一株抗香蕉枯萎病内生细菌的分离鉴定及其抗病促生作用

从巴西蕉中分离到一株能够明显抑制尖孢镰刀菌古巴专化型4 号生理小种(FOC4)的内生细菌DB09208, 通过生理生化、形态特征及16S rDNA 序列同源性比较, 初步认为它和类芽孢杆菌(Paenibacillus)比较接近。该菌株能够显著抑制FOC4 菌丝生长, 抑菌率为74.09%。盆栽试验表明,该菌株能够抑制香蕉枯萎病的发生, 防治效果达为65.2%, 该菌同时能显著促进香蕉苗生根, 促生效果与植物生长素IAA 相似, 显示出良好的开发前景。     

可可西里土壤样品中细菌多样性的分析

本实验采用纯培养和免培养相结合的方法对来源于可可西里的一份土壤样品中的细菌多样性进行了初步研究。纯培养实验使用了6种分离培养基, 共得到细菌19株, 其中放线菌7株, 非放线菌细菌12株。这些菌株分别属于叶杆菌属(Phyllobacterium)、贪食菌属(Variovorax)、假单胞菌属(Pseudomonas)、链霉菌属(Streptomyces)、小月菌属(Microlunatus)、原小单胞菌属(Promicromonospora)、韩国生工菌属(Kribbella)和芽孢杆菌属(Bacillus) 8个属。免培养分析采用基于通用引物PCR 扩增的细菌16S rRNA 基因文库的方法以及变性梯度凝胶电泳(DGGE)。16S rRNA基因文库分析结果表明, 该土壤样品细菌群落可划分为19个OTUs, 分属于5个不同纲, 优势顺序为β-Proteobacteria (75%), α-Proteobacteria (9%), γ-Proteobacteria (7%), Actinobacteria (7%), Firmicutes (2%)。变性梯度凝胶电泳分析表明, 该样品细菌多样性指数Shannon-wiener index为2.68, 表明其中微生物多样性较低, 这可能和其所处的极端环境有一定关系。比较纯培养和免培养的实验结果发现, 土壤中的一些优势细菌并没有被有效地分离, 需要在针对特定微生物设计特定培养基及培养条件进行选择性分离上做更多的探索研究。     

Enrichment and identification of large filamentous sulfur bacteria related to Beggiatoa species from brackishwater ecosystems of Tamil Nadu along the southeast coast of India

Beggiatoa species are filamentous sulfide-oxidizing bacteria belonging to the family Beggiatoaceae that contains several largest bacteria known today. These large sulfur bacteria occur in diverse ecosystems and play an important role in the global sulfur, nitrogen and phosphorus cycle. In this study, sediment samples from brackishwater shrimp culture ponds and other brackishwater ecosystems from Tamil Nadu, southeast coast of India, were enriched for Beggiatoa species. Extracted hay medium supplemented with catalase was used and were incubated for two weeks at 28°C. Out of seven set-ups, four yielded positive growth of filamentous sulfide-oxidizing bacteria. The filaments were several millimeters long, ranged in width between 2 and 15μm and exhibited typical gliding motility. The 16S rRNA gene of four single filaments representing the four positive enrichments was subjected to PCR-DGGE followed by sequencing. All four filaments were affiliated to the Beggiatoaceae, but showed less than 89% identity with the Beggiatoa type strain Beggiatoa alba and less than 93% identity with any other sequence of the family. One of the four filaments revealed a nearly full-length 16S rDNA sequence (1411bp) and it formed a monophyletic cluster with two of the partial DGGE-16S rRNA gene sequences (99-100% identity) within the Beggiatoa species cluster. These organisms could possibly represent a novel genus within the family Beggiatoaceae. The fourth partial sequence affiliated with less than 93% sequence identity to the genera Parabeggiatoa, Thioploca and Thiopilula, and was likewise strongly delineated from any sequence published in the family.     

Multiplex 16S rRNA haplotype-specific PCR, a rapid and convenient method for fish species identification: an application to West African Clupeiform larvae

A multiplex haplotype-specific polymerase chain reaction (MHS-PCR) method was developed, which identified seven Clupeiform species living in the tropical Eastern Atlantic region: Sardinella aurita, Sardinella maderensis, Ethmalosa fimbriata, Sardina pilchardus, Engraulis encrasicholus, Pellonula leonensis and Ilisha africana. 16S rRNA fragments were amplified using a species-specific set of primers, yielding species-specific size fragments, and then separated using agarose gel electrophoresis, enabling direct visual identification of targeted species. This method provides an accurate, easy and rapid tool for identifying species within large Clupeiform samples. It is suitable for investigations on early Clupeiform stages, species and identification in fishery management in the tropical Eastern Atlantic area.     

猪源绿色气球菌的分离鉴定与药敏特性研究

目的鉴定研究2006年和2008年本实验室分别从广东清远和广西北流两个猪场发病小猪的膝关节积液中分离到2株呈四联状或成对排列,在山羊血琼脂平板上能形成明显的α-溶血圈的G+球菌。方法采用小白鼠致病性试验、细菌常规生理生化鉴定、药敏试验、耐药基因检测及16S rRNA序列分析等方法对上述分离的2株细菌进行鉴定及药敏特性研究。结果确定这2株G+球菌为正常情况下非致病性绿色气球菌(登录号分别为:GQ161096;GQ161097)。系统发育分析表明,两分离株与绿色气球菌15MS(登录号:EU075039.1)同源性分别高达99.4%和98.7%。药敏试验及耐药基因检测发现,这2株绿色气球菌除了对新霉素、氟哌酸、恩诺沙星、菌必治和环丙沙星等5种药物呈高敏外,对先锋V等13种抗菌素均不敏感。耐药基因检测结果表明,GXBL-1可检出6个耐药基因,GDQY-1则可检出多达10个耐药基因。结论从这2株分离株耐药谱检测结果可以看出,正常菌株同样携带多重耐药基因,存在着通过质粒转座子和整合子将耐药基因转移给敏感菌,导致耐药性传播的可能,应引起足够重视。     

Molecular detection of Candidatus Rickettsia asembonensis in fleas collected from pets and domestic animals in Puducherry,India

Rickettsia are obligate intracellular pathogens transmitted by arthropod vectors. The re-emergence of several rickettsioses imposes severe global health burden. In addition to the well-established rickettsial pathogens, newer rickettsial species and their pathogenic potentials are being uncovered. There are many reports of spotted and typhus fever caused by rickettsiae in India. Hence, in this study we screened the ectoparasites of pet and domestic animals for the presence of rickettsia using polymerase chain reaction. Nine cat flea samples (Ctenocephalides felis felis), that tested positive for the presence of rickettsia were subjected to Multi Locus Sequence Typing. Nucleotide sequencing and Phylogenetic analysis of gltA, ompB and 16rrs genes revealed that the rickettsiae detected in cat fleas was Rickettsia asembonensis. Further studies are required to assess Rickettsia asembonensis pathogenic potential to human and its enzootic maintenance of in various hosts and vectors.     

A rapid,simple and sensitive loop-mediated isothermal amplification method to detect Anaplasma bovis in sheep and goats samples

A loop-mediated isothermal amplification (LAMP) technique has been widely used in detecting the nucleic acid of various pathogenic bacteria. In this study, a set of four LAMP primers was designed to specifically test Anaplasma bovis. The LAMP assay was performed at 62 °C for 60 min in a water bath. The specificity was confirmed by amplifying A. bovis isolate, while no cross reaction was observed with other five pathogens (Anaplasma bovis, Anaplasma phagocytophilum, Theileria luwenshuni, Babesia motasi and Schistosoma japonicum). The sensitivity of LAMP was 5 × 10⁰ copies/μL, 100 times more than that of conventional PCR (5 × 10² copies/μL). Of 120 blood DNA extracted from sheep and goats field samples, 81 (67.5%), 22 (18.3%) and 43 (35.8%) were positively detected by LAMP, conventional PCR and nested PCR, respectively. The findings indicated that the developed LAMP assay is a new convenient tool for rapid and cost-effective detection of A. bovis.     

Heteroduplex mobility assay for the identification of Listeria sp and Listeria monocytogenes strains: application to characterisation of strains from sludge and food samples

One hundred and ten Listeria sp. isolates from sewage sludge were identified according to phenotypic and genotypic methods. The Listeria sp. strains isolated from five types of sludge from three sewage treatment plants in Angers (France) and the surrounding area included L. monocytogenes (55.5%), L. innocua (29.1%), L. seeligeri (13.6%) and L. welshimeri (1.8%). The majority of L. monocytogenes strains belonged to serotypes 4b, 1/2b and 1/2a. Moreover, a heteroduplex mobility assay based on the 16S rRNA sequences was tested for its ability to identify the six species of the genus Listeria. This study, performed on 283 Listeria sp. strains from human, food and sewage sludge samples, showed that all the species were distinguishable from one another. L. innocua and L. seeligeri showed respectively three and two distinct banding patterns. Within L. monocytogenes, four groups (I-IV) were defined. The majority of food and environmental isolates were clustered in group I and it is noteworthy that group IV clustered epidemiologic isolates and strains belonging to serotypes 4b, 1/2a and 1/2b.     

A method for low volume and low Se concentration samples and application to paired cerebrospinal fluid and serum samples

The well-known beneficial health effects of Se have demanded the development of rapid and accurate methods for its analysis. A flow injection (FI) method with inductively coupled plasma mass spectrometry (ICP-MS) as a selenium-selective detector was optimized. Flow injection was carried out using a Knauer 1100 smartline inert series liquid chromatograph coupled with a Perkin Elmer DRC II ICP-mass spectrometer. For sample injection a Perkin Elmer electronic valve equipped with a 25 μL sample loop was employed. Before measurement, standards or samples were administered with 1 μg/L rhodium as internal standard for correction of changes in detector response according to changes in sample electrolyte concentration. The method characterization parameters are: LOD (3σ criterion): 26 ng/L, LOQ (10σ criterion): 86 ng/L, linearity: 0.05–>10 μg/L, r²=0.9999, serial or day-to-day precision at 2 μg/L: 4.48% or 5.6%. Accuracy was determined by (a) recovery experiments (CSF spiked with 2 μg/L Se); (b) comparison of FI-ICP-MS measurement with graphite furnace atomic absorption (GFAAS) measurements of 1:10 diluted serum samples; (c) Se determination in urine and serum control materials. Recovery (a) was 101.4%, measurement comparison with GFAAS (b) showed 98.8% (5 serum samples, 1:10 diluted in the range of 0.5–1.3 μg/L, compared to GFAAS determination, which was set to 100%), and accuracy was 96.8% or 105.6% for the serum or urine control material. Analysis time per sample was short and typically below 2 min for the complete measurement, including sample introduction, sample-line purge and quadruplicate Se determination.This method was used to determine Se in cerebrospinal fluid (CSF) and plasma (here parallel to GFAAS) in 35 paired serum and CSF samples. Se determination gave values in the range of 42–130 μg/L for serum and 1.63–6.66 μg/L for CSF. The median for Se in 35 individual CSF samples was 3.28 μg/L, the mean (±SD) was 3.67 (1.35) μg/L, whilst for individual serum samples the median was 81 μg/L and the mean (±SD) was 85 (26) μg/L. When relating the paired Se concentrations of CSF samples to respective serum samples it turned out that Se-CSF (behind blood brain barrier (BBB)) is independent on Se-serum concentration (before BBB).     

A universal method for the detection and identification of Aphidiinae parasitoids within their aphid hosts

Molecular methods are increasingly used to detect and identify parasites in their hosts. However, existing methods are generally not appropriate for studying complex host-parasite interactions because they require prior knowledge of species composition. DNA barcoding is a molecular method that allows identifying species using DNA sequences as an identification key. We used DNA amplification with primers common to aphid parasitoids and sequencing of the amplified fragment to detect and identify parasitoids in their hosts, without prior knowledge on the species potentially present. To implement this approach, we developed a method based on 16S rRNA mitochondrial gene and LWRh nuclear gene. First, we designed two primer pairs specific to Aphidiinae (Hymenoptera), the main group of aphid parasitoids. Second, we tested whether the amplified regions could correctly identify Aphidiinae species and found that 61 species were accurately identified of 75 tested. We then determined the ability of each primer pair to detect immature parasitoids inside their aphid host. Detection was earlier for 16S than for LWRh, with parasitoids detected, respectively, 24 and 48 h after egg injection. Finally, we applied this method to assess parasitism rate in field populations of several aphid species. The interest of this tool for analysing aphid-parasitoid food webs is discussed.     

16S-ARDRA, a tool for identification of lactic acid bacteria isolated from grape must and wine

Lactic acid bacteria (LAB) are found in a great variety of habitats, including grape must and wines. There is a close relationship between the species of LAB which develop during fermentation and the eventual quality of the wine. For these reasons analytical techniques allowing fast and reliable identification of wine LAB are needed. In this work a simple and accurate protocol for identifying species of LAB isolated from grape must and wine is presented. This protocol is based on the amplification, directly from colony, of 16S rDNA and later digestion with one of the following restriction enzymes BfaI, MseI and AluI. A sequential use of the three enzymes is proposed to simplify LAB wine identification, first MseI, then BfaI and finally, if necessary, AluI digestion. The technique was able to discriminate 32 of the 36 LAB reference species tested and allowed the identification of 342 isolates from musts and wines. The isolates belonged to the species: Lactobacillus brevis, L. collinoides, L. coryniformis, L. bilgardii, L. mali, L. paracasei, Leuconostoc mesenteroides, Oenococcus oeni, Pediococcus parvulus and P. pentosaceus.     

Development of real-time PCR and hybridization methods for detection and identification of thermophilic Campylobacter spp. in pig faecal samples

AIMS: To develop a real-time (rt) PCR for species differentiation of thermophilic Campylobacter and to develop a method for assessing co-colonization of pigs by Campylobacter spp. METHODS AND RESULTS: The specificity of a developed 5' nuclease rt-PCR for species-specific identification of Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis and of a hipO gene nucleotide probe for detection of C. jejuni by colony-blot hybridization were determined by testing a total of 75 reference strains of Campylobacter spp. and related organisms. The rt-PCR method allowed species-specific detection of Campylobacter spp. in naturally infected pig faecal samples after an enrichment step, whereas the hybridization approach enhanced the specific isolation of C. jejuni (present in minority to C. coli) from pigs. Conclusions: The rt-PCR was specific for Campylobacter jejuni, C. coli, C. lari, and C. upsaliensis and the colony-blot hybridization approach provided an effective tool for isolation of C. jejuni from pig faecal samples typically dominated by C. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Species differentiation between thermophilic Campylobacter is difficult by phenotypic methods and the developed rt-PCR provides an easy and fast method for such differentiation. Detection of C. jejuni by colony hybridization may increase the isolation rate of this species from pig faeces.     

Species-specific oligonucleotide probes for the detection and identification of Lactobacillus isolated from mouse faeces

AIMS: To develop species-specific monitoring techniques for rapid detection and identification of Lactobacillus isolated from mouse faeces. METHODS AND RESULTS: The specificity of oligonucleotide probes was evaluated by dot blot hybridization to 16S rDNA and 23S rDNA amplified by PCR from 12 Lactobacillus type strains and 100 strains of Lactobacillus isolated from mouse faeces. Oligonucleotide probes specific for each Lactobacillus species hybridized only with targeted rDNA. The Lactobacillus strains isolated from mouse faeces were identified mainly as Lactobacillus intestinalis, L. johnsonii, L. murinus and L. reuteri using species-specific probes. 16S rDNA of eight unidentified isolates were sequenced and two new probes were designed. Four of eight strains of unhybridized Lactobacillus were identified as L. johnsonii/gasseri group, and the remaining four strains as L. vaginalis. CONCLUSIONS: The species-specific probe set of L. intestinalis, L. johnsonii, L. murinus, L. reuteri and L. vaginalis in this study was efficient for rapid identification of Lactobacillus isolated from mouse faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The oligonucleotide probe set for Lactobacillus species harboured in the mouse intestine, can be used for rapid identification of lactobacilli and monitoring of the faecal Lactobacillus community.     

昆明犬肠道中乳杆菌的分离鉴定及16S rRNA同源性分析

目的 为了进一步开发动物微生态资源.方法 用生理生化表型及16SrRNA同源性分析,鉴定从昆明犬肠道内容物中分离到的7株乳杆菌,分别命名为LD1、LD2、LD3、LD4、LD5、LD6和LD7.结果 LD1、LD2、LD4为路氏乳杆菌(Lactobacillus reuteri),LD3、LD5、LD6、LD7为鼠乳杆菌(Lactobacillus murinus).结论 该研究为犬用微生态制剂及益生菌的开发应用奠定基础.     

Molecular application for identification of polycyclic aromatic hydrocarbons degrading bacteria (PAHD) species isolated from oil polluted soil in Dammam,Saud Arabia

Soil contamination with petroleum hydrocarbon products such as diesel and engine oil is becoming one of the major environmental problems. This study describes hydrocarbons degrading bacteria (PHAD) isolated from long-standing petrol polluted soil from the eastern region, Dammam, Saudi Arabia. The isolated strains were firstly categorized by accessible shape detection, physiological and biochemistry tests. Thereafter, a technique established on the sequence analysis of a 16S rDNA gene was used. Isolation of DNA from the bacterial strains was performed, on which the PCR reaction was carried out. Strains were identified based on 16S rDNA sequence analysis, As follows amplified samples were spontaneously sequenced automatically and the attained results were matched to open databases. Among the isolated bacterial strains, S1 was identified as Staphylococcus aureus and strain S1 as Corynebacterium amycolatum.     

Comparison of 16S rRNA sequencing with conventional and commercial phenotypic techniques for identification of enterococci from the marine environment

AIMS: To compare accuracy of genus and species level identification of presumptive enterococci isolates from the marine environment using conventional biochemical testing, four commercial identification systems and 16S rRNA sequence analysis. METHODS AND RESULTS: Ninety-seven environmental bacterial isolates identified as presumptive enterococci on mEI media were tested using conventional and Enterococcus genus screen biochemical tests, four commercial testing systems and 16S rRNA sequencing. Conventional and Enterococcus genus screen biochemical testing, 16S rRNA sequencing and two commercial test systems achieved an accuracy of > or = 94% for Enterococcus genus confirmation. Conventional biochemical testing and 16S rRNA sequencing achieved an accuracy of > or = 90% for species level identification. CONCLUSIONS: For confirmation of Enterococcus genus from mEI media, conventional or genus screen biochemical testing, 16S rRNA sequencing and the four commercial systems were correct 79-100% of the time. For speciation to an accuracy of 90% or better, either conventional biochemical testing or 16S rRNA sequencing is required. SIGNIFICANCE AND IMPACT OF THE STUDY: Accurate identification of presumptive environmental Enterococcus isolates to genus and species level is an integral part of laboratory quality assurance and further characterization of Enterococcus species from pollution incidents. This investigation determines the ability of six different methods to correctly identify environmental isolates.     

A new gel tube method for the direct detection, identification and susceptibility testing of bacteria in clinical samples

We recently developed a simple new method which is designed to separate and concentrate bacteria from a sample by centrifugation in a gel system. Bacterial enzyme activity is then detected inside the gel without further manipulation using a colorimetric or fluorogenic substrate. The method provides a rapid, direct means of detecting bacteria in clinical samples, dispensing with the 24-h period normally required to isolate colonies on agar. Various applications of the method are described below, e.g. screening of negative urine samples, identification of Escherichia coli in urine samples, identification of Staphylococcus aureus in blood culture broths and detection of oxacillin-resistant S. aureus in blood culture broths. The advantages of the gel system and other applications are discussed.     

A rapid general method for the identification of PCR products using a fibre-optic biosensor and its application to the detection of Listeria

A 200-mer fragment of the fla A gene from Listeria monocytogenes was amplified using the polymerase chain reaction (PCR) incorporating biotin- and fluorescein amadite (FAM-)labelled primers. Methods are described for isolating the single stranded FAM-labelled 200-mer. A central portion of this 200-mer was successfully hybridized onto a complementary sequence coated onto a fibre optic biosensor. Advantages in the specificity and speed of this approach compared to standard agarose gel electrophoresis and probing methods are discussed.     

A method for discovering common patterns from two RNA secondary structures and its application to structural repeat detection

We propose an ab initio method, named DiscoverR, for finding common patterns from two RNA secondary structures. The method works by representing RNA secondary structures as ordered labeled trees and performs tree pattern discovery using an efficient dynamic programming algorithm. DiscoverR is able to identify and extract the largest common substructures from two RNA molecules having different sizes without prior knowledge of the locations and topologies of these substructures. We also extend DiscoverR to find repeated regions in an RNA secondary structure, and apply this extended method to detect structural repeats in the 3'-untranslated region of a protein kinase gene. We describe the biological significance of a repeated hairpin found by our method, demonstrating the usefulness of the method. DiscoverR is implemented in Java; a jar file including the source code of the program is available for download at http://bioinformatics.njit.edu/DiscoverR.     

内蒙古原始森林桦树皮内生和表生乳酸菌分离鉴定及其特性

[背景]随着乳酸菌产业的快速发展,不同环境乳酸菌群落结构的分析和菌种资源的开发利用已经成为该领域的研究热点.从不同环境、不同来源分离、保藏乳酸菌对于优良菌种的筛选和开发利用具有重要意义.[目的]阐明内蒙古原始森林桦树皮中乳酸菌的组成,并获得丰富的野生型乳酸菌资源,为优良菌株的筛选和工业化应用提供核心菌株,从原始森林桦树...