干湿交替水分胁迫对水稻土细菌群落影响的研究

[目的]连续3次风干-湿润循环培养水稻土,在DNA和RNA水平下,探究细菌对干湿交替胁迫的响应机制,明确风干水稻土能否代替新鲜土壤进行细菌群落组成分析.[方法]针对我国江苏省常熟市水稻土,开展新鲜土壤的3次风干-湿润循环连续培养处理(每次循环中风干、湿润状态各维持7 d),在DNA和RNA水平应用16S rRNA基因高...     

Rapid diagnosis of leptospirosis in patients with different clinical manifestations by 16S rRNA gene based nested PCR

Leptospirosis, a zoonosis of global importance and it is underreported in India and more than 50,000 severe cases are reported each year. Here we present the evaluation of 16S rRNA based nested PCR assay for the rapid identification of human leptospires using serum and urine samples. The study includes 261 suspected cases for leptospirosis with different clinical manifestations. 16S rRNA based nested PCR assay was compared and evaluated against the conventional serological methods such as MAT and ELISA. The technique enabled amplification of a 289 bp product with notable percentage of positivity in all sample groups including 94.8 in pediatric cases, 93 in pregnant women, 94.2 in renal failure, 87.8 in jaundice and 94.6 in common febrile cases. The sensitivity and specificity was 94.4% and 100%, respectively. The technique proved to be prompt and effective for the diagnosis of leptospiral infection at the acute phase of the disease. PCR based approach detects leptospiral DNA from the clinical samples both at the acute and leptospiruria phase on comparison with its counter parts where detection is made possible only after 7 days or 7–30 days post-infection. In this regard PCR based diagnosis of leptospirosis should be made available for clinicians for the early diagnosis and prompt treatment of the disease.     

Rapid Aeromonas hydrophila identification by TaqMan PCR assay: comparison with a phenotypic method

Aims:  Aeromonas hydrophila is recognized as a human pathogen following wound exposure or ingestion of contaminated water and food. For rapid identification of this bacterium, a TaqMan-based real-time PCR assay has been developed.
Methods and Results:  Primers and probes that target specific sequences of the 16S rRNA gene and cytolytic enterotoxin gene ( aerA ) were combined in a duplex assay. Presence and size of PCR products were confirmed with microchannel fluidics electrophoresis analysis. After validation, using type strain CIP7614T DNA, the PCR assay was tested on 12 positive and negative controls. Twenty-one Aeromonas strains were isolated from environmental samples and were identified with biochemical tests as Aer. sobria , Aer. caviae and Aer. hydrophila . Only Aer. hydrophila strains tested positive by PCR assay.
Conclusions:  The PCR developed here was successfully applied for the identification of Aer. hydrophila from reference, clinical and environmental samples and showed a high discrimination between Aer. hydrophila and other Aeromonas species.
Significance and Impact of the Study:  This molecular method is convenient, rapid (2·5 h vs 24 h), specific to identify Aer. hydrophila and usable for diagnosis in medical and veterinary laboratories.     

实验动物CAR杆菌16SrRNA基因PCR监测方法的建立

目的建立CAR杆菌的PCR监测方法 ,筛查国内部分实验动物样本中CAR杆菌携带状况。方法利用CAR杆菌的特有16SrRNA基因序列片段267bp设计引物,通过从日本实验动物中央研究所获取的CAR标准株DNA,建立实验动物CAR杆菌16SrRNA基因PCR监测方法。结果利用建立的CAR杆菌16SrRNA基因PCR监测方法对国内455份实验动物样本进行筛查,未检出CAR杆菌感染。结论建立了敏感性好,特异性高的实验动物CAR杆菌PCR监测方法 ,未见动物携带CAR杆菌。     

宁夏地区春季奶牛场牛舍土壤细菌群落特征

【背景】现代规模化生产模式下,牛舍环境管理是影响奶牛高效健康生产的重要因素。【目的】探讨牛场不同牛舍土壤细菌群落特征,为奶牛健康生产提供理论依据。【方法】采集宁夏某规模化奶牛场的哺乳犊牛岛、断奶犊牛舍、育成牛舍、低产泌乳牛舍、高产头胎泌乳牛舍、高产经产泌乳牛舍、干奶牛舍和病牛舍这8个不同牛舍的土样,每个牛舍6个重复,共48份土样。利用16S rRNA基因扩增子测序分析细菌群落结构与多样性,并对细菌群落的功能进行预测。【结果】不同牛舍土样细菌群落组成存在差异,并且8个牛舍中高产头胎泌乳牛舍土样的细菌群落多样性最高。哺乳犊牛岛土壤与其他牛舍土壤细菌群落在门水平上差异较大;泌乳期牛舍土样之间的细菌群落结构相似度较高。在门的水平上,拟杆菌门(Bacteroidetes)、变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和厚壁菌门(Firmicutes)是这8个牛舍土样共有的优势菌门。在属的水平上,嗜盐碱的盐单胞菌属(Halomonas)、具有潜在降解特性的Fermentimonas和栖海面菌属(Aequorivita)及致病菌的鸟杆菌属(Ornithobacterium)是犊牛期牛舍土样的优势菌属;嗜盐碱的Truepera是育成牛舍土样的优势菌属;致病菌的不动杆菌属(Acinetobacter)和Parapedobacter、耐药菌的Pedobacter是泌乳期牛舍土样的优势菌属。【结论】致病菌和参与硝酸盐呼吸的细菌主要分布在哺乳犊牛岛,嗜盐碱菌主要分布在断奶犊牛舍和育成牛舍,产甲烷的细菌主要分布在高产头胎泌乳牛舍。本研究分析了不同牛舍土壤细菌群落多样性,为奶牛健康生产提供理论依据。     

Identification of mixed bacterial DNA contamination in broad-range PCR amplification of 16S rDNA V1 and V3 variable regions by pyrosequencing of cloned amplicons

Using a sensitive and rapid method combining broad-range PCR amplification of bacterial 16S rDNA fragments and pyrosequencing for detection, identification and typing, we have found contaminating bacterial DNA in our reagents used for PCR. Identified bacteria are the water-borne bacterial genera Pseudomonas, Stenotrophomonas, Xanthomonas, Ralstonia and Bacillus. Our results are in concordance with recent reports of contaminated industrial water systems. In light of this conclusion, we believe that there is a need for increased awareness of possible contamination in uncertified widely used molecular biology reagents, including ultra-pure water. Since sequence-based 16S rDNA techniques are used in a variety of settings for bacterial typing and the characterization of microbial communities, we feel that future certification of molecular biology reagents, as free of nucleic acids, would be advantageous.     

Use of Pyromark Q96 ID pyrosequencing system in identifying bacterial pathogen directly with urine specimens for diagnosis of urinary tract infections

Detection and identification of bacterial etiology in urine is critical for accurate diagnosis and subsequent rational treatment of urinary tract infections (UTIs). Urine culture followed by a series of biochemical reactions is currently the standard method for detecting and distinguishing microorganisms associated with UTIs. The whole procedure commonly takes more than 24 h. Here we developed a new system combining 16S rRNA gene broad-range PCR with pyrosequencing technology that allows for bacteria detection and identification in urine in 5 h. To evaluate this system for rapid diagnosis of bacteriuria, 768 urine specimens were collected from patients with suspected UTIs and were tested side-by-side using standard urine culture-based identification method and the pyrosequencing method. The results from pyrosequencing correlated well with those from traditional culture-based identification method. The overall agreement between these two methods reached 98.0% (753/768). In addition, we tested the sensitivity of pyrosequencing method and determined that urine bacterial numbers as low as 10⁴ cfu/ml could be accurately detected and identified. In conclusion, compared with traditional biochemical method, the PCR-pyrosequencing system significantly improved the detection and identification of bacteriuria with shorter time, higher accuracy, and higher throughput, thus allowing earlier pathogen-adapted antibiotic therapy for patients.     

Molecular bacterial diversity of a forest soil under residue management regimes in subtropical Australia

A major operational change in exotic pine plantations of subtropical Australia has been the decision to retain postharvest residues on site. A long-term field experiment was established in February 1996 to examine the impacts of residue management regimes [i.e. the postharvest residues removed (G0R), natural amount of residues retained (G1R) and residue quantity doubled and retained (G2R)] on tree growth (F1 hybrid pine) and sustainable soil management. Twelve soil samples, which included the above three residue regimes with four replicates, were collected at plantation age 6.4 years. A 16S rRNA gene clone library was established following soil community DNA extraction, polymerase chain reaction amplification and cloning. A total of 324 clones, including 27 from each sample, were randomly selected and sequenced to represent the bacterial composition and diversity of the clone library and thus the soil bacterial community under the residue management regimes. Phylogenetic analyses indicated that Acidobacteria (37.6%) and Proteobacteria (35.6%) were the dominant components of the soil bacterial community, followed by Actinobacteria (14.7%), Chlamydiae/Verrucomicrobia (7.3%), Unclassified Bacteria (3.8%) and Gemmatimonadetes (1.0%). Analysis of molecular variance revealed that there was no significant difference in bacterial composition and diversity among the residue management regimes or their replicated samples.     

Genetic variation in 16S-23S rDNA internal transcribed spacer regions and the possible use of this genetic variation for molecular diagnosis of Bacteroides species

The structural variation in 16S-23S rDNA internal transcribed spacer regions (ITS) among Bacteroides species was assessed by PCR amplification and sequencing analysis, and its possible use for molecular diagnosis of these species was evaluated. Ninety strains of the genus Bacteroides, including the species B. distasonis, B. eggerthii, B. fragilis, B. ovatus, B. thetaiotaomicron, B. uniformis and B. vulgatus, produced one to three ITS amplification products with sizes ranging from 615 to 810 bp. Some Bacteroides strains could be differentiated at species level on the basis of ITS amplification patterns and restriction fragment length polymorphism (RFLP) analysis using a four-nucleotide-recognizing enzyme, Msp I. The results of sequence analysis of ITS amplification products revealed genes for Ile-tRNA and Ala-tRNA in all strains tested. The nucleotide sequence, except for that in tRNA-coding regions, was highly variable and characteristic for each species, but a common sequence among B. fragilis, B. thetaiotaomicron and B. ovatus was observed. A digoxigenin-labeled oligonucleotide probe (named FOT1), which was designed from this conserved sequence, specifically hybridized to the ITS amplification products from B. fragilis, B. thetaiotaomicron and B. ovatus. These results suggest that the ITS region is a useful target for the development of rapid and accurate techniques for identification of Bacteroides species.     

Identification of dominant bacterial phylotypes in a cadmium-treated forest soil

The presence of heavy metals in soils can lead to changes in microbial community structure, characterized by the dominance of groups that are able to tolerate contamination. Such groups may provide good microbial indicators of heavy-metal pollution in soil. Through terminal restriction fragment length polymorphism (T-RFLP) profiling, changes in the bacterial community structure of an acidic forest soil that had been incubated with cadmium (Cd) for 30 days were investigated. T-RFLP revealed, in particular, three operational taxonomic units (OTUs) strongly dominating in relative abundance in the contaminated soil. By cloning of the amplified 16S rRNA genes and partial sequencing of 25 clones, these three dominant OTUs were phylogenetically characterized. One dominant OTU in the cadmium-contaminated soil was derived from Betaproteobacteria, genus Burkholderia, and the other two were from uncultured members of the class Actinobacteria, closely related to the genus Streptomyces. To confirm T-RFLP data, four primers were designed on the basis of this study's dominant sequences, targeting the OTUs corresponding to Burkholderia or Actinobacteria. Real-time PCR showed that Burkholderia target sequences were more abundant in cadmium-treated soil (7.8 x 10(7)+/- 3.0 x 10(7) targets g(-1) soil) than in untreated soil (4.0 x 10(6)+/- 8.9 x 10(5) targets g(-1) soil). It was concluded that the genus Burkholderia includes species that may be particularly dominant under cadmium contamination.     

胜利油田单12高温油藏非培养和富集培养样品的细菌组成特性

[目的]通过比较分析油藏样品的微生物群落结构特点,认识油藏微生物的生态功能.[方法]利用3种油藏微生物研究中常用的富集培养方法,对胜利油田单12区块S12-4油井产出水样品进行了选择性富集培养,运用构建16S rRNA基因文库的方法分析了富集样品和非培养样品的细菌多样性.[结果]通过16S rRNA基因序列比对发现,非培养样品、异养菌富集样品、烃降解菌富集样品和硫酸盐还原菌富集样品中的优势菌分别为Pseudomonas属,Thermotoga属,Thermaerobacter属和Thermotoga属的成员.多样性分析结果表明,非培养样品的微生物多样性最丰富,同时非培养样品和富集样品的微生物群落结构存在很大的差异,富集样品中的微生物包括优势菌在油藏原位环境中含量很低.[结论]细菌组成差异的比较结果,对油藏微生物的生态功能研究和微生物驱油潜力评估具有重要意义.     

Plant teratologies as a result of phytoplasma infections

The direct correlation between teratological cases and phytoplasma infections was ascertained in spontaneous and cultivated plant species. Plants, belonging to 31 species and 12 families, showing symptoms of growth abnormalities were collected and analysed. Attempted detection of Rhodococcus fascians by isolation, PCR indexing and 16S rRNA sequencing from fasciated tissues allowed to exclude its presence. Nested PCR by universal primers and 16S rRNA sequence analyses indicated the presence of phytoplasmas, belonging to six groups, in the 44% of symptomatic samples. Among the infected species, Austrocylindropuntia exaltata, Opuntia subulata, Euphorbia characias, Euphorbia dendroides, Euphorbia linifolia, Euphorbia myrsinites, Rumex buchephalophorus, Linaria multicaulis and Fedia cornucopiae represent new phytoplasma hosts world-wide. Moreover this is the first report of phytoplasma belonging to subgroup 16SrRNA II-I in Italy. These findings together with the known erratic distribution in plant tissues of these phloem-restricted prokaryotes indicate a close correlation between fasciation and similar growth disorders and phytoplasma infections.     

一株耐盐细菌的16S rRNA基因序列分析

从舟山群岛滩涂土壤中获得了海水和底泥样品,并从中提取到了一株耐盐细菌,通过用不同盐浓度的培养基培养,挑取单菌落,反复划线纯化,得到了耐盐菌的单菌落。通过菌株基因组DNA的提取、菌株的抗性实验、质粒的提取、16S rRNA的PCR扩增及克隆、16S rRNA的全序列分析等手段,对该菌株的16S rRNA的基因序列进行了研究。     

Identification of the emerging skin pathogen Corynebacterium amycolatum using PCR-amplification of the essential divIVA gene as a target

The actinomycete Corynebacterium amycolatum is a saprophytic bacterium usually associated with the human skin, but it is at present considered an emergent pathogen as it is isolated from nosocomial settings from samples of immunosuppressed patients. The conventional method to distinguish C. amycolatum from closely related species is mainly based on phenotypic or chemotaxonomic studies. We developed a molecular method to identify rapidly C. amycolatum based on the use of different primers for amplification of the cell division divIVA gene using conventional or real-time PCR. This technique was used for the first time to distinguish C. amycolatum from the closely related Corynebacterium striatum, Corynebacterium minutissimum and Corynebacterium xerosis, without the requirement of further molecular analysis. The suitability of the identification method was tested on 51 clinical isolates belonging to the nonlipophilic fermentative group of corynebacteria (cluster C. striatum/C. amycolatum), which were accurately characterized by sequencing a 0.8 kb fragment of the 16S rRNA gene.     

鼠棒状杆菌PCR检测方法的建立及其应用

目的建立鼠棒状杆菌PCR检测方法并应用于临床样本检测。方法用脑心浸出液培养基复苏、培养鼠棒状杆菌（corynebacteriumkutscheri,C．kutscheri）并提取基因组DNA作模板;根据GenBank中C．kutsche6的16S基因序列设计合成引物,建立鼠棒状杆菌PCR检测方法并进行敏感性和特异性的评价;人工感染昆明鼠,建立小鼠棒状杆菌感染模型,采集肝脏和肾脏,提取DNA进行检测。结果成功建立了鼠棒状杆菌PCR检测方法,该方法可检测到100个阳性质粒;对小鼠沙门氏菌、肺炎链球菌和巴氏杆菌无交叉反应;全部8个人工感染样本全部检测为阳性。结论建立的鼠棒状杆菌PCR检测方法灵敏度高、特异性好,可作为鼠棒状杆菌感染的快速检测方法。     

Diversity of endophytic bacterial communities in poplar grown under field conditions

Bacterial endophytes may be important for plant health and other ecologically relevant functions of poplar trees. The composition of endophytic bacteria colonizing the aerial parts of poplar was studied using a multiphasic approach. The terminal restriction fragment length polymorphism analysis of 16S rRNA genes demonstrated the impact of different hybrid poplar clones on the endophytic community structure. Detailed analysis of endophytic bacteria using cultivation methods in combination with cloning of 16S rRNA genes amplified from plant tissue revealed a high phylogenetic diversity of endophytic bacteria with a total of 53 taxa at the genus level that included Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. The community structure displayed clear differences in terms of the presence and relative proportions of bacterial taxa between the four poplar clones studied. The results showed that the genetic background of the hybrid poplar clones corresponded well with the endophytic community structure. Out of the 513 isolates and 209 clones identified, Actinobacteria, in particular the family Microbacteriaceae, made up the largest fraction of the isolates, whereas the clone library was dominated by Alpha- and Betaproteobacteria. The most abundant genera among the isolates were Pseudomonas and Curtobacterium, while Sphingomonas prevailed among the clones.     

Comparative sequence analysis of bacterial symbionts from the marine sponges Geodia cydonium and Ircinia muscarum

Marine sponges (Porifera) live in a symbiotic relationship with microorganisms, primarily bacteria. Recently, several studiesindicated that sponges are the most prolific source of biologically-active compounds produced by symbiotic microorganisms ratherthan by the sponges themselves. In the present study we characterized the bacterial symbionts from two Demospongiae, Irciniamuscarum and Geodia cydonium. We amplified 16S rRNA by PCR, using specific bacterial-primers. The phylogenetic analysisrevealed the presence of nine bacterial clones from I. muscarum and ten from G. cydonium. In particular, I. muscarum resultedenriched in Bacillus species and G. cydonium in Proteobacterium species. Since these bacteria were able to produce secondarymetabolites with potential biotechnological and biopharmaceutical applications, we hypothesized that I. muscarum and G. cydoniumcould be a considered as a “gold mine” of natural products.