Association between childhood obesity and gut microbiota:16S rRNA gene sequencing-based cohort study

BACKGROUND This study aimed to identify characteristic gut genera in obese and normal-weight children(8-12 years old)using 16S rDNA sequencing.The research aimed to provide insights for mechanistic studies and prevention strategies for childhood obesity.Thirty normal-weight and thirty age-and sex-matched obese children were included.Questionnaires and body measurements were collected,and fecal samples underwent 16S rDNA sequencing.Significant differences in body mass index(BMI)and body-fat percentage were observed between the groups.Analysis of gut microbiota diversity revealed lowerα-diversity in obese children.Differences in gut microbiota composition were found between the two groups.Prevotella and Firmicutes were more abundant in the obese group,while Bacteroides and Sanguibacteroides were more prevalent in the control group.AIM To identify the characteristic gut genera in obese and normal-weight children(8-12-year-old)using 16S rDNA sequencing,and provide a basis for subsequent mechanistic studies and prevention strategies for childhood obesity.METHODS Thirty each normal-weight,1:1 matched for age and sex,and obese children,with an obese status from 2020 to 2022,were included in the control and obese groups,respectively.Basic information was collected through questionnaires and body measurements were obtained from both obese and normal-weight children.Fecal samples were collected from both groups and subjected to 16S rDNA sequencing using an Illumina MiSeq sequencing platform for gut microbiota diversity analysis.RESULTS Significant differences in BMI and body-fat percentage were observed between the two groups.The Ace and Chao1 indices were significantly lower in the obese group than those in the control group,whereas differences were not significant in the Shannon and Simpson indices.Kruskal-Wallis tests indicated significant differences in unweighted and weighted UniFrac distances between the gut microbiota of normal-weight and obese children(P<0.01),suggesting substantial disparities in both the species and quantity of gut microbiota between the two groups.Prevotella,Firmicutes,Bacteroides,and Sanguibacteroides were more abundant in the obese and control groups,respectively.Heatmap results demonstrated significant differences in the gut microbiota composition between obese and normal-weight children.CONCLUSION Obese children exhibited lowerα-diversity in their gut microbiota than did the normal-weight children.Significant differences were observed in the composition of gut microbiota between obese and normal-weight children.

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.

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.

16S rDNA-RFLP技术鉴定西藏地区乳制品中的乳杆菌

将16S rDNA PCR技术和RFLP技术相结合,对分离自乳制品中的乳杆菌进行分类和鉴定。从我国西藏地区传统发酵乳中分离出51株乳杆菌,采用通用引物扩增16S rDNA,利用限制性内切酶AluI,HaeⅢ和HinfⅠ将16S rDNA扩增产物进行酶切后,经聚丙烯酰胺凝胶电泳图谱将菌种鉴定到种的水平。根据16S rDNA-RFLP的结果从中选出8株有代表性的菌株进行生理生化实验和16S rDNA序列的测定,实验结果与RFLP鉴定结果相吻合,表明此方法是一种快速、准确的可用于大量乳杆菌分类和鉴定的方法。

16S rDNA-RFLP法快速鉴定蒙古国传统乳制品中的乳酸菌

以17株标准菌株为参照,采用限制性片段长度多态性(RFLP)分析和16S rDNA序列分析相结合的技术,对分离自蒙古国5个地区传统乳制品中的55株乳酸菌进行了快速鉴定。结果表明,通过限制性内切酶AluⅠ、HaeⅢ、BsmaⅠ、TspRⅠ和HinfⅠ的指纹图谱分析,将49株乳杆菌和2株片球菌准确鉴定到种,其他4株肠球菌鉴定到属。采用16S rDNA-RFLP方法鉴定乳酸菌具有准确性,可靠性和高效性。

仙人掌丛枝病植原体检测和16SrDNA片段序列分析

对仙人掌丛生幼嫩组织进行超薄切片电镜观察 ,在韧皮部筛管中存在大量植原体 ;根据植原体 16 S r RNA基因保守序列设计的通用引物对 R16 F2 /R2 ,应用 PCR技术对仙人掌丛枝病进行分子检测 ,结果扩增到约 1.2 kb的特异性片段 ,而在健康组织中却没有此特异片段 ;通过 16 S r DNA片段核酸序列同源性比较 ,结果表明仙人掌丛枝病植原体与花生丛枝病植原体亲缘关系最近 ,据此可初步判断仙人掌丛枝病植原体是一种属于 16 Sr II组的植原体 ,基本确定了其分类地位。

黄华属根瘤菌的16S rDNA-RFLP分析

采用16S rDNA-RFLP及序列分析方法,对分离自黄华属的披针叶黄华、喀什黄华和光叶黄华根瘤菌进行分析研究.结果表明,分离得到的33株根瘤菌在种水平上具有丰富的遗传多样性,它们分别归属于中慢生根瘤菌属(Mesorhizobium)、中华根瘤菌属(Sinorhizobium)、根瘤菌属(Rhizobium)和土壤杆菌属(Agrobacterium).其中,以CCNWGS0011和CCNWGS0010-1为代表的5株根瘤菌构成独立的分支,可能为潜在的新种.

Isolation and identification of a sulfate reducing bacteria and sequence analysis of its dissimilatory sulfite reductase gene

A sulfate reducing bacteria was isolated from mining sewage of Daqing Oilfield by Hungate anaerobic technology. Physiological-biochemical analysis showed that the strain could utilize polyacrylamide as sole carbon and nitrogen source. The sequence analysis of 16S rDNA illustrated that the similarity of F8 and Desulfovibrio desulfuricans (AF192153) was 99%, and the similarity sequence of dissimilatory sulfite reductase gene (DSR) cloned from the strain and Desulfovibrio desulfuricans (AF273034) was 98%. Their phylogenitic analysis was basically anastomosed, and thus temporarily named as Desulfovibrio desulfuricans F8. The DSR cloned from F8 strain was 2740 bp in length consisting of three ORF, DSRA, DSRB and DSRD as a single operon (DSRABD) regulated by the same operator. DSRA contained typical conservative box of sulfate—sulfite reducing enzyme (SiteⅠand SiteⅡ), which could bind siroheme and [Fe4S4]. DSRB retained a [Fe4S4] binding site, with an uncomplimentary structure for siroheme binding. There was no conservative box in DSRD. Sequence analysis of DSR will provide a theoretical basis for quantitative detection, metabolic pathway modification through gene engineering, and sulfate reducing bacteria (SRB) suppression.

基于16 S rDNA分析复方蜥蜴散凝胶对UC模型大鼠肠道菌群的影响

目的通过16 S rDNA测序探究复方蜥蜴散凝胶对溃疡性结肠炎(UC)模型大鼠肠道菌群的影响。方法选取无特定病原体(SPF)级SD雄性大鼠,采取随机分组法,分为空白组、模型对照组、复方蜥蜴散凝胶高、中、低剂量组,柳氮磺吡啶组。采用TNBS-乙醇法制备大鼠UC模型,成模后进行结肠组织病理切片苏木素-伊红(HE)染色,取新鲜粪便提取DNA进行高通量16 S rDNA测序分析。结果与空白组比较,模型对照组结肠上皮细胞损伤、脱落,伴有腺体破坏、溃疡等病变,并可见大量的炎性细胞浸润;与模型对照组比较,复方蜥蜴散凝胶高、中、低剂量组和柳氮磺吡啶组结肠组织上皮细胞结构较清晰,炎性细胞浸润明显减少。测序结果显示:模型对照组肠道菌群多样性降低,经复方蜥蜴散凝胶和柳氮磺吡啶片治疗后,菌群多样性增加;菌群组成分析显示:与空白组比较,模型对照组中致病菌属丰度较高,治疗组有益菌占比增高。结论复方蜥蜴散凝胶可调节肠道菌群的多样性,通过增加有益菌,抑制致病菌进而起到治疗UC的作用。

Analysis of the intestinal flora in patientswith primary liver cancer

Objective To investigate the differences in intestinal flora of patients with primary liver cancer and ofhealthy individuals and to investigate the effect of the differential flora on the development of liver cancer.Methods Overall, 67 patients with primary liver cancer who received systematic and complete treatmentbetween January 2019 and December 2020 at the Sixth People’s Hospital of Qingdao and had completeclinical data were enrolled in this study, and 26 individuals who were healthy on physical examination inthe same period were used as healthy controls. Macro genome and 16s ribosome Deoxyribo Nucleic Acid(rDNA) high-throughput sequencing were performed on the stool flora of the enrolled patients and controls,and the differences in the intestinal flora were analyzed using the LEfSe bioinformatics software.Results Compared with the control samples, all the tested patient samples showed statistically significantdifferences in the number of colonies of 5 bacterial phyla, 5 orders, 8 families, 11 genera, and 14 species(P < 0.05).Conclusion Compared with healthy people, patients with primary liver cancer have significant differencesin the intestinal flora composition. The alteration of the intestinal flora may be correlated with the occurrenceof primary liver cancer, and the intestinal flora may become a novel target for the prevention and treatmentof primary liver cancer.

瘤胃甲硫氨酸降解菌的分离及其16 S rDNA全序列分析

为分析研究山羊瘤胃液中甲硫氨酸降解菌群的物种资源,对经分离纯化获得的一株甲硫氨酸降解菌MB6-1,采用PCR方法扩增其16 S rDNA基因,并测定其基因的核苷酸全序列。基于16 S rDNA序列的同源性比较和系统发育学分析(ribosomal database projectⅡ;简称RDPⅡ数据库),发现MB6-1可能是普罗威登斯菌属(Providencia)中的一个新种。菌株MB6-1的16 S rDNA序列已经被GenBank数据库收录,其序列号为DQ436917。

牦牛肠道与粪便乳酸菌的分离鉴定及PCR-16 S rDNA鉴定

以取自四川省不同地区的牦牛粪便、肠道内容物为材料,用MRS琼脂双层培养基进行厌氧培养,分离到50株乳酸菌,经生化鉴定为嗜热链球菌(2株)、乳酸乳球菌(1株)、保加利亚乳杆菌(5株)、嗜粪乳杆菌(10株)、嗜酸乳杆菌(8株)、乳酸乳杆菌(9株)、肠乳杆菌(10株)、弯曲乳杆菌(5株)。采用乳酸菌16 S rDNA通用引物,对分离的8种菌的16 S rDNA一段可变区序列进行扩增,均得到大小约470 bp的产物;扩增产物经纯化、测序后与GenBank中标准菌株的核甘酸序列比较,同源性均大于97．5％,同源性分析与生化试验的结果是一致的。证实,牦牛肠道和粪便的乳酸菌较为丰富,且乳杆菌的数量较多,这可能与牦牛复杂的生长环境有关。

Isolation and characterization of Acidithiobacillus caldus from several typical environments in China

Six strains of moderately thermophilic sulfur-oxidizing bacteria were isolated from several different typical environments in China. The identities of the isolates were confirmed by analyses of their 16S rRNA genes, and some key physiological traits. The isolates are Gram negative, rod-shaped bacteria, their optimal temperature and pH value for growth are 45-50℃ and 2.5-3.5 respectively. They are autotrophic and used elemental sulfur, sodium thiosulfate and potassium tetrathionate as electron donor, while a little glucose stimulated their growth. 16S rDNA sequences analysis reveals that the strains are phylogenetically clustered to Acidithiobacillus caldus.

The Impact of Irrigation on Bacterial Community Composition and Diversity in Liaohe Estuary Wetland

In this study, the sequencing of 16S ribosomal DNA was used to characterize the soil bacterial community composition and diversity in Liaohe estuarine wetland. Soil samples were taken from different locations in the wetland dominated by reed. Moreover, the soil quality parameters were evaluated(p H, moisture, organic matter, total nitrogen, available nitrogen, total phosphorus, available phosphorus). The results showed that the organic matter and nutrient contents were significantly higher in irrigated wetland than those in natural wetland. Major phylogenic groups of bacteria in soil samples including Proteobacteria, Acidobacteria, Gemmatimonadetes, Actinobacteria and Cyanobacteria were analyzed and we found that Proteobacteria was the most abundant in the community, and the phylum Acidobacteria was more abundant in irrigated wetland. Beta diversity analyses indicated that the soil bacterial community was mainly affected by sampling sites rather than seasons. In general, the bacterial community in natural wetland was not significantly different with that in artificial irrigated wetland. Artificial hydraulic engineering irrigated according to the water requirement rule of reed, increased the production of reeds, changed the way of wetland soil material input, but the diversity of bacterial community kept stable relatively.

Effect of Lycopus lucidus Turcz.supplementation on gut microflora and short chain fatty acid composition in Crj:CD-1 mice

We investigated the diversity and composition of microflora in feces of Lycopus lucidus Turcz.-fed mice.In addition,we evaluated the production of major cytokines(Interleukin-6 and-10)which are related to inflammation and fatty acid composition of several tissues.16S ribosomal DNA sequencing-based microbiome taxonomic profiling analysis was performed utilizing the EzBioCloud data base.Male mice fed on L.lucidus showed a significantly reduced number of lactic acid bacteria and coliform in the feces compared with the control group(p<0.05).16S rDNA sequencing analysis of fecal samples showed that L.lucidus supplementation decreased the community of harmful microflora(Enterobacteriaceae including Escherichia coli and Bacteroides sp.)in feces compared with the control group(p<0.05).There were no significant differences in mRNA expression of cytokine IL-6 and IL-10 between the control and L.lucidus fed groups.The fecal fatty acid composition in the L.lucidus group had percentages of 4:0,6:0,8:0 and 10:0 in the intestine but those short chain fatty acids were not detected in the control group.Our results showed that L.lucidus supplementation influenced gut environment by decreasing harmful microflora and increased the percentages of several short fatty acids.

Partial function prediction of sulfate-reducing bacterial community from the rhizospheres of two typical coastal wetland plants in China

Sulfate-reducing bacteria(SRB)are ubiquitous anaerobic microorganisms that play signifi cant roles in the global biogeochemical cycle.Coastal wetlands,one of the major habitats of SRB,exhibit high sulfate-reducing activity and thus play signifi cant roles in organic carbon remineralization,benthic geochemical action,and plant-microbe interactions.Recent studies have provided credible evidence that the functional rather than the taxonomic composition of microbes responds more closely to environmental factors.Therefore,in this study,functional gene prediction based on PacBio single molecular real-time sequencing of 16S rDNA was applied to determine the sulfate-reducing and organic substrate-decomposing activities of SRB in the rhizospheres of two typical coastal wetland plants in North and South China:Zostera japonica and Scirpus mariqueter.To this end,some physicochemical characteristics of the sediments as well as the phylogenetic structure,community composition,diversity,and proportions of several functional genes of the SRB in the two plant rhizospheres were analyzed.The Z.japonic a meadow had a higher dissimilatory sulfate reduction capability than the S.mariqueter-comprising saltmarsh,owing to its larger proportion of SRB in the microbial community,larger proportions of functional genes involved in dissimilatory sulfate reduction,and the stronger ability of the SRB to degrade organic substrates completely.This study confi rmed the feasibility of applying microbial community function prediction in research on the metabolic features of SRB,which will be helpful for gaining new knowledge of the biogeochemical and ecological roles of these bacteria in coastal wetlands.

Isolation and Genetic Analysis of Halophilous Bacteria from Marine Sediment Collected at Tianjin Port, China

[Objective] Marine sediment from Tianjin Port has a extremely high salinity.The bacteria which live in such habitats have evolved distinct physiological,metabolic,and morphological characteristics to survive.The objective of this study is to identify all the specific salt-tolerant characteristics and the genetic evolution of the bacteria in the sediment.[Methods] In this study,the total DNA of sediment from Tianjin Port was extracted,and 16S rDNA was used to conduct an analysis of the fauna of sediment bacteria. We also isolated sediment bacteria using beef extract-peptone media with seven different NaCl concentrations (0,0.5%,2%,5%,10%,15%,and 20%),aiming to analyze the dominant species of halophilous bacteria under different salinities.[Results] 1) With each stepwise increase of salinity from 0.5% to 20%,the total number of isolated bacterial colonies decreased.14 strains of bacteria were identified and classified by the16S rDNA sequencing analysis.Of these,four could tolerate 0~2% salinity,four could tolerate 0~5% salinity,one could tolerate 0~15% salinity,and one tolerated within the full 0~20% salinity range.Further four strains were only able to tolerate within a few narrow salinity ranges.such as 5%~10%,10%~15%,10%~20% and 15%~20%;2) The quantity of bacteria strains that can be isolated from the marine sediment decreased with the increase of salinity. Also, the Shannon wiener index and species richness index of marine sediment bacteria decreased significantly from 5% salinity.However,there were no significant differences in the species evenness index;3) When the salinity was 0~10%,the dominant species was Bacillus.When the salinity was 15%, Halomonas was the dominant species.When the salinity was 20%,there were no significant differences in the proportions of these species.[Conclusion] Our results showed that some bacteria could tolerate living conditions with high salinity,and we even found a species which can tolerate a wide range of salinities (0~20%).In further study,it would be valuable to analyze these bacteria's unique physiological and biochemical functions that allow them to adapt to environments with high salinity.It can provide theories to promote the development of microbial population resources in marine sediment and the reclaimation of salinized soil by salt tolerant microorganisms.

Structural and functional characteristics of microbiota in oropharynx of sub-healthy children with gastrointestinal heat retention syndrome differentiated by traditional Chinese medicine

Objective:To explore the structural and functional characteristics of microbiota in oropharynx of subhealthy children with gastrointestinal heat retention syndrome(GHRS)differentiated by traditional Chinese medicine,and screen the biometric operational taxonomic units(OTUs)to assist the clinical diagnosis.Methods:We recruited children according to the“GHRS diagnostic scale”,collected their oropharyngeal swabs,and sequenced the 16 SrDNA V4 region.We described the bacterial structure with alpha-indexes,beta-distances,and relative abundances;moreover,we screened the differential genera/OTUs with Wilcoxon rank-sum test,Metagenome Seq analysis,and linear discriminant analysis effect size(LEf Se)analysis,in which biometric OTUs were selected to construct the receiver operating characteristic curve to verify the diagnostic value.The bacterial function was predicted with Kyoto Encyclopedia of Genes and Genomes pathways according to 16S rDNA gene by using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States.Results:The study population was composed of 10 children with GHRS and 10 healthy control children.GHRS children were more likely to overeat(gluttony,P=.033).Alpha-indexes,such as Sobs,abundancebased coverage estimator,Bootstrap,and Qstat,were significantly higher in the GHRS group,while betadistances did not exhibit any significant intergroup differences.There were 9 differently distributed nonpredominant genera between the groups in Wilcoxon rank-sum test,as well as 13 non-predominant genera in Metagenome Seq analysis and 3 non-predominant OTUs in LEfSe analysis.OTU44 and OTU196 were used to construct the receiver operating characteristic curve,and the area under curve was 0.92.Predicted functions showed that pathways related to oxidative phosphorylation and carbon metabolism were enriched in healthy control samples,while the pathway related to renin secretion was remarkably enriched in GHRS samples.Conclusion:Unique oropharyngeal microbial structure and function were identified in GHRS children.OTU44 and OTU196 were specific OTUs,which could be used as biomarkers of GHRS to assist clinical diagnosis.

Study on the relationship between the structure of bacterial flora onthe tongue and types of tongue coating in patients with type 2diabetes mellitus

Objective:To analyze the characteristics of bacterial flora on the tongue and types of tongue coating between healthy individuals and patients with type 2 diabetes mellitus(T2DM)via detecting 16S rDNA of oral tongue coating microbial group.Methods:A total of 42 patients with T2DM were recruited,including 6 with thin white coating,9 with mirror-like coating,27 with yellow thick coating(YTC),and 28 healthy individuals.The V4 region of 16S rDNA from the tongue coating microbiota was sequenced using the Linux ubuntu sequencing platform,and the structure of bacterial flora in the tongue was analyzed.Treeplot construction,principal coordinates analysis,redundancy analysis,and linear discriminant analysis were conducted using R software to analyze the differences of bacterial flora in the tongue coating in each group.Results:A total of 8131 different operational taxonomic units(OTUs)were obtained via sequencing,of which 719 OTU samples showed significant differences(P<0.05).Comparing OTUs with the 16S rDNA database of known species and annotation of parallel species revealed 16 species with differences at the phylum level,31 at the class level,54 at the order level,88 at the family level,and 161 at the genus level.The dominant bacteria found in patients with T2DM included Lactobacillus,Streptophyta,Chloroplast,Cyanobacteria-Chloroplast,and Bifidobacteriaceae,etc.Dominant bacteria in the control group belonged to Pasteurellales,Pasteurellaceae,Leptotrichiaceae,Lachnoanaerobaculum,and Ignavibacteria among other bacterial families.All samples were clustered into three groups,each group characterized by dominant bacteria.The order of dominant bacteria in group 1 were Prevotella>Neisseria>Veillonella>Streptococcus>Fusobacterium>Leptotrichia and so on.The order of dominant bacteria of group 2 were Neisseria>Prevotella>Fusobacterium>Streptococcus>porphyromonas and so on.The dominant bacteria of group 3 were Prevotella>Fusobacterium>Streptococcus>Neisseria>Leptotrichia>Rothia>Veillonella>porphyromonas>f_Pasteurellaceae>Capnocytophaga>Actinomyces>Alloprevotella and so on.Single factor risk estimation:with group 1 used as the reference(OR=1),the risk of carrying group 2 and group 3 microflora with T2DM increased(OR values were 4.77 and 7.78,respectively).The proportion of group 1 microflora in the control group(25.9%)was higher than that in the T2DM group(5.4%)(χ2=3.873,P=0.049).Compared with group 1 and group 2,group 3 had a significantly dominant microflora structure in YTC group(χ2=7.120,P=0.008).RDA analysis indicated that Neisseria,Fusobacterium,and Prevotella were associated with HbA1c values>10 mmol/L,whereas Rothia,Streptococcus,and Veillonella were more correlated with HbA1c≤10 mmol/L.Conclusion:The incidence of T2DM is related to the imbalance of oral microflora in the human body.The tongue coating flora structure may influence the formation of different tongue coating types in this metabolic condition.Additionally,flora structures of Prevotella,Neisseria,and Fusobacterium were associated with elevated HbA1c.

The molecular biological characterization of a strain of biohydrogen-producing anaerobe in Clostridium Genus

The anaerobic process of biohydrogen production was developed recently. The isolation and identification of biohydrogen producing anaerobic bacteria with high evolution rate and yield is an important foundation of the fermented biohydrogen production process through which anaerobic bacteria digest organic wastewater. By considering physiological and biochemical traits, morphological characteristics and a 16S rDNA sequence, the isolated Rennanqilyf33 is shown to be a new species.