16S rRNA Gene-Based Metagenomic Analysis of Soil Bacterial Diversity in Brazzaville, Republic of the Congo

Soil contains a great diversity of microorganisms, among which are bacteria. This study aimed to explore bacterial diversity in soil samples in Brazzaville in the Republic of the Congo. Environmental DNA was extracted. The illumina MiSeq sequencing was held and the diversity indices have been computed. Illumina MiSeq sequencing revealed 21 Phyla, four of which were abundant: Proteobacteria, Acidobacteria, Actinobacteria and Bacteroidetes. Soil microbial communities in the studied samples were phylogenetically diverse but with a stable community structure. 17 classes are represented with relative abundances of Rihzobiales, Bacillales, Actinomycetales and Acidobacteriales. 40 families, the Alphaproteobacteria, the Bacilli and the 12 Actinobacteria. 83 orders among which the Rhizobiales are the most abundant followed by Bacillales and the least abundant followed by the Flavobacteriaceae. Of the 28 genera listed, the Bradyrhizobium is the most dominant in Mw3 and Mw4. 25 listed species, Bradyrhizobium, Bacillus, Actinoplanes, and Candidatu coribacter Acidobacterium are the most abundant species. The Shannon indices of Mw3 and Mw4 are equal, the H’max of Mw4 is greater than the H’max of Mw3. The Simpson index of Mw4 is equal to the Simpson index of Mw3, and the Pielou index (J) of Mw4 is less than the R of Mw3, but very close. This study opens interesting perspectives on the knowledge and exploitation of telluric bacteria in several areas of life.

Identification of Bacterial Fish Pathogens in Brazil by Direct Colony PCR and 16S rRNA Gene Sequencing

Intensive fish farming systems in Brazil have increased the disease incidence, mainly of bacterial origin, due to higher stocking density, high organic matter levels and poor quality of the aquatic environment that causes high mortality rates during outbreaks. The identification of pathogenic species using a fast and reliable method of diagnosis is essential for successful epidemiological studies and disease control. The present study evaluated the use of direct colony PCR in combination with 16S rRNA gene sequencing to diagnose fish bacterial diseases, with the goal of reducing the costs and time necessary for bacterial identification. The method was successful for all 178 isolates tested and produced bands with the same intensity as the standard PCR performed using pure DNA. In conclusion, the genetics methods allowed detecting the most common and important pathogens in Aquaculture, including 12 species of occurrence in Brazilian fish farms. The results of the present study constitute an advance in the available diagnostic methods for bacterial pathogens in fish farms.

16S-rRNA测序技术分析早产儿肠道细菌基因组指导新生儿坏死性小肠结肠炎手术时机选择的研究

目的探讨16S-rRNA测序技术分析早产儿肠道细菌基因组指导新生儿坏死性小肠结肠炎(NEC)手术时机选择。方法前瞻性选择2021年1月至2022年6月百色市人民医院需要手术治疗的NEC患儿30例为观察组,选择同期内科保守治疗的Ⅰ期15例和Ⅱa期15例患者为对照组。采用HiSeq测序平台,借助双端测序模式进行高通量二代测序,比较两组多样性指数、优势均属丰度及不同优势菌比值等;绘制受试者操作特征(ROC)曲线,分析16S-rRNA测序技术的指导价值。结果60例患者60份样本中共获得细菌84个,且两组样品均为副杆状菌属最高,其次为Ruminococcus、Blautia、Aeromonas和Fusobacterium;两组肠道菌群上述菌门丰度存在差异(P<0.05);从粪便标本中共获得有效序列7347481条,人均130857条,测序平均覆盖度为(92.15±5.61)%;观察组手术治疗的NEC患儿中香农-维纳(Shannon)及辛普森多样性(Simpson)指数低于对照组内科保守治疗患儿,差异有统计学意义(P<0.05);ROC曲线结果表明,16S-rRNA测序技术在NEC患儿手术时机选择中的指导AUC为0.846,指导灵敏度为87.51%,特异度为83.16%。结论NEC患儿常伴有肠道细菌基因组改变,且菌群结构的变化与患儿病情严重程度有关,通过16S-rRNA测序技术能指导NEC患儿手术治疗时机。

Application of 16s rDNA Sequencing in the Analysis of Pathogenic Bacteria in Sputum of Severe Bacterial Pneumonia

Objective: 120 patients with severe pneumonia who were kept in the comprehensive ICU of our hospital in 2018 were selected, and 16s rDNA sequencing was performed to analyze the composition of pathogenic bacteria in the sputum of severe pneumonia. Methods: The sputum samples of patients with severe bacterial pneumonia were collected, and the diversity of pathogens in the samples was analyzed by polymerase chain reaction (PCR) amplification and high-throughput sequencing (16s rDNA PCR-DGGE). Results: Sequence showed that sputum samples contained a relatively large number of species, and there were many species that were not detected by sequencing. The dominant bacteria were Streptococcus, Sphingomonas, Corynebacterium, Denatobacteria, Aquobacteria, Acinetobacteria, Prevotella, Klebsiella, Pseudomonas, etc. Conclusion: Bacteria caused by sputum of severe bacterial pneumonia are complex and diverse, which provides new methods and ideas for individualized treatment of patients with severe pneumonia.

High-throughput sequencing of 16S r RNA amplicons characterizes gut microbiota shift of juvenile sea cucumber Apostichopus japonicus feeding with three antibiotics

Sea cucumber Apostichopus japonicus is an important marine economic species in Asian countries due to its profound nutritional and medicinal value. So far, with the rapid development of intensifi ed artifi cial aquaculture of sea cucumbers, the use of antibiotics is still an inexpensive and dispensable way to treat pathogenic infections, especially during the nursery phase. However, there is little information on the eff ects of antibiotics on the intestinal microbiota of sea cucumber. Therefore an Illumina based sequencing method was used to examine the intestinal bacterial composition of juvenile A . japonicas following diets with three typical antibiotics (tetracycline, erythromycin, and norfl oxacin) under 15, 30, and 45 d. The fi ndings reveal that diff erent antibiotics have distinct eff ects on the growth performance of juvenile sea cucumbers. However, the richness and diversity of microbiota were barely aff ected by antibiotics but the community composition alterations indicated that the three antibiotics exhibited their respective patterns of reshaping the intestinal bacteria of juvenile sea cucumbers. In common, the abundance of some sensitive genera with helpful functions, such as Thalassotalea , Shewanella , Sulfi tobacter , and Halomonas decreased signifi cantly with exposure to antibiotics and the abundance of multiple potential pathogenic- and suspected antibiotic-resistant microorganisms like Arcobacter , Leucothrix , and Clostridium_sensu_stricto_1 was found increased signifi cantly in the antibiotic groups. These results suggest that low doses of antibiotics could aff ect the composition of the intestinal microbiota of sea cucumbers and might increase the risk of infection of the hosts. This study could help us to explore how antibacterial compounds modify the gut microbiota of sea cucumbers and provide theoretical guidance in hatchery management by scientifi c antibiotic use in sea cucumber mariculture.

Fecal microbiota of three bactrian camels(Camelus ferus and Camelus bactrianus) in China by high throughput sequencing of the V3-V4 region of the 16S rRNA gene

This study aimed to reveal the microbial diversity in the fecal samples of bactrian camels using the 16 S r RNA sequencing analysis on the Illumina Mi Seq platform. Three fecal samples were collected from two geographical regions in China. Operational taxonomic unit（OTU） clustering was performed by identifying an OTU at 97% sequence identity. The alpha and beta diversities were applied to estimate the differences in microbial diversity among the three fecal samples. Totally, 4409, 3151 and 4075 OTUs in the fecal samples were identified in the Lop Nor wild camel（Camelus ferus）, the domestic camel（C. bactrianus） and Dunhuang wild camel（C. ferus）, respectively. The majority of bactreria were affiliated with phylum Firmicutes and Bacteroidetes in the three samples. The wild camels had higher gastrointestinal tract microbial diversity than the domestic one, while the microbial composition of the Lop Nor wild camel shared higher similarity with domestic camel at the genus and family levels than that of the Dunhuang wild camel did. Our results may provide a theoretical basis for assessing their health conditions and may thus be useful for protecting the critically endangered species of C. ferus.

Characterization of root-associated bacterial community structures in soybean and corn using locked nucleic acid(LNA) oligonucleotide-PCR clamping and 454 pyrosequencing

supported in part by grants from the Strategic Priority Research Program of Chinese Academy of Sciences （XDB15010103）;the National Natural Science Foundation of China （41201247）

Correlation analysis of breast fibroadenoma and the intestinal flora based on 16S rRNA sequencing

Objective To analyze the characteristics of the intestinal microflora in patients with breast fibroadenoma using 16S ribosomal RNA(rRNA)high-throughput sequencing.Methods Fecal samples from 20 patients with breast fibroadenoma and 36 healthy subjects were randomly collected and analyzed using high-throughput sequencing technology for 16S rRNA V4 region sequencing,and the alpha diversity(Chao index,Shannon index)was calculated using Mothur(v.1.39.5)software.Beta diversity was analyzed using QIIME(v1.80).SPSS software(version 23.0)and the t-test of two independent samples were used to analyze differences in the abundance of bacteria between the two groups.Results Compared with that in the healthy control group,theαdiversity of the intestinal microflora in breast fibroadenoma patients increased,but the difference was not statistically significant(P>0.05).At the phylum level,significant differences were observed between the two groups.The abundance of Firmicutes was higher in the breast fibroadenoma group(P<0.05),whereas the abundance of Synergistetes was higher in the healthy control group(P<0.005).A total of five bacterial genera showed significant differences between the two groups:the breast fibroadenoma group showed higher levels of Bautia(P<0.005),Coprococcus(P<0.005),Roseburia(P<0.05),and Ruminococcus(P<0.005),whereas Sutterella was more abundant in the healthy control group than in the breast fibroadenoma group(P<0.05).Conclusion The diversity and abundance of the intestinal flora in patients with breast fibroadenoma are significantly different from those in healthy subjects,suggesting that an imbalance in the intestinal flora is correlated with the occurrence of breast fibroadenoma.

Evaluation of Real-Time 16S rDNA PCR and Pyrosequencing for Routine Identification of Bacteria in Joint Fluid and Tissue Specimens

16S rDNA PCR and sequencing are powerful tools for bacterial detection and identification, although their routine use is not currently widespread in the field of clinical microbiology. The availability of pyrosequencing now makes 16S rDNA assays more accessible to routine diagnostic laboratories, but this approach has had limited evaluation in general diagnostic practice. In this study we evaluated a real-time 16S rDNA PCR and pyrosequencing assay for use in a routine microbiology laboratory, by retrospectively testing joint fluid and joint tissue specimens received for conventional culture. We found that use of the real-time 16S rDNA assay was clinically valuable in this specimen type because it enabled us to identify a small number of culture-negative infections. Although faster and less labour-intensive, we found that the utility of pyrosequencing for pathogen identification is still hampered by shorter read lengths compared to conventional (Sanger) sequencing. Combining results from both molecular and conventional culture methods, bacteria were only detected in 11.8% specimens in this study. However, the detection rate was increased to 18.6% if specimens were only included from patients with a documented clinical suspicion of infection. In conclusion, while pyrosequencing had significant advantages in speed and ease-of-use over conventional sequencing, multiple reactions will be required to deliver comparable species-level identification, thus negating many of the benefits of using the technique. We found that 16S rDNA PCR and sequencing should be rationally targeted on the basis of good clinical information in the routine diagnostic setting, and not used as a general screening test for the exclusion of bacterial infection in joint specimens.

Application of 16S rDNA Sequencing Technology in the Analysis of Pathogenic Bacteria in Sputum of Severe Pneumonia

The diagnosis of pathogenic bacteria in severe pneumonia is difficult and the prognosis is poor. Its outcome is closely related to bacterial pathogenicity and the timeliness and pertinence of antibiotic treatment. Therefore, early diagnosis is of great significance to the prognosis of patients. Sputum examination and culture is the gold standard for the diagnosis of pathogens of severe pneumonia. However, due to the long time of bacterial culture, the early use of antibiotics, the change of bacteria species, mixed infection and other problems, the results of bacterial culture in sputum are often false negative. With the continuous application of new molecular biology techniques in clinical detection, the classification of bacteria and microorganisms has deepened from the identification of phenotypic characteristics to the classification of gene characteristics. Sequencing analysis with 16S rDNA sequencing technology has the characteristics of high sequencing flux, large amount of data obtained, short cycle, and can more comprehensively reflect the species composition of microbial community, real species distribution and abundance information. In this paper, 16S rDNA sequencing technology was used to analyze the bacterial population composition in the sputum of severe pneumonia, and to explore a new method of etiological diagnosis.

RETRACTED:Effect of Long-Term Inorganic Fertilization on Diversity and Abundance of Bacterial and Archaeal Communities at Tillage in Irrigated Rice Field

Short Retraction Notice The paper does not meet the standards of "Advances in Bioscience and Biotechnology". This article has been retracted to straighten the academic record. In making this decision the Editorial Board follows COPE's Retraction Guidelines. The aim is to promote the circulation of scientific research by offering an ideal research publication platform with due consideration of internationally accepted standards on publication ethics. The Editorial Board would like to extend its sincere apologies for any inconvenience this retraction may have caused. Editor guiding this retraction: Prof. Abass Alavi (EiC of ABB). Please see the article page for more details. The full retraction notice in PDF is preceding the original paper which is marked "RETRACTED".

Relationship between hyperlipidemia and the gut microbiome of rats, characterized using high-throughput sequencing

Objective:To determine the effects of a high-fat diet(HFD)on the gut microbiome in rats,to explore the relationship between the intestinal flora and blood lipid profile.Methods:SpragueeDawley rats were fed an HFD for four weeks to induce hyperlipidemia,then 16S rRNA sequencing was used to compare the intestinal flora between hyperlipidemic and control diet-fed rats.Results:The microbiome of rats fed an HFD for four weeks differed from that of control diet-fed rats.Bacterial species that were less abundant were most affected by HFD feeding,among which were many pathogenic species,which became significantly more abundant.Eighteen genera were present in significantly different numbers in hyperlipidemic and control rats,more than half of which have been linked to infection and inflammation,or energy intake and obesity.The results indicated a type of stress response of the flora to a high-fat environment.In addition,the age of the rats tended to influence the gut microbial composition.Conclusion:These findings suggest that HFD may induce hyperlipidemia by affecting the gut microbial composition.Changes in the abundance of pro-inflammatory and pathogenic bacteria,and those that influence energy intake and obesity,may be important mediators of this.

16S rRNA基因克隆文库用于菌群分析的效能研究和评价

目的建立16S rRNA基因克隆文库进行菌群相对定量的方法,评价其对细菌的检测效率以及对混合菌群中低含量细菌的分析能力。方法取脆弱类杆菌等9种细菌以相同及级差比例制备细菌悬液,分别用或不用变溶菌素处理后,用试剂盒提取核酸,16S rRNA基因通用引物进行PCR扩增、纯化、连接、克隆和测序,建立16S rRNA基因克隆文库,将序列与数据库进行比对分析。结果相同比例制备的菌悬液,加或不加变溶菌素提取的核酸,掺入的9种细菌均可检出。级差比例制备的菌悬液,加变溶菌素提取核酸,掺入的9种细菌均可检出;不加变溶菌素提取核酸,数量少难裂解的革兰阳性菌双歧杆菌未能检出。混合菌悬液中各种细菌的比例与文库反映的各种细菌的比例有数量对应关系,但不是线性对应关系。结论16S rRNA基因序列分析是一种较好的细菌菌群分析方法,它可以同时检出多种细菌,并能对混合细菌标本进行菌群相对定量,反映菌群中各种细菌的丰度,但不能准确定量菌群中各种细菌的数量。

基于16S rRNA基因序列分析家蚕肠道细菌的多样性

家蚕肠道菌群与蚕体对营养物质的消化吸收及健康性密切相关。为了解家蚕肠道细菌的多样性及雌、雄个体间肠道细菌类群的差异,通过应用454焦磷酸测序技术检测细菌16S rRNA基因序列的方法分析家蚕5龄第3天雌、雄幼虫中肠内容物中的细菌类群,共发现5 578个分类操作单元(operational taxonomic units),包括14个门、21个纲、30个目、71个科、120个属的细菌,雌、雄个体在上述分类阶元共有的肠道细菌菌群分别为8、9、20、38和46种。在属水平上对细菌菌群构成的分析显示,雄蚕肠道中的主要优势细菌为代尔夫特菌属Delftia、橙单胞菌属Aurantimonas、肠球菌属Enterococcus、嗜糖假单胞菌属Pelomonas、青枯菌属Ralstonia、台湾温单胞菌属Tepidimonas、假单胞菌属Pseudomonas、阿斯普罗单胞菌属Aspromonas、甲基杆菌属Methylobacterium和葡萄球菌属Staphylococcus;雌蚕肠道中的主要优势细菌为代尔夫特菌属Delftia、橙单胞菌属Aurantimonas、假单胞菌属Pseudomonas、嗜糖假单胞菌属Pelomonas、佩特罗菌属Petrobacter、肠球菌属Enterococcus、台湾温单胞菌属Tepidimonas、狭义的梭菌属Clostridium sensu stricto、阿斯普罗单胞菌属Aspromonas。其中,雄蚕肠道中有23个属的细菌丰度是雌蚕的1.5倍以上,在雌蚕肠道中有7个属的细菌丰度是雄蚕的1.5倍以上,表明雌性与雄性家蚕肠道细菌类群的组成和比率存在明显差异。基于16S rRNA基因序列的聚类分析显示,家蚕肠道中的优势细菌属可分为2个大类。家蚕肠道细菌的多样性研究结果可作为探讨雌蚕和雄蚕经济性状差异的新线索。

中国对虾16SrRNA基因序列多态性的研究

利用PCR技术扩增获得中国对虾 (Penaeuschinensis)线粒体DNA的 16SrRNA基因片段 ,通过测定该基因片段的序列 ,分析了取自我国烟台、长岛、青岛近海和宁波养殖的 17只中国对虾遗传多态性。结果发现 ,不同地理种群存在丰富的DNA序列多态性 ,17只个体具有 17种基因型 ,在扩增的长为 5 2 3bp的基因片段中 ,共检测到 3 7个多态性核苷酸位点 ( 7 0 7% )。UPGMA法构建的分子系统树表明不同地理种群中国对虾存在一定程度的遗传分化 ,长岛群体与烟台群体遗传关系较近 ,宁波群体次之 ,青岛群体为相对独立的一支。

福建华溪蟹线粒体DNA COI和16S rRNA基因序列的遗传多样性

目的探讨福建华溪蟹(Sinopotamon fukienense)的遗传多样性。方法采用PCR结合DNA测序技术,测定S.fukienense的线粒体COI和16SrRNA基因序列的组成。经比对获得639bp长度的COI基因序列和526bp的16SrRNA基因序列,以对比分析S.fukienense的遗传多样性。结果 S.fukienense基于COI基因核苷酸多样性(Pi)为0.048 4,高于其基于16SrRNA基因核苷酸多样性(Pi)为0.021 6。同时,S.fukienense基于COI基因单倍型间的平均遗传距离(P)为0.048,大于其基于16SrRNA基因单倍型间的平均遗传距离(P)0.026。结论 COI序列在分析S.fukienense遗传异变时的作用更优于16SrRNA基因序列。

5种经济海胆线粒体16S rRNA基因片段的序列分析

对光棘球海胆、中间球海胆、马粪海胆,海刺猬和紫海胆线粒体DNA 16SrRNA基因片段进行了PCR扩增和测序,得到395 bp的可信片段。通过ClustalX 1.83和Mega 3.0软件对所得线粒体16SrRNA片段序列进行比较,共检测到118个碱基变异,其中简约信息位点为33个。应用Mega 3.0计算各种间的相对遗传距离,以糙海参为外类群,得到NJ系统树和MP系统树,系统树各分支的置信度由“Bootstrap”1000次循环检验。结果表明,马粪海胆与光棘球海胆的亲缘关系较近,其次为中间球海胆,而紫海胆、海刺猬与这3种海胆的关系相对较远。

反相高效液相色谱法和16S rRNA序列分析法对分枝杆菌分型鉴别的比较研究

目的比较反相高效液相色谱法(RP-HPLC)和16S rRNA序列分析法对分枝杆菌分型鉴别的异同。方法将《伯杰细菌鉴定手册》中载入的49种分枝杆菌模式株接种于改良罗氏培养基上,置于最适温度下孵育。挑取生长良好且无污染的培养物,一部分经皂化和酸化提取分枝菌酸并衍生后,采用反相高效液相色谱法进行分枝菌酸指纹图谱构建;另一部分经裂解和PCR扩增,获得DNA,纯化后,采用核酸分析仪进行16S rRNA序列测定。结果 49种分枝杆菌模式株中,采用RP-HPLC分析时,单簇峰的结核分枝杆菌、牛分枝杆菌和胃分枝杆菌,双簇峰的爱知分枝杆菌和罗德岛分枝杆菌,三簇峰的南非分枝杆菌和母牛分枝杆菌共7种因各组内相对保留时间和相对峰高比值相近而难以进行鉴别;采用16S rRNA序列分析法分析时,产鼻疽分枝杆菌和塞内加尔分枝杆菌、溃疡分枝杆菌和海分枝杆菌、堪萨斯分枝杆菌和胃分枝杆菌、龟亚分枝杆菌和龟脓分枝杆菌以及结核分枝杆菌复合群(结核分枝杆菌、牛分枝杆菌、田鼠分枝杆菌和非洲分枝杆菌)共12种因各组内基因序列相似性百分比为100%而难以进行鉴别。通过两种分型鉴别方法的比较,可见除结核分枝杆菌和牛分枝杆菌外,两种分型方法相互补充,可将49种分枝杆菌模式株中的47种进行明确鉴别。结论分枝菌酸RP-HPLC和16S rRNA序列分析法均为分枝杆菌的分型鉴定提供了准确和有效的技术方法。两种方法相互借鉴能准确地将大多数分枝杆菌鉴定到种。

家兔胚胎胃肠道细菌分离及16S rRNA基因序列分析

为研究家兔胚胎胃肠道细菌菌群的种类,对常规饲养、正常妊娠的家兔胚胎后期胃肠道细菌进行分离,基于分离细菌的生理生化特性、16S rRNA基因序列的同源性比较及系统发育分析,初步确定家兔胚胎后期胃肠道中存在的微生物菌群有微球菌属(Micrococcus)、葡萄球菌属(Staphylococcus)、棒杆菌属(Corynebacterium)以及蜡样芽孢杆菌(Bacillus cereus)等细菌,其序列同源性均为97%～99%。分离细菌均为G+细菌,家兔胚胎胃肠道中细菌的平均数量达到0.8×102cfu/g～1.2×102cfu/g,本研究为哺乳动物胚胎时期存在于胃肠道中细菌的深入研究奠定了基础。

基于不同引物的16S rRNA序列分析法对SBP腹水细菌的鉴定及抗感染疗效评价的意义

目的:探讨联合单独或联合应用2对不同引物的16S r RNA序列分析法,能否提高自发性细菌性腹膜炎(spontaneous bacterial peritonitis,SBP)腹水细菌检测的敏感性及准确性,并且通过其检测细菌的结果来评价抗感染治疗效果.方法:收集77例肝硬化失代偿期合并腹水的标本,其中SBP 61例,非SBP 16例,应用2对不同引物(MSQ-F/MSQ-R与27F/1492R)的组合,对腹水细菌分别进行16S r RNA序列分析,对腹水进行常规化验、细菌培养及生化方法鉴定细菌,分析单独或联合应用2对不同引物的16S r RNA序列分析法在鉴定SBP腹水细菌中的应用价值,并对腹水细菌状态进行动态评价.结果:与腹水细菌培养、生化鉴定的细菌结果相比,16S r RNA序列分析法鉴定细菌的结果与之的符合率:单用引物MSQ-F/M S Q-R:属的符合率73.33%(11/15),种的符合率66.67%(10/15);单用27F/1492R引物:属的符合率80.00%(12/15),种的符合率73.3%(11/15);引物MSQ-F/MSQ-R联合27F/1492R:属的符合率86.67%(13/15),种的鉴定的符合率80.00%(12/15),应用2对引物的组合进行16S r RNA序列分析鉴定细菌,可以得到更高的准确率.5例SBP患者(腹水细菌培养阳性组2例,临床感染组3例)经抗感染治疗后,腹水细菌培养阴性,但临床感染控制不理想的患者,16S r RNA序列分析法检测腹水细菌仍为阳性.结论:联合应用2对不同引物的16S r RNA序列分析法,可以提高SBP腹水细菌的鉴定能力,并且可以动态应用于临床进行抗感染疗效评价.