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.

Cloning and Sequence Analysis of 16S rRNA and COI Gene in Mitochondrial DNA of Scortum barcoo

[Objective] The aim was to provide molecular biological basis for the researches on the genetic resources,genetic relationship among species and phyletic evolution of S.barcoo.[Method] PCR amplification and sequencing were used to study the 16S rRNA and COI gene fragments.[Result] As for 16S rRNA gene fragments,nucleotide sequences of 791 bp were obtained,and the A,T,G and C contents in this fragment were 31.6%,21.4%,20.4% and 26.7%respectively.As for the COI gene fragments,the size was 631 bp and the A,T,G And C contents were 27.7%,23.6%,29.8% and 18.9% respectively.Among these two gene fragments,the content of GC was lower than AT,and AT/GC of these two fragments was 1.13 and 1.05 respectively.[Conclusion] The genetic characteristics of gene fragments of 16S rRNA and COI of S.barcoo suggested that the variation in the same species was relatively low.The sequences of 16S rRNA gene in three samples the same,while the sequences of COI gene was also the same,indicating that these two gene of S.barcoo were conservative.

16S rRNA Gene-Based Analysis of Ileal Bacterial Community and Phylogeny in Nursing and Weaned Piglets

Weaning of piglets is generally considered as a stressor which changes intestinal ecosystem and leads to clinical implications. Microbiota inhabiting in small intestine （especially ileum） are assumed to promote health, but their functional properties are yet poody dascdbed. As indicated by the 16S rRNA gene sequences of ileal micrebiota in nursing piglets （at the age of 21 and 28 d） and 28-day-old weaned piglets （weaned at 21 d of age）, the microbiota were mainly comprised of gram-positive bacteria. There were 40 operational taxonomic units （OTUs） （from 171 clones） in the ileum of nursing piglets aged 21 d, 61 OTUs （from 194 clones） in the ileum of nursing piglets aged 28 d, and 56 OTUs （from 171 clones） in the ileum of weaned piglets aged 28 d. The flea of nursing piglets aged 21 d were dominantly occupied by Lactobacilli （87.7%） as well as Streptococ cus （ 3.5 % ）. Lactobacillus amy/ovorus （41.5 % ）, Lactobaci/lus sp. （ 19.3 % ）, Lactobaci/lus reuteri （ 12.3 % ）, Lactobacillus salivarius （ 9.4 % ） and L. mucosae （4.7%） were the predominant species among Lactobacil/L Similar results were obtained in the nursing piglets at 28 d of age ex- cept that Lactobaci/li decreased to 71.1% and Streptococcus increased to 21.1% significantly. Lactobacillus （52.0%） and Streptococcus （26.3%） were the two major groups in the ileum of weaned piglets aged 28 d. Lactobacillus amylovorus （31.6%） and Lactobaci/lus reuteri （ 16.4% ） was the two most important species in Lactobacillus. Therefore, Lactobacilli were predominant in the ileum of nursing and weaned piglets, and they had the highest diversity, followed by Streptococcus. The diversity of ileal microbiota was not different remarkably between the nursing piglets and the weaned piglets, but the composition changed significantly. These findings are helpful to understand ileal bacterial ecophysiology and further develop nutritional regimes to prevent or counteract complications during the weaning transition.

16S rRNA Gene Phylogenesis of Culturable Predominant Bacteria from Diseased Apostichopus japonicus(Holothuroidea,Echinodermata)

Cultured Apostichopus japonicus in China suffers from a kind of skin ulceration disease that has caused severe economic loss in recent years. The disease,pathogens of which are supposed to be bacteria by most researchers,is highly infectious and can often cause all individuals in the same culture pool to die in a very short time. The 16S rRNA gene phylogenesis of the culturable bacteria from the lesions of diseased individuals was conducted to study the biodiversity of the bacterial communities in the lesions and to identify probable pathogen(s) associated with this kind of disease. S. japonica samples were selected from a hatchery located in the eastern part of Qingdao,China. Bacterial universal primers GM5F and DS907R were used to amplify the 16S rRNA gene of bacteria colonies,and touchdown PCR was performed to amplify the target sequences. The results suggest that γ-proteobacteria(Alteromonadales and Vibrionales) of CFB group,many strains of which have been also determined as pathogens in other marine species,are the predominant bacterial genera of the diseased Apostichopus japonicus individuals.

Preliminary study on mitochondrial 16S rRNA gene sequences and phylogeny of flatfishes (Pleuronectiformes)

A 605 bp section of mitochondrial 16S rRNA gene from Paralichthys olivaceus, Pseudorhombus cinnamomeus, Psetta maxima and Kareius bicoloratus, which represent 3 families of Order Pleuronectiformes was amplified by PCR and sequenced to show the molecular systematics of Pleuronectiformes for comparison with related gene sequences of other 6 flatfish downloaded from GenBank. Phylogenetic analysis based on ge- netic distance from related gene sequences of 10 flatfish showed that this method was ideal to explore the rela- tionship between species, genera and families. Phylogenetic trees set-up is based on neighbor-joining, maximum parsimony and maximum likelihood methods that accords to the general rule of Pleuronectiformes evolution. But they also resulted in some confusion. Unlike data from morphological characters, P. olivaceus clustered with K. bicoloratus, but P. cinnamomeus did not cluster with P. olivaceus, which is worth further studying.

Genetic Diversity in Populations of Sepiella maindroni Using 16S rRNA Gene Sequence Analysis

Part of the 16S rRNA gene is amplified with PCR and sequenced for 5 populations of com-mon Chinese cuttlefish Sepiella maindroni: three from the South China Sea, one from East China Sea and one from Japan. The result shows that a total of 5 nucleotide positions are found to have gaps or insertions of base pairs among these individuals, and 13 positions are examined to be variable in all the sequences, which range from 494 to 509 base pairs. All of the individuals are grouped into 7 haplotypes (h1-h7). No marked genetic difference is observed among those populations. All of the individuals from Nagasaki belong to hl and the h3 haplotype is found only in the coastal waters of China. A(?)G transition in Nucleotide 255 is suggested to be taken as a kind of genetic marker to identify the populations distributed in East-South China Sea and the Nagasaki waters of Japan.

Isolation of Pectinase Producing Bacteria from the Rhizosphere of <i>Andrographis paniculata</i>Nees and 16S rRNA Gene Sequence Comparison of Some Potential Strains

Pectinases, the enzymes which break down pectic substances, have a wide range of applications in food, agriculture and environmental sectors. In the present study, attempts were made to isolate highly efficient pectinase producer from the rhizosphere of a medicinal plant, Andrographis paniculata Nees, known as the “King of bitters”. The total heterotrophic bacterial count of the rhizosphere soil of A. paniculata Nees ranged from 1.53 × 109 to 2.52 × 109 cfu/g. A total of 65 bacterial colonies were randomly selected from the nutrient agar plates, purified and assessed for pectinase activity. Out of the 65 isolates, 62 (95.38%) showed varying degree of pectinase activity in plate assay using pectin as a sole source of carbon. Among the pectinase producing strains, JBST36 showed best pectinase activity which is followed by the JBST22 and JBST27. Morphological characterization, biochemical tests and 16S rRNA gene sequencing were performed to identify the three most potential strains. Based on the morphological, biochemical and molecular data, JBST22 was identified as Bacillus flexus and the other two were identified as Bacillus subtilis. Furthermore, nucleotide sequences of the 16S rRNA gene of these 3 strains were compared and a phylogenetic tree was constructed. The study reveals that there are at least 66 base differences in the 16S rRNA gene sequences of B. flexus JBST22 and the B. subtilis JBST36.

Diversity of Microflora in Colonic Mucus from Severe Ulcerative Colitis Patients Analyzed by Terminal Restriction Fragment Length Polymorphism and Clone Libraries of Bacterial 16S rRNA Gene Sequences

Although the gut microflora is thought to be an essential factor in the development of ulcerative colitis (UC), the entire gut microflora occurring in UC remains unknown. Most studies use feces to represent the microflora distribution;however, here we analyzed the bacterial diversity in colonic mucus from UC patients receiving colectomy surgery and control patients. The diversity of microflora was investigated using a combination of terminal restriction fragment length polymorphism (T-RFLP) and clone library analyses of the 16S rRNA gene sequences. In the T-RFLP analysis, the number of terminal restriction fragments (T-RFs) decreased significantly in UC patients when compared to control samples. Also in the clone library analysis, the number of operational taxonomic units (OTU) and the Shannon diversity index were reduced significantly in UC patients. These molecular analyses reveal an overall dysbiosis in UC patients. No specific pathogen was found, and a strong negative correlation in relative abundance of bacterial populations was observed between the phyla Bacteroidetes and Firmicutes in the UC patients. This is the first report showing a significant correlation between these two phyla, which may be important characteristics in the pathogenesis of UC.

Comparison of 16S rRNA Gene Primers on Studying Microbial Community Composition in Bottom Water and Sediment of Artificial Reefs in Laoshan Bay, China

Marine microorganisms are indispensable regulators of nutrient cycling and energy flow,which are crucial for artificial reefs(ARs)ecosystems.However,little is known about the microbial communities in the bottom water and sediment of ARs.Studies of microbial diversities have greatly advanced due to the development of high-throughput sequencing technologies,whereas the results may vary significantly due to the primers’choice.This study investigated the influences of two 16S ribosomal RNA gene primer choices(V4 and V3-V4)on microbial community compositions and structures.The results showed that the taxonomic assignment de-tected by primer V3-V4 was higher compared with that obtained by primer V4,whereas microbial community compositions had strong correlations between the two primers.Microbial beta diversities of ARs can be uncovered by both primers,but the relation-ships between communities and environmental parameters were inconsistent.The performances of the two primers for water samples were highly consistent,but the inconformity was evident for sediment samples.Given the relatively lower taxonomic classification of primer V4 for sediment samples,primer V3-V4 was recommended for later studies.With the development and advancement of ARs in China,our findings provide a meaningful reference for ecologists focusing on the microbial diversities and ecological functions of these artificial habitats in the future.

16S rRNA Gene Sequence Analysis of Snow Leopard, Gray Wolf, Horse and Bactrian Camel in Mongolia

In this study, mitochondrial 16S rRNA sequences of snow leopard, gray wolf, domestic horse and Bactrian camel inhabited or domesticated in Mongolian territory were obtained by using polymerase chain reaction （PCR） based on universal primers for 16S rRNA （F-5＇-AACGAGCCTGGTGATA-3＇ and R-5＇-CTCCGGTCTGAACTCAGATCACGTA-3＇）. The 16S rRNA sequence was 1,048 bp to 1,086 bp in length, and each sequence was compared to other related species （Felidae, Camelidae, Equidae and Canidae） by using NCBI Basic Local Alignment Search Tool （BLAST）. Results showed that sequences were highly similar to sequences in GenBank database （93%-99%）. Then phylogenetic analysis was performed based on about 1,100 bp sequence of 16S rRNA for Panthera uncia, Canis lupus, Equus caballus, Camelus bactrianus and other related species. The result revealed that P. uncia and P. leo were sister species, C. bactrianus and C. ferus were more closely related species, and wolf and dog were the almost similar species. This finding could be important for designing species specific primers for PCR based analysis of animal species identification and forensic veterinary medicine.

Phylogenetic Relationships among 12 Species of Tetrigidae (Orthoptera:Tetrigoidea) Based on Partial Sequences of 12S and 16S Ribosomal RNA

Mitochondrial 12S and 16S ribosomal RNA genes sequences were sequenced using dye-labeled terminator on an ABI 377 automated sequencer in 11 individuals and 1 species' sequences were gained from GenBank,representing 6 genera of family Tetrigidae.The collated sequences were aligned using Clustal X version 1.81 and then,the sequence variability and heredity distances based on Kimura 2-parameter model were calculated using Mega 2.1.In obtained sequences (736 bp),the average A+T content is 73.9%,ranging from 71.2% to 77.5%;the overall G+C content is 26.1%,ranging from 22.5% to 28.8%.Based on alignment of the combined sequences,185 parsimony-informative sites were revealed in 755 available base pairs.Phylogenetic trees were reconstructed using NJ,MP and ML methods with Cylindraustralia kochii as outgroup.The results indicated that the monophyletic nature of Tetrix is questioned and suggest that T.tubercarina may be given tribal rank.Our results also show that Coptltettix huanjiangensis and C.gongshanensis are the same species,i.e.Coptltettix gongshanensis Zheng,and C.huanjiangensis is the synonyms of C.gongshanensis.

Phylogenetic diversity of planktonic bacteria in the Chukchi Borderland region in summer

Planktonic bacteria are abundant in the Chukchi Borderland region. However, little is known about their di- versity and the roles of various bacteria in the ocean. Seawater samples were collected from two stations K2S and K4S where sea ice was melting obviously. The analysis of water samples with fluorescence in situ hybridization （FISH） showed that DMSP-degrading bacteria accounted for 13% of the total bacteria at the station K2S. No aerobic anoxygenic phototrophic （AAP） bacteria were detected in both samples. The bacterial communities were characterized by two 16S rRNA gene clone libraries. Sequences fell into four major lineages of the domain Bacteria, including Proteobacteria （Alpha, Beta and Gamma subclasses）, Bac- teroidetes, Actinobacteria and Firmicutes. No significant difference was found between the two clone li- braries. SAR11 and Rhodobacteraceae clades of Alphaproteobacteria and Pseudoalteromonas of Gammapro- teobacteria constituted three dominant fractions in the clone libraries. A total of 191 heterotrophic bacterial strains were isolated and 76% showed extracellular proteolytic activity. Phylogenetic analysis reveals that the isolates fell into Gammaproteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. The most common genus in both the bacterial isolates and protease-producing bacteria was Pseudoalteromonas. UniFrac data showed suggestive differences in bacterial communities between the Chukchi Borderland and the northern Bering Sea.

Impact of Different Rates of Nitrogen Supplementation on Soil PhysicochemicalProperties and Microbial Diversity in Goji Berry

Goji berry(Lycium barbarum L.)is substantially dependent on nitrogen fertilizer application,which can signifi-cantly enhance fruit yield and Goji berry industrial development in Ningxia,China.This study aimed to analyze the functions of differential nitrogen application rates including low(N1),medium(N2),and high(N3)levels in soil microbial community structure(bacterial and fungal)at 2 diverse soil depths(0-20,20-40 cm)through high-throughput sequencing technology by targeting 16S RNA gene and ITS1&ITS2 regions.All the observed physicochemical parameters exhibited significant improvement(p<0.05)with increased levels of nitrogen and the highest values for most parameters were observed at N2.However,pH decreased(p<0.05)gradually.The alpha and beta diversity analyses for bacterial and fungal communities’metagenome displayed more similarities than differences among all groups.The top bacterial and fungal phyla and genera suggested no obvious(p>0.05)differences among three group treatments(N1,N2,and N3).Furthermore,the functional enrichment analysis demonstrated significant(p<0.05)enrichment of quorum sensing,cysteine and methionine metabolism,and transcriptional machinery for bacterial communities,while various saprotrophic functional roles for fungal communities.Conclusively,moderately reducing the use of N-supplemented fertilizers is conducive to increasing soil nitrogen utilization rate,which can contribute to sustainable agriculture practices through improved soil quality,and microbial community structure and functions.

Impact of preoperative antibiotics and other variables on integrated microbiome-host transcriptomic data generated from colorectal cancer resections

BACKGROUND Integrative multi-omic approaches have been increasingly applied to discovery and functional studies of complex human diseases.Short-term preoperative antibiotics have been adopted to reduce site infections in colorectal cancer(CRC)resections.We hypothesize that the antibiotics will impact analysis of multi-omic datasets generated from resection samples to investigate biological CRC risk factors.AIM To assess the impact of preoperative antibiotics and other variables on integrated microbiome and human transcriptomic data generated from archived CRC resection samples.METHODS Genomic DNA(gDNA)and RNA were extracted from prospectively collected 51 pairs of frozen sporadic CRC tumor and adjacent non-tumor mucosal samples from 50 CRC patients archived at a single medical center from 2010-2020.The 16S rRNA gene sequencing(V3V4 region,paired end,300 bp)and confirmatory quantitative polymerase chain reaction(qPCR)assays were conducted on gDNA.RNA sequencing(IPE,125 bp)was performed on parallel tumor and non-tumor RNA samples with RNA Integrity Numbers scores≥6.RESULTS PERMANOVA detected significant effects of tumor vs nontumor histology(P=0.002)and antibiotics(P=0.001)on microbialβ-diversity,but CRC tumor location(left vs right),diabetes mellitus vs not diabetic and Black/African Ancestry(AA)vs not Black/AA,did not reach significance.Linear mixed models detected significant tumor vs nontumor histology*antibiotics interaction terms for 14 genus level taxa.QPCR confirmed increased Fusobacterium abundance in tumor vs nontumor groups,and detected significantly reduced bacterial load in the(+)antibiotics group.Principal coordinate analysis of the transcriptomic data showed a clear separation between tumor and nontumor samples.Differentially expressed genes obtained from separate analyses of tumor and nontumor samples,are presented for the antibiotics,CRC location,diabetes and Black/AA race groups.CONCLUSION Recent adoption of additional preoperative antibiotics as standard of care,has a measurable impact on-omics analysis of resected specimens.This study still confirmed increased Fusobacterium nucleatum in tumor.

16-23S rRNA Spacer Region Polymorphism in Gangetic River Water Isolates of Salmonella

Salmonella is one of the major pathogenic bacteria present in contaminated water. 16-23S rRNA spacer region has been reported to be polymorphic at serovar level in Salmonella. Salmonella isolates obtained from Ganges river water were studied for 16-23S rRNA spacer region polymorphism. Thirty three isolates belonging to eight serovars (S. Typhimurium, S. Abuja, S. Pantypridd, S. Lagos, S. Chinkual, S. Zwickau, S. Goldenberg and S. Oritamerin) were studied for the polymorphism. Out of 33 isolates, 15 different profiles were observed no serovar specific profile. Our findings indicate that 16-23S rRNA spacer region is not specific at serovar level, but can be used for differentiation of different Salmonella isolates.

Isolation of Swine Corynebacteria and Analysis of 16S rDNA Gene

A strain of gram-positive bacillus was isolated from suppurative lung organs of nursery pigs in a pig farm, which was further characterized by morphological observation, cultivation test, biochemical test, drug sensitivity test, pathogenicity test and 16S rDNA gene cloning and sequence analysis. The results showed that the isolate grew well in rabbit blood agar plate and horse serum tryptone soybean agar （TSA） plate under aerobic condition, which could lead to mortality of mice and were susceptible to cephalosporin antibiotics and fluoroquinolone antibiotics. Phylogenetic analysis showed that the isolate had close genetic evolutionary relationships with Corynebacterium bacteria, and the sequence of 16S rRNA gene shared the homology of 91.7% -98.3% with the representative strain of coryne- bacteria, indicating the isolated strain was eorynebactefium.

Development of a 16S r RNA gene-based microarray for the detection of marine bacterioplankton community

A better understanding of bacterioplankton community shifts following change in marine environments is critical to predict the marine ecosystem function. In order to get a snapshot of the microbial taxonomy profiling of a wide range marine area, a quick, convenient and low cost method would be favorable. In this study, we developed a 16S rRNA gene-based microarray using ARB software, which contained 447 probes targeting 160 families of marine bacteria. The specificity, sensitivity and quantitative capability of this microarray were assessed by single cloned16S rRNA genes. The reliability of this microarray was tested by eight environmental samples. The results showed that the microarray was specific, only 1.16% false results were detected in five single-clone hybridization tests. The microarray could detect DNA samples as few as 1 ng/μL and the signal intensity could reflect the relative abundance of the bacteria in the range of 1 ng/μL to 100 ng/μL of DNA concentration. Hybridization with environmental samples showed that it can discriminate bacterioplankton communities by sites and time. High throughput sequencing results from the eight samples confirmed the hybridization results. It indicated that this developed microarray could be used as a convenient tool to monitor the bacterioplankton community in marine environment.

奶牛子宫内膜炎化脓隐秘杆菌16SrRNA基因的鉴定与分析

对内蒙古呼和浩特地区奶牛子宫内膜炎化脓隐秘杆菌进行了分离鉴定。经生化实验鉴定的化脓隐秘杆菌利用PCR技术扩增其16S rRNA基因,得到1.5 kb目的片段;将目的片段与T载体连接,测定目的片段的基因序列,与GenBank公布的化脓隐秘杆菌16S rRNA基因序列进行比较分析,其同源率为93%～100%。本试验共分离到化脓隐秘杆菌32株。

基于线粒体12S和16S rRNA基因部分序列的角蟾亚科部分属种的系统发育关系

测定了角蟾亚科 2属 8种 (亚种 )和外群 3种的线粒体 12S和 16SrRNA基因部分DNA序列 ,比对后序列长共 94 9bp ,其中变异位点数 32 0 ,简约位点数 2 0 6。邻接法和最大简约法分析的系统关系树一致表明内群为一单系群 ,其中腺角蟾首先与其他物种分开 ;沙坪角蟾与宽头短腿蟾聚为一支 ;余下的 5种 (亚种 )角蟾组成一支 ,其中小角蟾短肢亚种的广西种群和香港种群聚为一亚支 ,另一亚支包括峨眉角蟾、小角蟾指名亚种、尾凸角蟾和重庆武隆的角蟾种 ,后两种角蟾进化关系最近。本结果支持短肢角蟾为有效种 ,同时提示腺角蟾、沙坪角蟾与宽头短腿蟾可能隶属 3个不同的亚属或属。

16S-23S rRNA基因区间细菌鉴定实验研究

目的 :探讨 16 S- 2 3S r RNA基因区间对细菌鉴定的应用。方法 :以 16 S- 2 3S r RNA基因区间为靶序列设计引物 ,采用聚合酶链反应检测标准菌株及临床菌株 DNA。结果 :对 2 7株代表 2 7个菌种的标准菌株进行 PCR扩增 ,分别出现不同的 DNA图谱 ,该图谱能直接或经核酸内切酶处理后用于细菌分类 ,敏感性可达 2 .5 CFU/ml的细菌 ,与人外周血白细胞 DNA和真菌及病毒无交叉。 32株临床细菌标本均扩增出与相应标准菌株一致的图谱。结论 :16 S- 2 3S r RNA基因区间 PCR扩增技术检测细菌 ,具有敏感、特异、快速、准确的优点 。