋内志贺菌外所有细菌均成功鉴定到单一种的水平。结论 16S-23S rRNA 基因可作为血流感染细菌检测较好的分子靶标。     

基于高通量测序技术的稽留流产绒毛遗传学分析

目的应用高通量测序技术检测稽留流产绒毛染色体,探讨稽留流产与染色体异常的关系,为患者的下一次妊娠提供临床指导。方法借助高通量测序技术,对111例稽留流产绒毛进行染色体检测,统计染色体异常的类型及所占比例,并将检测结果与临床诊断指征结合,做进一步分析。结果 111例稽留流产绒毛样本检测成功率100. 0%;高通量测序结果显示:111例稽留流产绒毛样本中染色体正常46例(41. 4%);染色体异常65例,异常率为58. 6%。其中以染色体数目异常占比最高,有50例(45. 0%);染色体结构异常15例(13. 5%);初次流产患者组31例,染色体异常率为54. 8%(17/31),两次及两次以上流产患者组73例,染色体异常率为61. 6%(45/73),7例患者信息不详,排除在外。结论染色体异常是孕妇稽留流产的主要危险因素,其中最主要为染色体数目异常;流产次数与稽留流产绒毛染色体异常之间无相关性;高通量测序技术检测绒毛染色体具有高效、快速、准确及可以实现全基因组覆盖等优点,能检测出稽留流产组织的染色体异常具体情况,对孕妇的再次妊娠具有重要的临床指导意义。     

高通量测序技术在病原生物学方面的研究进展

高通量测序技术(high-throughput sequencing),又称为下一代测序技术(next-generation se-quencing,NGS),具有高准确性、高通量、高灵敏度和低运行成本等突出优势。随着高通量测序技术的迅猛发展,科研工作者开始越来越多地应用高通量测序技术来解决各种生物学问题。本文即对高通量测序技术在病原生物学的应用以及研究进展方面作以综述。     

基于高通量测序的输卵管妊娠患者生殖道菌群研究

目的 探讨输卵管妊娠术前后及正常妊娠者生殖道菌群的结构差异,以便了解生殖道菌群对输卵管妊娠及手术治疗的影响。方法 选取输卵管妊娠术前后及正常妊娠者共60例为本次研究对象,其中输卵管妊娠术前为BTP组（n=20）,输卵管妊娠术后为ATP组（n=20）,并选取同期在本院门诊产检的正常宫内早孕（孕6～7周）女性20例作为（EP组）。采用16S rDNA测序分析三组生殖道菌群的多样性和结构差异,并对肠道菌群功能进行预测。结果 三组OTU丰度聚类热图提示,三组研究对象的OTU具有明显的差别,但三组菌群的多样性差异无统计学意义（P>0.05）;三组研究对象生殖道菌群以乳酸杆菌内酯丰度最高,且BTP组丰度最高;其次是脆乳杆菌,以EP组丰度最高;LDA分析结果提示：三组研究对象生殖道菌群的种类不一致,提示输卵管妊娠及手术对研究对象生殖道菌群的种类有一定的影响;龙胆科、杆菌科、麻风科、细脉藻科、普雷沃特拉科等细菌群类与输卵管妊娠关系密切。结论 输卵管妊娠患者输卵管妊娠术前后和正常妊娠者生殖道菌群种类具有明显的差别,表明生殖道菌群对输卵管妊娠能产生重要的影响。     

基于16S rRNA测序的肺癌不同阶段中痰液菌群差异分析

目的 初步评估不同分期肺癌患者痰液菌群分布的差异。方法 收集2019年6月—2021年8月本院肺癌早期、晚期患者及正常对照各20例,取痰液进行16S rRNA测序,分析群落丰富度指数ace和chao,以及群落多样性指数coverage、shannon及simpson,并计算相对丰度。结果 群落多样性指数shannon在各组之间差异均有统计学意义（P<0.01）,群落多样性指数simpson在早期和晚期组均高于对照组,差异有统计学意义（P<0.01）。放线菌门在晚期肺癌组（26.86±13.63）、早期肺癌组（10.42±4.50）及对照组（5.08±3.16）中的相对丰度差异两两之间差异均有统计学意义（P<0.01）;变形菌门在早期肺癌组（22.08±30.25）中的相对丰度高于对照组（7.21±12.08）,差异有统计学意义（P<0.01）;互养菌门在晚期肺癌组（0.33±0.28）、早期肺癌组（0.10±0.27）和对照组（0.06±0.09）中的相对丰度差异均有统计学意义（P<0.01）;蓝细菌门在晚期肺癌组（0.17±0.57）、早期肺癌组（0.0...     

基于16S rRNA测序分析CRE定植对ICU患者肠道微生物的影响

目的 采用16S rRNA测序方法分析耐碳青霉烯类肠杆菌(CRE)定植对重症监护病房(ICU)患者肠道微生物的影响。 方法 收集ICU患者新鲜粪便或肛拭子标本, 接种于含有2 μg/mL厄他培南的MacConkey平板上培养。采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)对菌株进行鉴定, 纸片扩散法对菌株进行药敏分析, 采用聚合酶链式反应(PCR)对初步筛查出的CRE菌株进行16S rDNA片段扩增, 采用微量肉汤稀释法进一步确定CRE。最后, 分别提取CRE定植组与非定植组患者的粪便或肛拭子标本基因组DNA在HiSeq平台进行16S rRNA测序分析。 结果 共收集ICU患者粪便或肛拭子标本241份, 其中CRE阳性标本17份。16S扩增子V3~V4区测序共分离到726 OTUs, CRE未定植组有631 OTUs, CRE定植组有480 OTUs, 两组间共有385 OTUs。这些共有OTUs绝大多数属于厚壁菌门、变形菌门、拟杆菌门、疣微菌门及放线菌门。在纲水平上, CRE定植前后γ-变形菌纲(W=193.000, P＜0.001, FDR=0.004)和互营养菌(W=37.500, P=0.001, FDR=0.018)相对丰度差异有统计学意义。在种水平上, 两组间肺炎克雷伯菌(W=195.000, P＜0.001)、解糖肠球菌(W=153.000, P=0.038)及弯曲杆菌(W=63.500, P=0.050)比较差异有统计学意义。未发现肠道CRE定植对ICU患者肠道微生物KEGG功能通路有显著影响。 结论 CRE定植会降低ICU患者肠道微生物的多样性, CRE定植组在肺炎克雷伯菌、解糖肠球菌及弯曲杆菌的丰度上与非定植组存在显著差异。     

Habitual Diet Pattern Associations with Gut Microbiome Diversity and Composition: Results from a Chinese Adult Cohort

The influence of long-term diet on gut microbiota is an active area of investigation. The present work aimed to explore the associations between habitual diet patterns and gut microbiota in a large sample of asymptomatic Chinese adults. The gut microbiome was profiled through the sequencing of the 16S rRNA gene in stool samples from 702 Chinese adults aged 50–75 years who underwent colonoscopies and were diagnosed to be free of colorectal neoplasm. Long-term dietary consumption was assessed through a food-frequency questionnaire. The microbial associations with specific food groups and the posteriori dietary pattern were tested using the Kruskal–Wallis H test, permutational ANOVAs, and multivariate analyses with linear models. The Shannon indexes generally shared similar levels across different food intake frequency groups. Whole grain and vegetable intakes totally explained 1.46% of the microbiota compositional variance. Using the data-driven posteriori approach, a general dietary pattern characterized by lower intakes of refined grains was highlighted to be associated with higher abundances of the genus Anaerostipes and a species of it. We also observed 17 associations between various food group intakes and specific genera and species. For instance, the relative abundances of the genus Weissella and an uncultured species of it were negatively associated with red meat intake. The results of this study support the idea that the usual dietary consumption measured by certain food items or summary indexes is associated with gut microbial features. These results deepen the understanding of complex relationships of diet and gut microbiota, as well as their implications for gut microbiome studies of human chronic diseases.     

Rice- or pork-based diets with similar calorie and content result in different rat gut microbiota

Rice is the most important food crop, and pork is the most widely eaten meat in the world. In this study, we compared the gut microbiota of the rats fed with rice or pork mixed diets, which have similar caloric contents. The physiological indices (body weights, hematology, serum chemistry, organ weights and histopathology) of two groups were all within the normal range. Two diets did not induce difference in the diversity of gut bacteria. However, Firmicutes were significantly higher in rice diet group, while Bacteroidetes were enriched in pork diet group. Butyrate and the bacteria enzymes β-glucuronidase, β-glucosidase and nitroreductase in the feces were all drastically higher in pork diet group. This study indicates that different diets with similar calorie and nutritional composition could change the community structure but not the diversity of rat fecal microbiota.     

5687例高通量基因测序产前筛查指征及结果分析

目的 探讨孕期开展高通量基因测序产前筛查技术的临床应用价值.方法 回顾性分析在甘肃省妇幼保健院就诊的自愿接受高通量基因测序产前筛查单胎妊娠孕妇5687例,采用高通量测序平台对孕妇外周血游离胎儿DNA进行测序分析,对测序结果进行生物信息分析,提示高风险及性染色体异常者,进一步行羊膜腔穿刺进行胎儿染色体核型分析.结果 5687例孕妇中游离胎儿DNA高通量基因测序技术检出37例21-三体高风险,5例18-三体高风险,2例13-三体高风险,性染色体异常30例.羊水染色体核型分析证实其中30例胎儿21-三体,3例18-三体,性染色体异常12例,高通量基因测序技术对于21-三体阳性预测值可达97%.结论 高龄孕妇是胎儿染色体异常的高危人群.高通量基因测序技术是胎儿21-三体的有效产前筛查方法,它具有无创、准确的特点,且筛查结果不受孕周限制,具有较高的临床应用价值.     

Diet Is a Stronger Covariate than Exercise in Determining Gut Microbial Richness and Diversity

Obesity is a common metabolic disorder caused by a sedentary lifestyle, and a high-fat and a high-glucose diet in the form of fast foods. High-fat diet-induced obesity is a major cause of diabetes and cardiovascular diseases, whereas exercise and physical activity can ameliorate these disorders. Moreover, exercise and the gut microbiota are known to be interconnected, since exercise can increase the gut microbial diversity and contribute to the beneficial health effects. In this context, we analyzed the effect of diet and exercise on the gut microbiota of mice, by next-generation sequencing of the bacterial V4 region of 16S rRNA. Briefly, mice were divided into four groups: chow-diet (CD), high-fat diet (HFD), high-fat diet + exercise (HFX), and exercise-only (EX). The mice underwent treadmill exercise and diet intervention for 8 weeks, followed by the collection of their feces and DNA extraction for sequencing. The data were analyzed using the QIIME 2 bioinformatics platform and R software to assess their gut microbial composition, richness, and diversity. The Bacteroidetes to Firmicutes ratio was found to be decreased manifold in the HFD and HFX groups compared to the CD and EX groups. The gut microbial richness was comparatively lower in the HFD and HFX groups and higher in the CD and EX groups (ACE, Chao1, and observed OTUs). However, the Shannon alpha diversity index was higher in the HFD and HFX groups than in the CD and EX groups. The beta diversity based on Jaccard, Bray–Curtis, and weighted UniFrac distance metrics was significant among the groups, as measured by PERMANOVA. Paraprevotella, Desulfovibrio, and Lactococcus were the differentially abundant/present genera based on the intervention groups and in addition to these three bacteria, Butyricimonas and Desulfovibrio C21c20 were differentially abundant/present based on diet. Hence, diet significantly contributed to the majority of the changes in the gut microbiota.     

Metagenomic Changes of Gut Microbiota following Treatment of Helicobacter pylori Infection with a Simplified Low-Dose Quadruple Therapy with Bismuth or Lactobacillus reuteri

Background: Probiotic supplementation to antibiotic regimens against Helicobacter pylori infection has been proposed to improve eradication rate and to decrease detrimental effects on gut microbiota. Aims: To evaluate microbiota modifications due to a low-dose quadruple therapy with bismuth or Lactobacillus reuteri. Methods: Forty-six patients infected with H. pylori were prospectively enrolled in a single-centre, randomized controlled trial to receive b.i.d. with meals for 10 days low-dose quadruple therapy consisting of rabeprazole 20 mg and bismuth (two capsules of Pylera^® plus 250 mg each of tetracycline and metronidazole), or the same dose of rabeprazole and antibiotics plus Gastrus^® (L. reuteri), one tablet twice-a-day for 27 days. Stool samples were collected at the enrolment, at the end and 30–40 days after the treatment. Gut microbiota composition was investigated with 16S rRNA gene sequencing. Results: Eradication rate was by ITT 78% in both groups, and by PP analysis 85.7% and 95.5% for Gastrus^® and bismuth group, respectively. Alpha and beta diversity decreased at the end of treatment and was associated with a reduction of bacterial genera beneficial for gut homeostasis, which was rescued 30–40 days later in both groups, suggesting a similar impact of the two regimens in challenging bacterial community complexity. Conclusions: Low-dose bismuth quadruple therapy proved to be effective with lower costs and amount of antibiotics and bismuth. Gastrus^® might be an option for patients with contraindications to bismuth. L. reuteri was unable to significantly counteract dysbiosis induced by antibiotics. How to administer probiotics to prevent gut microbiota alterations remains an open question.     

Towards dynamic monitoring of cell cultures using high throughput sequencing

We used a combination of DOP-PCR with high throughput sequencing (HTS) to study infected cell cultures over time to assess the feasibility of using this technique to provide a read-out other than cytopathic effect in cell culture infectivity assays. Because DOP-PCR primers feature a short constant sequence at their 3′ terminus, the procedure yields a reproducible representational library of products from a given PCR template, including viral nucleic acids. Using SV40- and MVM-infected cultures harvested at different times, we show that the number of viral matches among DOP-PCR products parallels the quantity of virus as shown by real-time PCR, and further show that HTS analysis of specific DOP-PCR products that increase in quantity over time could be used to identify the infecting virus with a sensitivity similar to that of typical cell-culture assays that rely on cytopathic effect.     

Association of Gut Microbiota with Atherogenic Dyslipidemia,and Its Impact on Serum Lipid Levels after Bariatric Surgery

Gut microbiota has been suggested to modulate circulating lipids. However, the relationship between the gut microbiota and atherogenic dyslipidemia (AD), defined as the presence of both low HDL-C and hypertriglyceridemia, is not fully understood. Moreover, because obesity is among the main causes of secondary AD, it is important to analyze the effect of gut microbiota composition on lipid profiles after a weight loss intervention. We compared the microbial diversity and taxonomic composition in patients with AD (n = 41) and controls (n = 38) and sought correlations of genera abundance with serum lipid levels in 20 patients after weight loss induced by Roux-en-Y gastric bypass (RYGB) surgery. Gut microbiota composition was profiled using next-generation sequencing of 16S rRNA. Gut microbiota diversity was significantly lower in atherogenic dyslipidemia. Moreover, relative abundance of two genera with LDA score >3.5 (Megasphaera and LPS-producing Escherichia-Shigella), was significantly higher in AD subjects, while the abundance of four short chain fatty acids (SCFA) producing-genera (Christensenellaceae R-7, Ruminococcaceae UCG-014; Akkermansia and [Eubacterium] eligens group) was significantly higher in controls. Notably, [Eubacterium] eligens group abundance was also significantly associated with higher HDL-C levels in RYGB patients one year after surgery. Although dietary polyunsaturated fatty acid/saturated fatty acid (PUFA/SFA) ratio and PUFA intake were higher in controls than in AD subjects, of the four genera differentiated in cases and controls, only Akkermansia abundance showed a positive and significant correlation with PUFA/SFA ratio. Our results suggest that SCFA-producing bacteria promote a healthy lipid homeostasis, while the presence of LPS-producing bacteria such Escherichia-Shigella may contribute to the development of atherogenic dyslipidemia.     

1型糖尿病儿童肠道菌群谱分析研究

目的 通过16SrRNA基因测序方法探索1型糖尿病(T1DM)儿童肠道菌群结构变化的特征,并比较T1DM儿童与健康儿童间肠道菌群的差异,为临床应用益生菌进行TIDM早期干预提供理论基础和实验依据。方法 选取2018年10月-2019年10月在昆明市儿童医院内分泌遗传代谢科住院并初诊为T1DM的5~14岁儿童18例,同时选取19例性别、年龄相近的健康儿童为研究对象,收集两组儿童粪便标本后进行16SrRNA基因测序实验,并采用QIIME 2分析流程进行生物信息学分析,比较两组间肠道菌群的差异。结果 1)利用QIIME2软件将相似度100%的序列聚类分析后共获得3 248个Feature数;2)经物种鉴定及注释,绝大部分菌群都分类到属级和种级;3)Alpha多样性分析说明本次研究测序深度充分,并且T1DM儿童肠道菌群的丰富度及多样性较健康儿童降低 (P<0.05);4)Beta多样性分析中,PCoA图说明T1DM儿童和健康儿童间肠道菌群结构存在明显差异;5)肠道菌群组成差异分析中,在门水平上,T1DM儿童放线菌门、拟杆菌门和蓝细菌门丰度显著增高,而变形菌门、杆菌门的丰度降低(P<0.05);在属水平上,T1DM儿童粪杆菌属、双歧杆菌属和拟杆菌属的丰度较健康儿童增高,而埃希氏杆菌属-志贺氏杆菌、肠球菌属、Blautia菌属丰度降低(P<0.05)。结论 T1DM儿童存在肠道菌群生态失衡,并且菌群物种的丰富度及多样性降低。此外,T1DM儿童与健康儿童肠道菌群结构分布存在明显差异。