Characterization of Bacterial Community Structure and Diversity in Rhizosphere Soils of Three Plants in Rapidly Changing Salt Marshes Using 16S rDNA

The structure and diversity of the bacterial communities in rhizosphere soils of native Phragmites australis and Scirpus rnariqueter and alien Spartina alterniflora in the Yangtze River Estuary were investigated by constructing 16S ribosomal DNA （rDNA） clone libraries. The bacterial diversity was quantified by placing the clones into operational taxonomic unit （OTU） groups at the level of sequence similarity of 〉 97%. Phylogenetic analysis of the resulting 398 clone sequences indicated a high diversity of bacteria in the rhizosphere soils of these plants. The members of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria of the phylum Proteobacteria were the most abundant in rhizobacteria. Chao 1 nonpaxametric diversity estimator coupled with the reciprocal of Simpson＇s index （l/D） was applied to sequence data obtained from each library to evaluate total sequence diversity and quantitatively compare the level of dominance. The results showed that Phragmites, Scirpus, and Spartina rhizosphere soils contained 200, 668, and 382 OTUs, respectively. The bacterial communities in the Spartina and Phragraites rhizosphere soils displayed species dominance revealed by 1/D, whereas the bacterial community in Scirpus rhizosphere soil had uniform distributions of species abundance. Overall, analysis of 16S rDNA clone libraries from the rhizosphere soils indicates that the changes in bacterial composition may occur concomitantly with the shift of species composition in plant communities.

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.

Establishment of Molecular Biological Method for Identification of Bacteria by 16S rDNA and gyrB Gene

[Objectives]The paper was to establish a molecular biological method for identification of bacterial strains.[Methods]The thalli of standard bacterial strains existing in the laboratory were collected and genomic DNA was extracted for amplification of 16S rDNA and gyrB gene.The 16S rDNA and gyrB gene sequences were obtained after sequencing.Sequences were aligned and analyzed via EzBioCloud and NCBI database,and phylogenetic trees were constructed to determine the species relationship of strains.Meantime,they were compared with known strains.[Results]This method could identify 5 standard strains accurately to the species level.The 16S rDNA and gyrB gene sequences were aligned and analyzed in EzBioCloud database and NCBI database.The strain with the max score was consistent with the known strain.And the query cover and ident were both above 99%.[Conclusions]The established molecular biological method for identification of bacterial strains by 16S rDNA and gyrB gene has good accuracy,which effectively solves the problem that the laboratory identification of bacteria relies on traditional methods and the accuracy can not be guaranteed,and further improves the identification ability of laboratory bacterial strains.

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.

沈抚石油污水灌区稻田土壤细菌遗传多样性——16S rDNA-PCR-DGGE分析

以不依赖于培养的16S rDNA-PCR-DGGE技术，评价了石油污染对我国最大的石油污水灌区——沈抚灌区稻田土壤细菌遗传多样性的影响，并对微生物群落中的优势菌群进行了研究。结果表明，沈抚灌区土壤总石油烃（Total petroleum hydrocarbon，TPH）含量为277—5213mgkg^-1干土，TPH在灌区干渠和支渠中的积累和分布趋势大体上是上游地区较严重，下游地区较轻，并且与土壤中有机质含量呈显著正相关（r=0．691，P〈0．05）。在目前的污染程度下，石油污水能够刺激土壤好氧异养细菌（Aerobic heterotrophic bacteria，AHB）的生长，其数量与TPH含量呈显著正相关（r=0．928，P〈0．001），而细菌遗传多样性与TPH含量呈显著负相关（r=-0．715，P=0．013）。DGGE图谱优势条带测序结果表明沈抚灌区土壤细菌群落中的优势菌群为变形细菌（Proteobacteria）β-亚群和γ-亚群的菌种，这些优势菌群的形成可能与石油烃的生物降解有关。

Bacterial biota in reflux esophagitis and Barrett's esophagus

AIM： To identify the bacterial flora in conditions such as Barrett＇s esophagus and reflux esophagitis to determine if they are similar to normal esophageal flora. METHODS： Using broad-range 16S rDNA PCR, esophageal biopsies were examined from 24 patients [9 with normal esophageal mucosa, 12 with gastroesophageal reflux disease （GERD）, and 3 with Barrett＇s esophagus]. Two separate broad-range PCR reactions were performed for each patient, and the resulting products were cloned. In one patient with Barrett＇s esophagus, 99 PCR clones were analyzed. RESULTS： Two separate clones were recovered from each patient （total = 48）, representing 24 different species, with 14 species homologous to known bacteria, 5 homologous to unidentified bacteria, and 5 were not homologous （〈97% identity） to any known bacterial 16S rDNA sequences. Seventeen species were found in the reflux esophagitis patients, 5 in the Barrett＇s esophagus patients, and 10 in normal esophagus patients. Further analysis concentrating on a single biopsy from an individual with Barrett＇s esophagus revealed the presence of 21 distinct bacterial species. Members of four phyla were represented, including Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria. Microscopic examination of each biopsy demonstrated bacteria in intimate association with the distal esophageal epithelium, suggesting that the presence of these bacteria is not transitory. CONCLUSION： These findings provide evidence for a complex, residential bacterial population in esophageal reflux-related disorders. While much of this biota is present in the normal esophagus, more detailed comparisons may help identify potential disease associations.

Bacterial and cyanobacterial diversities determined by T-RFLP analyses in the Jiaozhou Bay

The methods of DAPI staining epifluorescence microscopy and T-RFLP analysis were used to analyze the microbial abundance and diversity in surface seawater sampled from 12 stations inside and outside of the Jiaozhou Bay during a survey on 12 and 13 September 2004. The abundance of total microbes is in the range of 10^6～ 10^7 cells/cm^3, similar to those of most semi-enclosed bays in the temperate areas in the world. The highest microbial densities occur in the northeastern part of the Jiaozhou Bay, around the mouths of Loushan and Licun Rivers and the Hongdao aquacultural farming areas, suggesting that the degree and characteristics of pollutions, along with geographical and hydrological effects, may be important determinants affecting the abundance and distribution of bacteria in the Jiaozhou Bay. Bacterial communities inside and outside of the Jiaozhou Bay can be grouped into three classes based on T-RFLP patterns and cluster analyses. Stations at the water channel of the bay mouth and outside, such as D1, D3, D5, D6 and D7, are grouped together to stand for the outside bacterial community interacting with the environment outside of the Jiaozhou Bay. Stations of the innermost side of the Jiaozhou Bay, such as A3, A5, B2 and Y1, are grouped together to stand for the residential bacteria community. Stations C1, C3 and CA are grouped together and may stand for the transitional bacterial assemblage between the residential community and the outside community. However, there is no such a defined relationship for the case of cyanobacterial diversity, indicating the fact that cyanobacteria are more flexible and adaptable to all kinds of conditions.

Bacterial diversity in activated sludge from a consecutively aerated submerged membrane bioreactor treating domestic wastewater

The bacterial diversity of activated sludge from submerged membrane bioreactor (SMBR) was investigated. A 16S rDNA clone library was generated, and 150 clones were screened using restriction fragment length polymorphism (RFLP). Of the screened clones, almost full-length 16S rDNA sequences of 64 clones were sequenced. Phylogenetic tree was constructed with a database containing clone sequences from this study and bacterial rDNA sequences from NCBI for identification purposes. The 90.6% of the clones were a?l...

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.

基于16S rDNA快速鉴定细菌的PCR测序方法的建立及其进化关系分析

目的建立基于16S rDNA快速鉴定细菌的PCR测序方法。方法通过一组16S rDNA通用引物扩增得到基因组全长,PCR产物经纯化后直接测序分析。利用BLAST软件从GenBank数据库中搜索相关菌株的16S rDNA全序列,采用Clustal X软件进行多序列比对和同源性分析,确定细菌的种属。结果实验利用引物建立了16S rDNA全长序列分析方法,未知菌种和已知菌株通过PCR方法获得约1500bp的全长序列。比对分析已知菌株测序结果与预期标准序列完全一致,证实建立的PCR方法结果可靠。利用建立的PCR法,对实验室从污染物中分离得到不同未知菌株进行鉴定,成功确定了这些菌株的种属。结论建立的基于16S rDNA的PCR方法是可行的,可快速准确地检测及鉴定细菌种类,尽早发现细菌污染,对污染制品输注后的针对性治疗方面具有潜在的应用价值。

Effect of Inoculation with Alcaligenes faecalis on the Bacterial Community in Paddy Soils

The rifampin resistant Alcaligenes faecalis strain A1501R survived at a high, initially stable and later slowly declining population size (10 8 to 10 6 CFU per g dry soil) for 60 days in microcosm experiments. Introduction of strain A1501R did not cause great changes in the community profiles generated via microbial community level 16S rDNA based PCR followed by denaturing gradient gel electrophoresis (DGGE) and the Biolog substrate utilization system. The introduction of A1501R had no impact on the dominant bacterial populations in soil. Significant differences in the utilization of some substrates were observed between the control and inoculated soils using a combination of DGGE and Biolog analysis. Results of DGGE and BIOLOG analysis show that introduction of A1501R can affect the lactic acid utilizing bacterial community in case of selection by lactic acid. A specific probe based on the 16S rDNA variable region V6 was constructed via PCR amplification of 16S rDNA using the 971(f) and 1057(r) primers for the detection of A. faecalis strains introduced into soils. Dot blot hybridization of strains or soil DNA and aligment of sequences of the V6 region of 16S rDNA from different bacterial strains show that the V6 probe is very specific for A. faecalis A1501R.

Bacterial Biofilm in Water Bodies of Cherrapunjee: The Rainiest Place on Planet Earth

Bacterial attachment is influenced by the cell surface, attachment media and other environmental factors. Bacterial community composition involved in biofilm formation in extremely high rainfall areas like Cherrapunjee has not been reported. The present study was undertaken to characterize bacteria involved in biofilm formation on different substrata in water bodies of Cherrapunjee, the highest rainfall receiving place on planet earth and to assess if the continuous rainfall has an effect on nature and colonization of biofilm bacteria. We developed the biofilm bacteria on stainless steel and glass surfaces immersed in water bodies of the study sites. Isolation of biofilm bacteria were performed on different culture media followed by estimation of protein and carbohydrate content of bacterial exopolysaccharides. 16S rRNA gene sequences were amplified for molecular characterization. The results showed that the biofilm bacterial diversity in water bodies of Cherrapunjee was influenced by substratum and was observed more in stainless steel than glass surface. Scanning electron microscopy images revealed that biofilm microstructure may represent a key determinant of biofilm growth and physiology of associated bacteria. The overall protein content of the extracted EPS of all the isolates were relatively higher than the carbohydrate content. Diverse bacteria proliferated on the substrata regardless of each other's presence, with more diverse bacteria colonizing the substrata on 7th day compared to 15th day of incubation. The biofilm bacteria compositions in the highest rainfall receiving habitat were not distinctly different from reports available, hence not unique from other water bodies.

Analysis of the Bacterial Communities in Lime Concretion Black Soil upon the Incorporation of Crop Residues

To analyze the bacterial communities in lime concretion black soil upon the incorporation of crop residues for two years in wheat-maize system, total DNA was directly extracted and PCR-amplified with the F357GC and R518 primers targeting the 16S rRNA genes of V3 region. The amplified fragments were analyzed by perpendicular DGGE. Analyzing of species richness index S and Shannon diversity index H revealed that there was a high diversity of soil bacterial community compositions among all treatments after incorporation of crop residues and fertilizing under field conditions. Eleven DGGE bands recovered were re-amplified, sequenced. Phylogenetic analysis of the representative DGGE fingerprints identified four groups of the prokaryotic communities in the soil by returning wheat residues and fertilizing under field conditions. The bacterial communities belonged to gamma proteobacterium, Cupriavidus sp, halophilic eubacterium, Acidobacterium sp, Sorangium sp, delta proteobacterium, Streptococcus sp and Streptococcus agalactiae were main bacterial communities. Principal Component Analysis (PCA) showed that there were the differences in DNA profiles among the six treatments. It showed that wheat residue returning, maize residue returning and fertilizing all can improve bacterial diversity in varying degrees. As far as improvement of bacterial diversity was concerned, wheat residue returning was higher than fertilizing, and fertilizing higher than maize residue returning.

Molecular characterization and diversity analysis of bacterial communities associated with Dialeurolonga malleswaramensis （Hemiptera： Aleyrodidae） adults using 16S rDNA amplicon pyrosequencing and FISH

Dialeurolonga malleswaramensis Sundararaj （Hemiptera： Aleyrodidae） is a phytophagous sap sucking insect. It infests Polyalthia longifolia, an important avenue tree of India, effective in alleviating noise pollution and having immense medicinal importance. Samples of this insect were collected from Polyalthia longifolia. The cytochrome c oxidase subunit I gene （mtCOl） helped in the molecular characterization of the insect. This study reports the bacterial diversity in D. malleswararnensis adults by high throughput 16S rDNA amplicon pyrosequencing. The major genera identified were Portiera and Arsenophonus. Other bacterial genera detected were uncultured alpha proteobacterium, Sphingopyxis and Methylobacterium. We also employed fluorescence in situ hybridization （FISH） in whole mount samples to confirm the presence of dominant endosymbionts Portiera and Arsenophonus to the bacteriocyte of D. malleswaramensis. This study concludes that combining techniques like 16S rDNA amplicon pyrosequencing and FISH reveal both dominant and rare bacteria. The data also predict the evolutionary position of this pest with respect to other whitefly species using a mitochondrial marker.

基于16S rDNAs的快速检测血液细菌污染的荧光定量PCR研究

目的探讨快速筛选血液及血液制品中细菌污染的方法。方法对20余种常见致病性细菌的16SrDNAs进行多序列比对,设计扩增细菌16SrDNAs基因的荧光定量PCR(FQ-PCR)通用引物。以12种致病菌、2种真菌、1种支原体、1种病毒和人基因组DNA为模板,验证通用引物检测细菌的特异性。通用引物扩增金黄色葡萄球菌16SrDNA靶片段,构建标准质粒pMDT-Bfr,并建立FQ-PCR定量标准曲线。分别以金黄色葡萄球菌和大肠埃希菌作为革兰阳性和革兰阴性细菌代表菌,以其不同浓度的DNA为模板进行FQ-PCR反应,检验该方法的敏感性。抽提梯度稀释的金黄色葡萄球菌和大肠埃希菌模拟血液细菌污染标本的总DNA作为FQ-PCR反应模板,检验基于16SrDNAs的FQ-PCR作为血液细菌污染检测方法的敏感性。结果设计的FQ-PCR通用引物有较高的特异性,仅能检测出细菌的16SrDNAs;用该方法检测革兰阳性和革兰阴性菌,对于浓度在103拷贝/μl以上的16SrDNAs基因都可以准确定量;以该方法检测血液细菌污染模拟标本,灵敏度可达到105CFU/ml。结论初步建立了以16SrDNAs作为FQ-PCR靶基因快速检测血液细菌污染的方法。该方法特异性强、灵敏度高、成本低廉,具有很好的研究价值与应用前景。

Soil fungistasis and its relations to soil microbial composition and diversity:A case study of a series of soils with different fungistasis

Fungistasis is one of the important approaches to control soil-borne plant pathogens.Some hypotheses about the mechanisms for soil fungistasis had been established,which mainly focused on the soil bacterial community composition,structure,diversity as well as function.In this study,the bacterial community composition and diversity of a series of soils treated by autoclaving,which coming from the same original soil sample and showing gradient fungistasis to the target soil-borne pathogen fungi Fusarium grami...

16S rDNA快速鉴定血液制品中污染细菌的测序方法的建立

目的建立基于16SrDNA快速鉴定细菌的PCR测序方法（PCR-SBT）。方法通过一组16SrDNA通用引物扩增得到基因组全长，PCR产物经纯化后直接测序分析。利用BLAST软件从GenBank数据库中搜索相关菌株的16SrDNA全序列，采用Clustal X软件进行多序列比对和同源性分析，确定细菌的种属，并与常规生化鉴定结果比较。利用大肠杆菌基因组一系列稀释度标本进行PCR扩增，检测方法的敏感性。结果实验利用多对引物建立了16SrDNA全长序列分析方法，13个标准菌株通过PCR．SBT方法获得约1400bp的全长序列。比对分析13个标准菌株测序结果与预期标准序列完全一致，证实建立的PCR—SBT方法结果可靠。利用建立的PCR—SBT法，对实验室从血小板制品和脐带血中分离得到不同未知菌株进行鉴定，成功确定了这些菌株的种属。以大肠杆菌DNA为模板，方法的最低检测限为反应体系DNA含量0．2ng。结论建立的基于16SrDNA的PCR—SBT方法是可行的，可快速准确地检测及鉴定细菌种类，尽早发现细菌污染血制品，对污染血制品输注后的针对性治疗方面具有潜在的应用价值。

Depth-related distribution of bacterial community in sediments of eutrophic Guanting reservoir

In this study, DNAs were extracted from sediment samples at depths of 5, 35, and 69 cm from eutrophic Guanting reservoir, China. 16S rDNAs were amplified by PCR and clone libraries were constructed. The depth-related distribution of bacterial community in the sediment was characterized by using amplified 16S rDNA restriction analysis (ARDRA) and sequencing of the dominant clones. The results indicated that species diversity in the sediment of Guanting reservoir was rather high with the Shannon-Wiener index about 5.8. Bacterial richness varied in different depths: the highest in the sample of 35 cm in depth; followed by the sample of 5 cm in depth; and the lowest bacterial richness in the sample of 69 cm. Dominant species from the three samples were different although there were some common clones. Phylogenetic analysis showed that all of the dominant clones in the three layers were uncultured bacteria and distantly related to the previously reported species in beta or gamma subclass of proteobacteria, including bacterial groups that have the ability to degrade aromatic hydrocarbons, n-alkanes, chlorinated organic compounds, or to accumulate polyphosphate, etc. Changes of depth-related bacterial community in the Guanting reservoir sediment might reflect the pollution history and the water quality of the reservoir. In addition, the cloned sequences from the Guanting reservoir sediment were all different from the presently reported ones, indicating that there were some particular bacteria in that environment.

Succession of bacterial community during composting:dissimilarity between compost mixture and biochar additive

Previous research showed that biochar addition facilitated composting and elevated nutrient retention.However,few of these studies explored bacterial structure and abundance in the compost mixture and biochar additive.Thus,this study aims to distinguish bacterial communities in both compost and bamboo biochar(BB)additive.Results indicated that the dynamics of nutrient contents in compost and BB samples were in a similar pattern,although there were lower levels of nutrients and metals(i.e.,Cu and Zn)in BB additives.The total number of operational taxonomic units(OTUs)in both compost and BB additives peaked on day 7 and then gradually reduced during composting.There was more abundance of bacteria in compost,whereas the diversity of bacteria was more in BB additives.Furthermore,the dominant bacteria in compost and BB samples were distinct at the different stages of composting.The Firmicutes steadily decreased in compost samples(from 34.78%to 7.65%),while it was the dominant phylum in BB additives during the whole composting period.Furthermore,Ruminofilibacter,Pseudoxanthomonas,and Actinomadura were the most abundant genera in compost samples than Pseudoxanthomonas,Azoarcus,and Paenibacillus in BB additives at the final stage of composting.Results from this study could provide a theoretical reference for the sound performance of biochar-added composting.

口腔综合治疗台水路污染现状及水样16S rDNA高通量测序分析

目的调查口腔综合治疗台水路(DUWLs)污染现状,探讨预防控制措施。方法选取北京市某医院13台独立水源口腔综合治疗台,采用7.5‰的次氯酸钠消毒管路,采集消毒前、后三用枪和高速手机水样,计算菌落总数,采集其中两台牙椅三用枪和高速手机水样进行16S rDNA高通量测序进行物种相对丰度、β多样性分析。结果DUWLs三用枪位点消毒前菌落总数为(1621±853)CFU/ml,消毒后菌落总数为(3±7)CFU/ml;高速手机位点消毒前菌落总数为(2787±1239)CFU/ml,消毒后菌落总数为(3±8)CFU/ml;DUWLs消毒前后三用枪和高速手机水样菌落总数比较均有统计学差异(P<0.05)。DUWLs水样中主要门是变形菌门,相对丰度超过65%,其他门包括浮霉菌门、拟杆菌门、蓝藻门、放线菌门、螺旋菌门等,消毒后,物种相对丰度降低,尤其是鞘氨醇单胞菌属、浮霉菌属。Beta多样性分析显示,消毒前后两组间β多样性差异有统计学意义(P<0.05)。结论DUWLs存在微生物污染,其内壁有生物膜,存在引起医院感染的潜在风险。使用7.5‰的次氯酸钠消毒剂对DUWLs具有较好的消毒效果,可常规应用于临床口腔综合治疗台。