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Sample records for bacterial isolates dna

  1. Comparison of different methods for isolation of bacterial DNA from retail oyster tissues

    Science.gov (United States)

    Oysters are filter-feeders that bio-accumulate bacteria in water while feeding. To evaluate the bacterial genomic DNA extracted from retail oyster tissues, including the gills and digestive glands, four isolation methods were used. Genomic DNA extraction was performed using the Allmag™ Blood Genomic...

  2. Comparison of Mycoplasma ovipneumoniae isolates using bacterial restriction endonuclease DNA analysis and SDS-PAGE.

    Science.gov (United States)

    Mew, A J; Ionas, G; Clarke, J K; Robinson, A J; Marshall, R B

    1985-12-01

    Sixteen isolates of Mycoplasma ovipneumoniae recovered from the nasal tract or lungs of sheep from different flocks in New Zealand were examined by bacterial restriction endonuclease DNA analysis (BRENDA) using EcoR1 and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). All isolates gave BRENDA patterns which differed entirely from one another. Following 20 serial passages (corresponding to approximately 67 generations) of an isolate, no change was detected in the BRENDA pattern. When eight isolates were examined by SDS-PAGE most bands were common but, nevertheless, each isolate was unique in the sense that they differed from one another in one or more bands. The marked heterogeneity of patterns observed when strains of M. ovipneumoniae are compared by BRENDA, together with the stability of such patterns over many generations, will enable this approach to be used to study the epidemiology of individual strains of M. ovipneumoniae within a flock.

  3. Isolation of DNA from bacterial samples of the human gastrointestinal tract

    NARCIS (Netherlands)

    Zoetendal, E.G.; Heilig, G.H.J.; Klaassens, E.S.; Booijink, C.C.G.M.; Kleerebezem, M.; Smidt, H.; Vos, de W.M.

    2006-01-01

    The human gastrointestinal (GI) tract contains a complex microbial community that develops in time and space. The most widely used approaches to study microbial diversity and activity are all based on the analysis of nucleic acids, DNA, rRNA and mRNA. Here, we present a DNA isolation protocol that i

  4. Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen suppressive soil

    Energy Technology Data Exchange (ETDEWEB)

    Hjort, K.; Bergstrom, M.; Adesina, M.F.; Jansson, J.K.; Smalla, K.; Sjoling, S.

    2009-09-01

    Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal-restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal-restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF{sup 103} of the isolate, Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

  5. Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils

    Energy Technology Data Exchange (ETDEWEB)

    Mahmoudi, Nagissa; Slater, Greg F.; Fulthorpe, Roberta R.

    2011-07-01

    Polycyclic aromatic hydrocarbons (PAHs) are in the environment and are carcinogenic, teratogenic and mutagenic. Their hydrophobic structure gives them low water solubility and makes them readily absorbed onto soils and sediments, where they persists until they are degraded. Microbial degradation of PAHs has been well documented and is thought to be an important process in remediating contaminated sediments and soils. Obtaining high quality purified DNA is an essential requirement for the successful DNA amplifications that underlie all subsequent procedures. Several commercial DNA extraction kits exist that provide consistent solutions for the central problems - cell lysis and humic acid removal. This study compared four commercial DNA extraction kits to extract pure, high quality bacterial and eukaryotic DNA from PAH contaminated soils and concluded that they can be used on a wide variety of soils, including heavily contaminated soils. The PowerSoil kit was the most effective and reliable.

  6. Bacterial diversity in spent mushroom compost assessed by amplified rDNA restriction analysis and sequencing of cultivated isolates.

    Science.gov (United States)

    Ntougias, Spyridon; Zervakis, Georgios I; Kavroulakis, Nektarios; Ehaliotis, Constantinos; Papadopoulou, Kalliope K

    2004-11-01

    Spent mushroom compost (SMC) is the residual by-product of commercial Agaricus spp. cultivation, and it is mainly composed of a thermally treated cereal straw/animal manure mixture colonized by the fungal biomass. Research on the valorization of this material is mainly focusing on its use as soil conditioner and plant fertilizer. An investigation of the bacterial diversity in SMC was performed using molecular techniques in order to reveal the origin of SMC microflora and its potential effect on soil microbial communities after incorporation into agricultural soils. The bacterial population was estimated by the plate count method to a mean of 2.7 10(9) colony forming units (cfu) per g of dry weight, while the numbers of Gram-positive and Gram-negative bacteria were 1.9 10(9) and 4.9 10(8) cfu per g dw respectively as estimated by enumeration on semi-selective media. Fifty bacterial isolates were classified into 14 operational taxonomic units (OTUs) following ARDRA-PCR of the 16S rDNA gene. Sequencing of the 16S rDNA amplicon assigned 12 of the 14 OTUs to Gram-positive bacteria, associated with the genera Bacillus, Paenibacillus, Exiguobacterium, Staphylococcus, Desemzia, Carnobacterium, Brevibacterium, Arthrobacter and Microbacterium of the bacterial divisions Firmicutes and Actinobacteria. Two bacterial groups have phylogenetic links with the genera Comamonas and Sphingobacterium, which belong to beta-Proteobacteria and Bacteroidetes respectively. Two potentially novel bacteria are reported, which are associated with the genera Bacillus and Microbacterium. Most of the bacteria identified are of environmental origin, while strains related to species usually isolated from insects, animal and clinical sources were also detected. It appears that bacterial diversity in SMC is greatly affected by the origin of the initial material, its thermal pasteurization treatment and the potential unintended colonization of the mushroom substrate during the cultivation process.

  7. Radiation Induced DNA Double Strand Break Studies of a Metal Sensitive Novel Bacterial Isolate from East Calcutta Wetland

    Directory of Open Access Journals (Sweden)

    Sanhita Chowdhury

    2009-01-01

    uncultivable anaerobic bacterial isolate and predict the growth conditions for the isolate. On irradiation with 60Co γ rays the isolate showed maximum repair following 60 Gray damage. DNA polymerase inhibitor arabinose CTP inhibited the repair mechanism completely. This indicated that DNA polymerase took active part in repair process and thus the mechanism was that of homologous recombination repair.

  8. Electromagnetic Signals from Bacterial DNA

    CERN Document Server

    Widom, A; Srivastava, Y N; Sivasubramanian, S

    2011-01-01

    Chemical reactions can be induced at a distance due to the propagation of electromagnetic signals during intermediate chemical stages. Although is is well known at optical frequencies, e.g. photosynthetic reactions, electromagnetic signals hold true for muck lower frequencies. In E. coli bacteria such electromagnetic signals can be generated by electric transitions between energy levels describing electrons moving around DNA loops. The electromagnetic signals between different bacteria within a community is a "wireless" version of intercellular communication found in bacterial communities connected by "nanowires". The wireless broadcasts can in principle be of both the AM and FM variety due to the magnetic flux periodicity in electron energy spectra in bacterial DNA orbital motions.

  9. Bacterial isolates degrading aliphatic polycarbonates.

    Science.gov (United States)

    Suyama, T; Hosoya, H; Tokiwa, Y

    1998-04-15

    Bacteria that degrade an aliphatic polycarbonate, poly(hexamethylene carbonate), were isolated from river water in Ibaraki. Prefecture, Japan, after enrichment in liquid medium containing poly(hexamethylene carbonate) suspensions as carbon source, and dilution to single cells. Four of the strains, 35L, WFF52, 61A and 61B2, degraded poly(hexamethylene carbonate) on agar plate containing suspended poly(hexamethylene carbonate). Degradation of poly(hexamethylene carbonate) was confirmed by gel permeation chromatography. Besides poly(hexamethylene carbonate), the strains were found to degrade poly(tetramethylene carbonate). The strains were characterized morphologically, physiologically, and by 16S rDNA sequence analysis. Strains 35L and WFF52 were tentatively identified as Pseudomonas sp. and Variovorax sp., respectively, while strains 61A and 61B2 constitute an unidentified branch within the beta subclass of the Proteobacteria.

  10. Identification of Bacterial Species in Kuwaiti Waters Through DNA Sequencing

    Science.gov (United States)

    Chen, K.

    2017-01-01

    With an objective of identifying the bacterial diversity associated with ecosystem of various Kuwaiti Seas, bacteria were cultured and isolated from 3 water samples. Due to the difficulties for cultured and isolated fecal coliforms on the selective agar plates, bacterial isolates from marine agar plates were selected for molecular identification. 16S rRNA genes were successfully amplified from the genome of the selected isolates using Universal Eubacterial 16S rRNA primers. The resulted amplification products were subjected to automated DNA sequencing. Partial 16S rDNA sequences obtained were compared directly with sequences in the NCBI database using BLAST as well as with the sequences available with Ribosomal Database Project (RDP).

  11. Oral bacterial DNA findings in pericardial fluid

    Directory of Open Access Journals (Sweden)

    Anne-Mari Louhelainen

    2014-11-01

    Full Text Available Background: We recently reported that large amounts of oral bacterial DNA can be found in thrombus aspirates of myocardial infarction patients. Some case reports describe bacterial findings in pericardial fluid, mostly done with conventional culturing and a few with PCR; in purulent pericarditis, nevertheless, bacterial PCR has not been used as a diagnostic method before. Objective: To find out whether bacterial DNA can be measured in the pericardial fluid and if it correlates with pathologic–anatomic findings linked to cardiovascular diseases. Methods: Twenty-two pericardial aspirates were collected aseptically prior to forensic autopsy at Tampere University Hospital during 2009–2010. Of the autopsies, 10 (45.5% were free of coronary artery disease (CAD, 7 (31.8% had mild and 5 (22.7% had severe CAD. Bacterial DNA amounts were determined using real-time quantitative PCR with specific primers and probes for all bacterial strains associated with endodontic disease (Streptococcus mitis group, Streptococcus anginosus group, Staphylococcus aureus/Staphylococcus epidermidis, Prevotella intermedia, Parvimonas micra and periodontal disease (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, Fusobacterium nucleatus, and Dialister pneumosintes. Results: Of 22 cases, 14 (63.6% were positive for endodontic and 8 (36.4% for periodontal-disease-associated bacteria. Only one case was positive for bacterial culturing. There was a statistically significant association between the relative amount of bacterial DNA in the pericardial fluid and the severity of CAD (p=0.035. Conclusions: Oral bacterial DNA was detectable in pericardial fluid and an association between the severity of CAD and the total amount of bacterial DNA in pericardial fluid was found, suggesting that this kind of measurement might be useful for clinical purposes.

  12. 16S rDNA sequence as measure for identification of bacterial isolates in bulk%16S rDNA序列在大批量细菌分离物分子鉴定中的应用研究

    Institute of Scientific and Technical Information of China (English)

    陈源源; 沈微; 樊游; 陈献忠; Suren Singh; 石贵阳; 王正祥

    2013-01-01

    The feasibility of 16S rDNA sequence-based analysis for systemic classification of bacterial isolates was extensively evaluated. The 16S rDNA sequences of 3 726 bacterial strains collected in CICIM-CU were amplified by PCR and sequenced. The sequencing results were compared to reference data available at the GenBank database by using BLAST. Most of bacterial isolates (82.5% ) were assigned to species level successfully. 653 strains (17.5% ) were only could be assigned to genus level. Some closely-related species belonged to genus Bacillus and Geobacillus had highly similar 16S rDNA sequences, making 16S rDNA sequence analysis-based identification problematic. This study showed that 16S rDNA sequences were sufficiently sensitive for identifying most of bacterial isolates. It could be powerfully and ordinarily used as first-round molecular marker to systemic identification of bacterial isolates in bulk.%利用16S rDNA序列同源性分析法对保藏于中国高校工业微生物资源与信息中心(CICIM-CU)的3 726株细菌分离物进行了分子鉴定.实验结果显示:其中3 073株细菌分离物可被成功鉴定至种一级水平,分属于210个种,45个属,占整个细菌分离物的82.5%;其余653株细菌分离物可鉴定到属一级,占整个细菌分离物的17.5%.研究中也发现,16S rDNA序列在芽孢杆菌属(Bacillus)和土芽孢杆菌属(eobacillus)等少数菌属中的部分种间鉴定的敏感性不高.上述结果表明16S rDNA序列在大多数细菌种属鉴定中具有较高的特异性和敏感性,可以作为第一轮分子标识用于大批量细菌分离物的鉴定.

  13. Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

    Science.gov (United States)

    Croteau, Rodney Bruce; Crock, John E.

    2005-01-25

    A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-famesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-famesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

  14. Bacterial community analysis of activated sludge: an evaluation of four commonly used DNA extraction methods

    NARCIS (Netherlands)

    Vanysacker, L.; Declerck, S.A.J.; Hellemans, B.; De Meester, L.; Vankelecom, I.; Declerck, P.

    2010-01-01

    The effectiveness of three commercially available direct DNA isolation kits (Mobio, Fast, Qiagen) and one published direct DNA extraction protocol (Bead) for extracting bacterial DNA from different types of activated sludge was investigated and mutually compared. The DNA quantity and purity were det

  15. Genomic DNA fingerprint analysis of biotype 1 Gardnerella vaginalis from patients with and without bacterial vaginosis.

    Science.gov (United States)

    Wu, S R; Hillier, S L; Nath, K

    1996-01-01

    Of the 20 biotype 1 Gardnerella vaginalis isolates analyzed, 10 from patients with bacterial vaginosis and 10 from patients without bacterial vaginosis, none shared the same DNA fingerprint. However, a 1.18-kb HindIII fragment was common among 18 of the 20 biotype 1 isolates in a restriction fragment length polymorphism analysis with a 7.9-kb G. vaginalis DNA probe. PMID:8748302

  16. ANTIMICROBIAL ACTIVITY OF LACTIC ACID BACTERIAL ISOLATES

    Directory of Open Access Journals (Sweden)

    Utkarsha S. Shivsharan

    2013-08-01

    Full Text Available Micro-organisms have tendency to produce antimicrobial substances which show biological activity against other kind of micro-organisms. This phenomenon of bacterial antagonism is observed in lactic acid bacteria with competitive advantages. The lactic acid bacteria are commonly present in many fermented products, fruits and milk products. The variety of antimicrobial substances produced by lactic acid bacteria showing good inhibition capacity include production of lactic acid, acetic acid, hydrogen peroxide, carbon dioxide, diacetyl and bacteriocin. Bacteriocins produced by lactic acid bacteria are the subject of intense research because of their antimicrobial activity against food born bacteria such as Listeria monocytogenes, staphylococcus aureus, Bacillus cereus, Clostridium botulinum and several others .Bacteriocins may be bacteriostatic or bactericidal with narrow or broad range of activity. The main of the study was to study the antimicrobial activity of such lactic acid bacterial isolates.

  17. Detection of Bacterial Wilt Pathogen and Isolation of Its Bacteriophage from Banana in Lumajang Area, Indonesia

    Directory of Open Access Journals (Sweden)

    Hardian Susilo Addy

    2016-01-01

    Full Text Available Bacterial wilt disease on banana is an important disease in Lumajang District and causes severe yield loss. Utilizing bacteriophage as natural enemy of pathogenic bacteria has been widely known as one of the control strategies. This research was aimed at determining the causing agent of bacterial wilt on banana isolated from Lumajang area, to obtain wide-host range bacteriophages against bacterial wilt pathogen and to know the basic characteristic of bacteriophages, particularly its nucleic acid type. Causative agent of bacterial wilt was isolated from symptomatic banana trees from seven districts in Lumajang area on determinative CPG plates followed by rapid detection by PCR technique using specific pair-primer. Bacteriophages were also isolated from soil of infected banana crop in Sukodono District. Morphological observation showed that all bacterial isolates have similar characteristic as common bacterial wilt pathogen, Ralstonia solanacearum. In addition, detection of FliC region in all isolates confirmed that all isolates were R. solanacearum according to the presence of 400 bp of FliC DNA fragment. Moreover, two bacteriophages were obtained from this experiment (ϕRSSKD1 and ϕRSSKD2, which were able to infect all nine R. solanacearum isolates. Nucleic acid analysis showed that the nucleic acid of bacteriophages was DNA (deoxyribonucleic acid.

  18. Simple & Safe Genomic DNA Isolation.

    Science.gov (United States)

    Moss, Robert; Solomon, Sondra

    1991-01-01

    A procedure for purifying DNA using either bacteria or rat liver is presented. Directions for doing a qualitative DNA assay using diphenylamine and a quantitative DNA assay using spectroscopy are included. (KR)

  19. Preparation of high molecular weight gDNA and bacterial artificial chromosome (BAC) libraries in plants.

    Science.gov (United States)

    Biradar, Siddanagouda S; Nie, Xiaojun; Feng, Kewei; Weining, Song

    2014-01-01

    Bacterial artificial chromosome (BAC) libraries are extremely valuable large-insert DNA libraries for physical mapping, positional cloning, comparative genomic analysis, complete genome sequencing, and evolutionary studies. Due to their stability and relative simplicity BAC libraries are most preferred over other approaches for cloning large genomic DNA fragments for large-insert libraries. Isolation of intact high molecular weight (HMW) DNA is a critical step underlying the success of large-insert genomic DNA library construction. It requires the isolation of purified nuclei, embedding them into LMP agarose plugs, restriction digestion of the plugs, and quite often size selection using PFGE and electro-elution of insert DNA. The construction of BAC libraries is complex and challenging for most molecular laboratories. To facilitate the construction of BAC libraries, we present a step-by-step protocol for isolation of HMW DNA and construction of plant BAC libraries.

  20. DMPD: The actions of bacterial DNA on murine macrophages. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 10534106 The actions of bacterial DNA on murine macrophages. Sester DP, Stacey KJ, ... Show The actions of bacterial DNA on murine macrophages. PubmedID 10534106 Title The actions of bacterial DNA on murine macrophage

  1. Production and characterization of bacterial cellulose by Leifsonia sp. CBNU-EW3 isolated from the earthworm, Eisenia fetida

    Science.gov (United States)

    A total of five bacterial strains were isolated from earthworm, Eisenia fetida and examined for bacterial cellulose (BC) production in Hestrin–Schramm medium (HS). Among the five strains tested, CBNU-EW3 exhibited excellent BC production and was identified as Leifsonia sp. by 16S rDNA sequence analy...

  2. In vitro activity of difloxacin against canine bacterial isolates

    NARCIS (Netherlands)

    Hoven, van den J.R.; Wagenaar, J.A.; Walker, R.D.

    2000-01-01

    The in vitro activity of difloxacin against canine bacterial isolates from clinical cases was studied in the United States and The Netherlands. Minimal inhibitory concentrations (MIC), the postantibiotic effect, the effect of pH on antimicrobial activity, and the bacterial killing rate tests were de

  3. A model capturing novel strand symmetries in bacterial DNA

    Energy Technology Data Exchange (ETDEWEB)

    Sobottka, Marcelo, E-mail: sobottka@mtm.ufsc.br [Departamento de Matematica, Universidade Federal de Santa Catarina (Brazil); Hart, Andrew G., E-mail: ahart@dim.uchile.cl [Departamento de Ingenieria Matematica and Centro de Modelamiento Matematico, Universidad de Chile (Chile)

    2011-07-15

    Highlights: {yields} We propose a simple stochastic model to construct primitive DNA sequences. {yields} The model provide an explanation for Chargaff's second parity rule in primitive DNA sequences. {yields} The model is also used to predict a novel type of strand symmetry in primitive DNA sequences. {yields} We extend the results for bacterial DNA sequences and compare distributional properties intrinsic to the model to statistical estimates from 1049 bacterial genomes. {yields} We find out statistical evidences that the novel type of strand symmetry holds for bacterial DNA sequences. -- Abstract: Chargaff's second parity rule for short oligonucleotides states that the frequency of any short nucleotide sequence on a strand is approximately equal to the frequency of its reverse complement on the same strand. Recent studies have shown that, with the exception of organellar DNA, this parity rule generally holds for double-stranded DNA genomes and fails to hold for single-stranded genomes. While Chargaff's first parity rule is fully explained by the Watson-Crick pairing in the DNA double helix, a definitive explanation for the second parity rule has not yet been determined. In this work, we propose a model based on a hidden Markov process for approximating the distributional structure of primitive DNA sequences. Then, we use the model to provide another possible theoretical explanation for Chargaff's second parity rule, and to predict novel distributional aspects of bacterial DNA sequences.

  4. DNA isolation from rat tail or ear

    NARCIS (Netherlands)

    Cuppen, E.

    2010-01-01

    This protocol describes a rapid procedure for isolating DNA from rat tail or ear punches. The simplest version of the protocol can be scaled for use in 96-well (deep-well) plates. The quality of the DNA is sufficient for any polymerase chain reaction (PCR)-based genotyping approach.

  5. Binding and entry of DNA in bacterial transformation

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.A.

    1976-01-01

    Bacterial transformation in relation to DNA transport and competence in Streptococcus pneumoniae (also called Diplococcus pneumoniae) is discussed. This species will serve as a model with which to compare transformation in other bacterial species, particularly Bacillus subtilis and Haemophilus influenzae, with emphasis on the many similarities as well as differences.

  6. Purification of transfection-grade plasmid DNA from bacterial cells with superparamagnetic nanoparticles

    Science.gov (United States)

    Chiang, Chen-Li; Sung, Ching-Shan

    2006-07-01

    The functionalized magnetic nanobeads were used to develop a rapid protocol for extracting and purifying transfection-grade plasmid DNA from bacterial culture. Nanosized superparamagnetic nanoparticles (Fe 3O 4) were prepared by chemical coprecipitation method using Fe 2+, Fe 3+ salt, and ammonium hydroxide under a nitrogen atmosphere. The surface of Fe 3O 4 nanoparticles was modified by coating with the multivalent cationic agent, polyethylenimine (PEI). The PEI-modified magnetic nanobeads were employed to simplify the purification of plasmid DNA from bacterial cells. We demonstrated a useful plasmid, pRSETB-EGFP, encoding the green fluorescent protein with T7 promoter, was amplified in DE3 strain of Escherichia coli. The loaded nanobeads are recovered by magnetically driven separation and regenerated by exposure to the elution buffer with optimal ionic strength (1.25 M) and pH (9.0). Up to approximately 819 μg of high-purity (A 260/A 280 ratio=1.86) plasmid DNA was isolated from 100 ml of overnight bacterial culture. The eluted plasmid DNA was used directly for restriction enzyme digestion, bacterial cell transformation and animal cell transfection applications with success. The PEI-modified magnetic nanobead delivers significant time-savings, overall higher yields and better transfection efficiencies compared to anion-exchange and other methods. The results presented in this report show that PEI-modified magnetic nanobeads are suitable for isolation and purification of transfection-grade plasmid DNA.

  7. Conjunctival sac bacterial flora isolated prior to cataract surgery

    Directory of Open Access Journals (Sweden)

    Suto C

    2012-01-01

    Full Text Available Chikako Suto1,2, Masahiro Morinaga1,2, Tomoko Yagi1,2, Chieko Tsuji3, Hiroshi Toshida41Department of Ophthalmology, Saiseikai Kurihashi Hospital, Saitama; 2Department of Ophthalmology, Tokyo Women's Medical University, Tokyo; 3Department of Clinical Laboratory, Saiseikai Kurihashi Hospital, Saitama; 4Department of Ophthalmology, Juntendo University Shizuoka Hospital, Izunokuni, Shizuoka, JapanObjective: To determine the trends of conjunctival sac bacterial flora isolated from patients prior to cataract surgery.Subjects and methods: The study comprised 579 patients (579 eyes who underwent cataract surgery. Specimens were collected by lightly rubbing the inferior palpebral conjunctival sac with a sterile cotton swab 2 weeks before surgery, and then cultured for isolation of bacteria and antimicrobial sensitivity testing. The bacterial isolates and percentage of drug-resistant isolates were compared among age groups and according to whether or not patients had diabetes mellitus, hyperlipidemia, dialysis therapy, oral steroid use, dry eye syndrome, or allergic conjunctivitis.Results: The bacterial isolation rate was 39.2%. There were 191 strains of Gram-positive cocci, accounting for the majority of all isolates (67.0%, among which methicillin-sensitive coagulase-negative staphylococci was the most frequent (127 strains, 44.5%, followed by methicillin-resistant coagulase-negative staphylococci (37 strains, 12.7%. All 76 Gram-positive bacillary isolates (26.7% were from the genus Corynebacterium. Among the 16 Gram-negative bacillary isolates (5.9%, the most frequent was Escherichia coli (1.0%. The bacterial isolation rate was higher in patients >60 years old, and was lower in patients with dry eye syndrome, patients under topical treatment for other ocular disorders, and patients with hyperlipidemia. There was no significant difference in bacterial isolation rate with respect to the presence/absence of diabetes mellitus, steroid therapy, dialysis, or

  8. Superstatistical model of bacterial DNA architecture

    Science.gov (United States)

    Bogachev, Mikhail I.; Markelov, Oleg A.; Kayumov, Airat R.; Bunde, Armin

    2017-02-01

    Understanding the physical principles that govern the complex DNA structural organization as well as its mechanical and thermodynamical properties is essential for the advancement in both life sciences and genetic engineering. Recently we have discovered that the complex DNA organization is explicitly reflected in the arrangement of nucleotides depicted by the universal power law tailed internucleotide interval distribution that is valid for complete genomes of various prokaryotic and eukaryotic organisms. Here we suggest a superstatistical model that represents a long DNA molecule by a series of consecutive ~150 bp DNA segments with the alternation of the local nucleotide composition between segments exhibiting long-range correlations. We show that the superstatistical model and the corresponding DNA generation algorithm explicitly reproduce the laws governing the empirical nucleotide arrangement properties of the DNA sequences for various global GC contents and optimal living temperatures. Finally, we discuss the relevance of our model in terms of the DNA mechanical properties. As an outlook, we focus on finding the DNA sequences that encode a given protein while simultaneously reproducing the nucleotide arrangement laws observed from empirical genomes, that may be of interest in the optimization of genetic engineering of long DNA molecules.

  9. Extraction of ribosomal RNA and genomic DNA from soil for studying the diversity of the indigenous bacterial community

    NARCIS (Netherlands)

    Duarte, G.F.; Rosado, A.S.; Keijzer-Wolters, A.C.; Elsas, van J.D.

    1998-01-01

    A method for the indirect (cell extraction followed by nucleic acid extraction) isolation of bacterial ribosomal RNA (rRNA) and genomic DNA from soil was developed. The protocol allowed for the rapid parallel extraction of genomic DNA as well as small and large ribosomal subunit RNA from four soils

  10. Biosynthesis and structural characterization of silver nanoparticles from bacterial isolates

    Energy Technology Data Exchange (ETDEWEB)

    Zaki, Sahar, E-mail: saharzaki@yahoo.com [Environmental Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, Mubarak City for Scientific Research and Technology Applications, Alexandria, 21934 New Burgelarab City (Egypt); El Kady, M.F. [Fabrication Technology Department, Advanced Technology and New Materials Research Institute (ATNMRI), Mubarak City for Scientific Research and Technology Applications, Alexandria (Egypt); Abd-El-Haleem, Desouky [Environmental Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, Mubarak City for Scientific Research and Technology Applications, Alexandria, 21934 New Burgelarab City (Egypt)

    2011-10-15

    Graphical abstract: In this study five bacterial isolates belong to different genera were found to be able to biosynthesize silver nanoparticles. Biosynthesis and spectral characterization are reported here. Highlights: {yields} About 300 bacterial isolates were screened for their ability to produce nanosilvers {yields} Five of them were potential candidates for synthesis of silver nanoparticles {yields} Production of silver nanoparticles was examined using UV-Vis, XRD, SEM and EDS. {yields} The presence of nanoparticles with all five bacterial isolates was confirmed. -- Abstract: This study aimed to develop a green process for biosynthesis of silver nanomaterials by some Egyptian bacterial isolates. This target was achieved by screening an in-house culture collection consists of 300 bacterial isolates for silver nanoparticle formation. Through screening process, it was observed that strains belonging to Escherichia coli (S30, S78), Bacillus megaterium (S52), Acinetobacter sp. (S7) and Stenotrophomonas maltophilia (S54) were potential candidates for synthesis of silver nanoparticles. The extracellular production of silver nanoparticles by positive isolates was investigated by UV-Vis spectroscopy, X-ray diffraction (XRD), transmission electron microscope (TEM), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). The results demonstrated that UV-visible spectrum of the aqueous medium containing silver ion showed a peak at 420 nm corresponding to the plasmon absorbance of silver nanoparticles. Scanning electron microscopy micrograph showed formation of silver nanoparticles in the range of 15-50 nm. XRD-spectrum of the silver nanoparticles exhibited 2{theta} values corresponding to the silver nanocrystal that produce in hexagonal and cubic crystal configurations with different plane of orientation. In addition, the signals of the silver atoms were observed by EDS-spectrum analysis that confirms the presence of silver nanoparticles (Ag

  11. Bacterial natural transformation by highly fragmented and damaged DNA

    DEFF Research Database (Denmark)

    Overballe-Petersen, Søren; Harms, Klaus; Orlando, Ludovic Antoine Alexandre

    2013-01-01

    of DNA from a 43,000-y-old woolly mammoth bone, we further demonstrate that such natural transformation events include ancient DNA molecules. We find that the DNA recombination is RecA recombinase independent and is directly linked to DNA replication. We show that the adjacent nucleotide variations......DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often DNA is recognized as nutrient source...... for microbes, but not as potential substrate for bacterial evolution. Here, we show that fragmented DNA molecules (≥20 bp) that additionally may contain abasic sites, cross-links, or miscoding lesions are acquired by the environmental bacterium Acinetobacter baylyi through natural transformation. With uptake...

  12. Isolation and characterization of organic-sulfur degradation bacterial strain

    Institute of Scientific and Technical Information of China (English)

    YANG Yu; DIAO Meng-xue; SHI Wu-yang; LI Li; DAI Qin-yun; QIU Guan-zhou

    2007-01-01

    A bacterial strain that was capable of degrading organic sulfur (dibenzothiophene) was isolated by enrichment techniques from the petroleum-contaminated soil collected from Zhongyuan Oil Field. The strain is named ZYX and is gram-positive.This strain undergoes bacilus-coccus morphological change, and forms yellow-pigment glossy circular colonies with 1.5 mm in diameter on average after 2 d incubation on Luria-Bertani(LB) plates. The full-length of 16S rDNA sequence of strain ZYX was determined and analyzed. Strain ZYX is found most relative with the genus of Arthrobacter. The similarity values between ZYX and Arthrobacter sp. P2 is 99.53%. The main morphological, biochemical and physiological features of strain ZYX accord with those of Arthrobacter. It is found that the optimal initial pH for growth is about 7.0, and the optimal concentration of dibenzothiophene(DBT)for growth is 0.10 g/L. Additionally, the results show that the best carbon source and nitrogen source are glycerol and glutamine,respectively.

  13. Antibiogram of bacterial species isolated from canine pyometra

    Directory of Open Access Journals (Sweden)

    Madhu Swamy

    2013-06-01

    Full Text Available Aim: The aim of the present work was to ascertain the bacterial flora causing pyometra in female dogs and their antibiotic sensitivity. Materials and Methods: A study was conducted to determine the antibiogram of bacterial species isolated from 20 female dogs diagnosed with pyometra. The vaginal discharge was collected by sterile swab and streaked smoothly over Mueller Hinton medium and sensitivity towards antibiotics was determined by measuring the zone of inhibition using a Hi-media scale. Results: The antobiogram showed that Gentamicin was the most sensitive (85% antibiotic followed by Enrofloxacin, Ciprofloxacin and Amoxicillin (65%, 65% and 55%, respectively. The isolates were most resistant to Oxytetracycline (85% followed by Tetracycline, Ampicillin, Chloramphenicol, Cloxacillin and Erythromycin (80%, 80%, 75%, 70% and 70%, respectively. Conclusion: Gentamicin was found to be most effective antibiotic against the bacterial species isolated from canine pyometra. [Vet World 2013; 6(8.000: 546-549

  14. Heterogeneity in restriction patterns of Gardnerella vaginalis isolates from individuals with bacterial vaginosis.

    Science.gov (United States)

    Nath, K; Devlin, D; Beddoe, A M

    1992-02-01

    This study was undertaken to resolve the genetic make up of Gardnerella vaginalis present in bacterial vaginosis (BV). DNA from several G. vaginalis isolates from within and between individual BV patients were compared by BamHI, ClaI and EcoRI restriction endonuclease analysis (REA) followed by a restriction fragment length polymorphism (RFLP) study, utilizing a 5.7-kb BamHI G. vaginalis ATCC14018 DNA probe. Four G. vaginalis isolates from one patient (GVP-062) were composed of 3 different biotypes (biotypes 3, 5 and 8), and while the REA mirrored the biotype, in RFLP studies at least 3 isolates had DNA fragments in common. All of the isolates from 2 other patients (GVP-063 and GVP-072) represented a single biotype (biotype 2), but under REA and in RFLP studies, the isolates GVP-063 differed from GVP-072. An opposite case existed with the isolates GVP-072 (biotype 2) and GVP-065 (biotype 5), which appeared similar under REA and in RFLP studies. Finally, reisolates after 8 weeks (GVP-080) from a BV patient (isolates GVP-065) representing the same biotype (biotype 5) differed under REA and in RFLP studies. Thus, lacking any unique DNA fingerprint, G. vaginalis occurring in BV represents a (genetically) mixed population.

  15. Biodegradation of carcinogenic textile azo dyes using bacterial isolates of mangrove sediment

    Institute of Scientific and Technical Information of China (English)

    Guru Prasad Srinivasan; Asnar Sikkanthar; Anandajothi Elamaran; Caroline R Delma; Kumaran Subramaniyan

    2014-01-01

    Objective:To evaluate the biodegrading property against carcinogenic azo dyes using bacterial isolates of mangrove sediment. Methods: The bacterial isolates were subjected to submerged fermentation and their growth kinetics were studied. The potential strain was characterized using 16S rDNA sequencing. Results:In the present study, dye degrading bacterial colonies were isolated from the mangrove sediment samples of Parangipettai estuarine area, Tamil Nadu. Of the 30 morphologically different strains isolated, 5 showed antagonistic property. The growth kinetics of the two strains, P1 and G1, which showed potent activity were calculated. One particular isolate (P1) showing promising dye degrading potential in the submerged fermentation was further characterized. The strain was identified as Paenibacillus sp. by 16S rDNA sequencing. Conclusions:This study reveals the less explored microflora of mangrove sediments. The novel strain may further be analyzed and used in the treatment of effluent from dye industry so as to reduce the impact of carcinogenic contaminants.

  16. Biodegradation of carcinogenic textile azo dyes using bacterial isolates of mangrove sediment

    Directory of Open Access Journals (Sweden)

    Guru Prasad Srinivasan

    2014-02-01

    Full Text Available Objective: To evaluate the biodegrading property against carcinogenic azo dyes using bacterial isolates of mangrove sediment. Methods: The bacterial isolates were subjected to submerged fermentation and their growth kinetics were studied. The potential strain was characterized using 16S rDNA sequencing. Results: In the present study, dye degrading bacterial colonies were isolated from the mangrove sediment samples of Parangipettai estuarine area, Tamil Nadu. Of the 30 morphologically different strains isolated, 5 showed antagonistic property. The growth kinetics of the two strains, P1 and G1, which showed potent activity were calculated. One particular isolate (P1 showing promising dye degrading potential in the submerged fermentation was further characterized. The strain was identified as Paenibacillus sp. by 16S rDNA sequencing. Conclusions: This study reveals the less explored microflora of mangrove sediments. The novel strain may further be analyzed and used in the treatment of effluent from dye industry so as to reduce the impact of carcinogenic contaminants.

  17. Isolation of biologically active nanomaterial (inclusion bodies from bacterial cells

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    Peternel Špela

    2010-09-01

    Full Text Available Abstract Background In recent years bacterial inclusion bodies (IBs were recognised as highly pure deposits of active proteins inside bacterial cells. Such active nanoparticles are very interesting for further downstream protein isolation, as well as for many other applications in nanomedicine, cosmetic, chemical and pharmaceutical industry. To prepare large quantities of a high quality product, the whole bioprocess has to be optimised. This includes not only the cultivation of the bacterial culture, but also the isolation step itself, which can be of critical importance for the production process. To determine the most appropriate method for the isolation of biologically active nanoparticles, three methods for bacterial cell disruption were analyzed. Results In this study, enzymatic lysis and two mechanical methods, high-pressure homogenization and sonication, were compared. During enzymatic lysis the enzyme lysozyme was found to attach to the surface of IBs, and it could not be removed by simple washing. As this represents an additional impurity in the engineered nanoparticles, we concluded that enzymatic lysis is not the most suitable method for IBs isolation. During sonication proteins are released (lost from the surface of IBs and thus the surface of IBs appears more porous when compared to the other two methods. We also found that the acoustic output power needed to isolate the IBs from bacterial cells actually damages proteins structures, thereby causing a reduction in biological activity. High-pressure homogenization also caused some damage to IBs, however the protein loss from the IBs was negligible. Furthermore, homogenization had no side-effects on protein biological activity. Conclusions The study shows that among the three methods tested, homogenization is the most appropriate method for the isolation of active nanoparticles from bacterial cells.

  18. Two-dimensional DNA displays for comparisons of bacterial genomes

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    Malloff Chad

    2003-01-01

    Full Text Available We have developed two whole genome-scanning techniques to aid in the discovery of polymorphisms as well as horizontally acquired genes in prokaryotic organisms. First, two-dimensional bacterial genomic display (2DBGD was developed using restriction enzyme fragmentation to separate genomic DNA based on size, and then employing denaturing gradient gel electrophoresis (DGGE in the second dimension to exploit differences in sequence composition. This technique was used to generate high-resolution displays that enable the direct comparison of > 800 genomic fragments simultaneously and can be adapted for the high-throughput comparison of bacterial genomes. 2DBGDs are capable of detecting acquired and altered DNA, however, only in very closely related strains. If used to compare more distantly related strains (e.g. different species within a genus numerous small changes (i.e. small deletions and point mutations unrelated to the interesting phenotype, would encumber the comparison of 2DBGDs. For this reason a second method, bacterial comparative genomic hybridization (BCGH, was developed to directly compare bacterial genomes to identify gain or loss of genomic DNA. BCGH relies on performing 2DBGD on a pooled sample of genomic DNA from 2 strains to be compared and subsequently hybridizing the resulting 2DBGD blot separately with DNA from each individual strain. Unique spots (hybridization signals represent foreign DNA. The identification of novel DNA is easily achieved by excising the DNA from a dried gel followed by subsequent cloning and sequencing. 2DBGD and BCGH thus represent novel high resolution genome scanning techniques for directly identifying altered and/or acquired DNA.

  19. DNA repair in bacterial cultures and plasmid DNA exposed to infrared laser for treatment of pain

    Science.gov (United States)

    Canuto, K. S.; Sergio, L. P. S.; Marciano, R. S.; Guimarães, O. R.; Polignano, G. A. C.; Geller, M.; Paoli, F.; Fonseca, A. S.

    2013-06-01

    Biostimulation of tissues by low intensity lasers has been described on a photobiological basis and clinical protocols are recommended for treatment of various diseases, but their effects on DNA are controversial. The objective of this work was to evaluate effects of low intensity infrared laser exposure on survival and bacterial filamentation in Escherichia coli cultures, and induction of DNA lesions in bacterial plasmids. In E. coli cultures and plasmids exposed to an infrared laser at fluences used to treat pain, bacterial survival and filamentation and DNA lesions in plasmids were evaluated by electrophoretic profile. Data indicate that the infrared laser (i) increases survival of E. coli wild type in 24 h of stationary growth phase, (ii) induces bacterial filamentation, (iii) does not alter topological forms of plasmids and (iv) does not alter the electrophoretic profile of plasmids incubated with exonuclease III or formamidopyrimidine DNA glycosylase. A low intensity infrared laser at the therapeutic fluences used to treat pain can alter survival of E. coli wild type, induce filamentation in bacterial cells, depending on physiologic conditions and DNA repair, and induce DNA lesions other than single or double DNA strand breaks or alkali-labile sites, which are not targeted by exonuclease III or formamidopyrimidine DNA glycosylase.

  20. Segrosome complex formation during DNA trafficking in bacterial cell division

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    Maria A. Oliva

    2016-09-01

    Full Text Available Bacterial extrachromosomal DNAs often contribute to virulence in pathogenic organisms or facilitate adaptation to particular environments. The transmission of genetic information from one generation to the next requires sufficient partitioning of DNA molecules to ensure that at least one copy reaches each side of the division plane and is inherited by the daughter cells. Segregation of the bacterial chromosome occurs during or after replication and probably involves a strategy in which several protein complexes participate to modify the folding pattern and distribution first of the origin domain and then of the rest of the chromosome. Low-copy number plasmids rely on specialised partitioning systems, which in some cases use a mechanism that show striking similarity to eukaryotic DNA segregation. Overall, there have been multiple systems implicated in the dynamic transport of DNA cargo to a new cellular position during the cell cycle but most seem to share a common initial DNA partitioning step, involving the formation of a nucleoprotein complex called the segrosome. The particular features and complex topologies of individual segrosomes depend on both the nature of the DNA binding protein involved and on the recognized centromeric DNA sequence, both of which vary across systems. The combination of in vivo and in vitro approaches, with structural biology has significantly furthered our understanding of the mechanisms underlying DNA trafficking in bacteria. Here, I discuss recent advances and the molecular details of the DNA segregation machinery, focusing on the formation of the segrosome complex.

  1. Hyperchromicity and strand separation in bacterial DNA.

    Science.gov (United States)

    FREIFELDER, D; DAVISON, P F

    1962-05-01

    Studies of the per cent of strand separation of N(14)-N(15) hybrid coli DNA heated to various temperatures in formaldehyde have shown that the process of strand separation is a function of temperature and formaldehyde concentration and is directly related to the measured hyperchromicity. No strands separate until about 75 per cent of full hyperchromicity is obtained, and even at apparently full hyperchromicity a large fraction of the strands may be held together, possibly by guanine-cytosine-rich regions.

  2. Hyperchromicity and Strand Separation in Bacterial DNA

    Science.gov (United States)

    Freifelder, David; Davison, Peter F.

    1962-01-01

    Studies of the per cent of strand separation of N14-N15 hybrid coli DNA heated to various temperatures in formaldehyde have shown that the process of strand separation is a function of temperature and formaldehyde concentration and is directly related to the measured hyperchromicity. No strands separate until about 75 per cent of full hyperchromicity is obtained, and even at apparently full hyperchromicity a large fraction of the strands may be held together, possibly by guanine-cytosine-rich regions. PMID:13894962

  3. Towards understanding the molecular basis of bacterial DNA segregation

    DEFF Research Database (Denmark)

    Leonard, Thomas A.; Møller-Jensen, Jakob; Löwe, Jan

    2005-01-01

    Bacteria ensure the fidelity of genetic inheritance by the coordinated control of chromosome segregation and cell division. Here, we review the molecules and mechanisms that govern the correct subcellular positioning and rapid separation of newly replicated chromosomes and plasmids towards the cell...... poles and, significantly, the emergence of mitotic-like machineries capable of segregating plasmid DNA. We further describe surprising similarities between proteins involved in DNA partitioning (ParA/ParB) and control of cell division (MinD/MinE), suggesting a mechanism for intracellular positioning...... common to the two processes. Finally, we discuss the role that the bacterial cytoskeleton plays in DNA partitioning and the missing link between prokaryotes and eukaryotes that is bacterial mechano-chemical motor proteins. Udgivelsesdato: Mar 29...

  4. High prevalence of biofilm synergy among bacterial soil isolates in cocultures indicates bacterial interspecific cooperation

    DEFF Research Database (Denmark)

    Ren, Dawei; Madsen, Jonas Stenløkke; Sørensen, Søren Johannes;

    2015-01-01

    of single-species biofilms, indicating that all the individual strains benefit from inclusion in the multispecies community. Our results show a high prevalence of synergy in biofilm formation in multispecies consortia isolated from a natural bacterial habitat and suggest that interspecific cooperation...

  5. Probiotic Activity of a Bacterial Strain Isolated from Ancient Permafrost Against Salmonella Infection in Mice.

    Science.gov (United States)

    Fursova, O; Potapov, V; Brouchkov, A; Pogorelko, G; Griva, G; Fursova, N; Ignatov, S

    2012-09-01

    Bacillus cereus strain F, collected from relict permafrost located in Siberia, was analyzed for probiotic activity in the mouse Salmonella enterica model. Viable bacterial cells were found in frozen soils taken at Mammoth Mountain in Yakutia from a depth below the level of seasonal thawing. Geological data indicated the absence of a thawing within millions of years of deposited soils, which helped to ensure the ancient origin of our sample. According to DNA analysis, bacterial cells collected from the relict permafrost appeared to be B. cereus strain F. The morphology of these bacteria was analyzed using atomic force microscopy. B. cereus strain F was assessed as a nonpathogenic bacterium by evaluation of its pathogenicity. A S. enterica model is described in mice after per oral inoculation and serves as a model for the human carrier state. Using this model, probiotic activity by the bacterial strain isolated from the ancient permafrost has been shown against Salmonella infection in mice.

  6. Spectrum and Sensitivity of Bacterial Keratitis Isolates in Auckland

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    S. Marasini

    2016-01-01

    Full Text Available Background. The bacteria isolated from severe cases of keratitis and their antibiotic sensitivity are recognised to vary geographically and over time. Objectives. To identify the most commonly isolated bacteria in keratitis cases admitted over a 24-month period to a public hospital in Auckland, New Zealand, and to investigate in vitro sensitivity to antibiotics. Methods. Hospital admissions for culture-proven bacterial keratitis between January 2013 and December 2014 were identified. Laboratory records of 89 culture positive cases were retrospectively reviewed and antibiotic sensitivity patterns compared with previous studies from other NZ centres. Results. From 126 positive cultures, 35 species were identified. Staphylococcus was identified to be the most common isolate (38.2%, followed by Pseudomonas (21.3%. Over the last decade, infection due to Pseudomonas species, in the same setting, has increased (p≤0.05. Aminoglycosides, cefazolin, ceftazidime, erythromycin, tetracycline, and doxycycline were 100% effective against tested isolates in vitro. Amoxicillin (41.6%, cefuroxime (33.3%, and chloramphenicol (94.7% showed reduced efficacy against Gram-negative bacteria, whereas penicillin (51% and ciprofloxacin (98.8% showed reduced efficacy against Gram-positive bacteria. Conclusions. Despite a shift in the spectrum of bacterial keratitis isolates, antibiotic sensitivity patterns have generally remained stable and show comparability to results within the last decade from NZ centres.

  7. Spectrum and Sensitivity of Bacterial Keratitis Isolates in Auckland

    Science.gov (United States)

    Swift, S.; Dean, S. J.; Ormonde, S. E.

    2016-01-01

    Background. The bacteria isolated from severe cases of keratitis and their antibiotic sensitivity are recognised to vary geographically and over time. Objectives. To identify the most commonly isolated bacteria in keratitis cases admitted over a 24-month period to a public hospital in Auckland, New Zealand, and to investigate in vitro sensitivity to antibiotics. Methods. Hospital admissions for culture-proven bacterial keratitis between January 2013 and December 2014 were identified. Laboratory records of 89 culture positive cases were retrospectively reviewed and antibiotic sensitivity patterns compared with previous studies from other NZ centres. Results. From 126 positive cultures, 35 species were identified. Staphylococcus was identified to be the most common isolate (38.2%), followed by Pseudomonas (21.3%). Over the last decade, infection due to Pseudomonas species, in the same setting, has increased (p ≤ 0.05). Aminoglycosides, cefazolin, ceftazidime, erythromycin, tetracycline, and doxycycline were 100% effective against tested isolates in vitro. Amoxicillin (41.6%), cefuroxime (33.3%), and chloramphenicol (94.7%) showed reduced efficacy against Gram-negative bacteria, whereas penicillin (51%) and ciprofloxacin (98.8%) showed reduced efficacy against Gram-positive bacteria. Conclusions. Despite a shift in the spectrum of bacterial keratitis isolates, antibiotic sensitivity patterns have generally remained stable and show comparability to results within the last decade from NZ centres. PMID:27213052

  8. A clinical Acanthamoeba isolate harboring two distinct bacterial endosymbionts.

    Science.gov (United States)

    Müller, Anneliese; Walochnik, Julia; Wagner, Martin; Schmitz-Esser, Stephan

    2016-10-01

    Acanthamoebae feed on bacteria but are also frequent hosts of bacterial symbionts. Here, we describe the stable co-occurrence of two symbionts, one affiliated to the genus Parachlamydia and the other to the candidate genus Paracaedibacter (Alphaproteobacteria), within a clinical isolate of Acanthamoeba hatchetti genotype T4. We performed fluorescence in situ hybridization (FISH) and transmission electron microscopy (TEM) to describe this symbiosis. Our study adds to other reports of simultaneous co-occurrence of two symbionts within one Acanthamoeba cell.

  9. Isolation of bacterial plasmids by density gradient centrifugation in cesium trifluoroacetate (CsTFA) without the use of ethidium bromide.

    Science.gov (United States)

    Andersson, K; Hjorth, R

    1985-01-01

    Plasmids extracted from bacterial cells by alkaline extraction can easily be isolated from linear DNA by isopycnic centrifugation in CsTFA. This is a fast and simple method which circumvents the use of the intercalating dye, ethidium bromide, and consequently the problems associated with its removal. The buoyant densities for covalently closed circular DNA and linear DNA in CsTFA are 1.60 g/ml and 1.65 g/ml, respectively. The isolation is achieved regardless of plasmid size and can be accomplished at temperatures of between 4 and 30 degrees C. Plasmid DNA isolated in gradients of CsTFA are of a high purity and have been found to be intact when cleaved with restriction enzymes and ligated with T4 DNA ligase.

  10. Bacterial Genotoxins: Merging the DNA Damage Response into Infection Biology

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    Francesca Grasso

    2015-08-01

    Full Text Available Bacterial genotoxins are unique among bacterial toxins as their molecular target is DNA. The consequence of intoxication or infection is induction of DNA breaks that, if not properly repaired, results in irreversible cell cycle arrest (senescence or death of the target cells. At present, only three bacterial genotoxins have been identified. Two are protein toxins: the cytolethal distending toxin (CDT family produced by a number of Gram-negative bacteria and the typhoid toxin produced by Salmonella enterica serovar Typhi. The third member, colibactin, is a peptide-polyketide genotoxin, produced by strains belonging to the phylogenetic group B2 of Escherichia coli. This review will present the cellular effects of acute and chronic intoxication or infection with the genotoxins-producing bacteria. The carcinogenic properties and the role of these effectors in the context of the host-microbe interaction will be discussed. We will further highlight the open questions that remain to be solved regarding the biology of this unusual family of bacterial toxins.

  11. Isolation of Bacterial Agents from the Lungs of Cattle with Pneumonia and Detection of Pasteurella Spp. by Polymerase Chain Reaction

    OpenAIRE

    KILIÇ, Ayşe; MUZ, Adile

    2004-01-01

    Lungs from 8222 cattle slaughtered at an abattoir in Elazığ were examined macroscopically, and pneumonia was detected in 500 (6.1%) lungs. These samples were inoculated onto blood agar supplemented with 7% sheep blood for isolation of bacterial agents. A polymerase chain reaction (PCR) based upon the use of species-specific primers was carried out on DNA samples extracted from suspected Pasteurella spp. isolates. In addition, a mouse inoculation test was carried out on suspected Pasteurella m...

  12. Plasmid profiling of bacterial isolates from confined environments

    Science.gov (United States)

    van Houdt, Rob; Provoost, Ann; Coninx, Ilse; Leys, Natalie; Mergeay, Max

    Plasmid profiling of bacterial isolates from confined environments R. Van Houdt, I. Coninx, A. Provoost, N. Leys, and M. Mergeay Expertise group for Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK•CEN), Boeretang 200, B-2400 Mol, Belgium. Human exploration of extreme and isolated hostile environments such as space requires special confined small volume habitats to protect and house the crew. However, human confinement in such small volume habitats has restrictions on waste disposal and personal hygiene and inevitably generates a particular community of microorganisms within the habitat. These microorganisms are mainly originating from the crew (skin, mucous membranes, upper respiratory tract, mouth, and gastrointestinal tract) but also include the residing environmental microorganisms. Earth-based confined habitats such as the Antarctic Research Station Concordia are used as test beds for long-duration spaceflights to study the physiologic and psychological adaptation to isolated environments. The dynamics of the environmental microbial population in such a test bed could render additional insights in assessing the potential health risks in long-duration space missions. Not only total bacterial contamination levels are important, but it is essential to identify also the predominant microbial taxa and their mobile genetic elements (MGE). These MGEs could be exchanged between bacteria by horizontal gene transfer and may alter the pathogenic potential since they often carry antibiotic resistance or more in general adaptation-enhancing traits. In this study several bacterial strains isolated in the Concordia research station were examined for their plasmid content. An optimized protocol for extraction of large plasmids showed the present of at least one plasmid in 50% of the strains. For all strains the minimal inhibitory concentration of a range of antibiotics was determined indicating resistance to

  13. Isolation of cell-free bacterial inclusion bodies

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    Rodríguez-Carmona Escarlata

    2010-09-01

    Full Text Available Abstract Background Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for inclusion body isolation from Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of inclusion bodies in biological interfaces. Results Using an appropriate combination of chemical and mechanical cell disruption methods we have established a convenient procedure for the recovery of bacterial inclusion bodies with undetectable levels of viable cell contamination, below 10-1 cfu/ml, keeping the particulate organization of these aggregates regarding size and protein folding features. Conclusions The application of the developed protocol allows obtaining bacterial free inclusion bodies suitable for use in mammalian cell cultures and other biological interfaces.

  14. Molecular characterization of conjugative plasmids in pesticide tolerant and multi-resistant bacterial isolates from contaminated alluvial soil.

    Science.gov (United States)

    Anjum, Reshma; Grohmann, Elisabeth; Malik, Abdul

    2011-06-01

    A total of 35 bacteria from contaminated soil (cultivated fields) near pesticide industry from Chinhat, Lucknow, (India) were isolated and tested for their tolerance/resistance to pesticides, heavy metals and antibiotics. Bacterial isolates were identified by 16S rDNA sequencing. Gas Chromatography analysis of the soil samples revealed the presence of lindane at a concentration of 547 ng g(-1) and α-endosulfan and β-endosulfan of 422 ng g(-1) and 421 ng g(-1) respectively. Atomic Absorption Spectrophotometry analysis of the test sample was done and Cr, Zn, Ni, Fe, Cu and Cd were detected at concentrations of 36.2, 42.5, 43.2, 241, 13.3 and 11.20 mg kg(-1) respectively. Minimum inhibitory concentrations of all the isolates were determined for pesticides and heavy metals. All the multi-resistant/tolerant bacterial isolates were also tested for the presence of incompatibility (Inc) group IncP, IncN, IncW, IncQ plasmids and for rolling circle plasmids of the pMV158-family by PCR. Total community DNA was extracted from pesticide contaminated soil. PCR amplification of the bacterial isolates and soil DNA revealed the presence of IncP-specific sequences (trfA2 and oriT) which was confirmed by dot blot hybridization with RP4-derived DIG-labelled probes. Plasmids belonging to IncN, IncW and IncQ group were neither detected in the bacterial isolates nor in total soil DNA. The presence of conjugative or mobilizable IncP plasmids in the isolates indicate that these bacteria have gene transfer capacity with implications for dissemination of heavy metal and antibiotic resistance genes. We propose that IncP plasmids are mainly responsible for the spread of multi-resistant bacteria in the contaminated soils.

  15. Efficacy of a marine bacterial nuclease against biofilm forming microorganisms isolated from chronic rhinosinusitis.

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    Robert C Shields

    Full Text Available BACKGROUND: The persistent colonization of paranasal sinus mucosa by microbial biofilms is a major factor in the pathogenesis of chronic rhinosinusitis (CRS. Control of microorganisms within biofilms is hampered by the presence of viscous extracellular polymers of host or microbial origin, including nucleic acids. The aim of this study was to investigate the role of extracellular DNA in biofilm formation by bacteria associated with CRS. METHODS/PRINCIPAL FINDINGS: Obstructive mucin was collected from patients during functional endoscopic sinus surgery. Examination of the mucous by transmission electron microscopy revealed an acellular matrix punctuated occasionally with host cells in varying states of degradation. Bacteria were observed in biofilms on mucosal biopsies, and between two and six different species were isolated from each of 20 different patient samples. In total, 16 different bacterial genera were isolated, of which the most commonly identified organisms were coagulase-negative staphylococci, Staphylococcus aureus and α-haemolytic streptococci. Twenty-four fresh clinical isolates were selected for investigation of biofilm formation in vitro using a microplate model system. Biofilms formed by 14 strains, including all 9 extracellular nuclease-producing bacteria, were significantly disrupted by treatment with a novel bacterial deoxyribonuclease, NucB, isolated from a marine strain of Bacillus licheniformis. Extracellular biofilm matrix was observed in untreated samples but not in those treated with NucB and extracellular DNA was purified from in vitro biofilms. CONCLUSION/SIGNIFICANCE: Our data demonstrate that bacteria associated with CRS form robust biofilms which can be reduced by treatment with matrix-degrading enzymes such as NucB. The dispersal of bacterial biofilms with NucB may offer an additional therapeutic target for CRS sufferers.

  16. Validation of hierarchical cluster analysis for identification of bacterial species using 42 bacterial isolates

    Science.gov (United States)

    Ghebremedhin, Meron; Yesupriya, Shubha; Luka, Janos; Crane, Nicole J.

    2015-03-01

    Recent studies have demonstrated the potential advantages of the use of Raman spectroscopy in the biomedical field due to its rapidity and noninvasive nature. In this study, Raman spectroscopy is applied as a method for differentiating between bacteria isolates for Gram status and Genus species. We created models for identifying 28 bacterial isolates using spectra collected with a 785 nm laser excitation Raman spectroscopic system. In order to investigate the groupings of these samples, partial least squares discriminant analysis (PLSDA) and hierarchical cluster analysis (HCA) was implemented. In addition, cluster analyses of the isolates were performed using various data types consisting of, biochemical tests, gene sequence alignment, high resolution melt (HRM) analysis and antimicrobial susceptibility tests of minimum inhibitory concentration (MIC) and degree of antimicrobial resistance (SIR). In order to evaluate the ability of these models to correctly classify bacterial isolates using solely Raman spectroscopic data, a set of 14 validation samples were tested using the PLSDA models and consequently the HCA models. External cluster evaluation criteria of purity and Rand index were calculated at different taxonomic levels to compare the performance of clustering using Raman spectra as well as the other datasets. Results showed that Raman spectra performed comparably, and in some cases better than, the other data types with Rand index and purity values up to 0.933 and 0.947, respectively. This study clearly demonstrates that the discrimination of bacterial species using Raman spectroscopic data and hierarchical cluster analysis is possible and has the potential to be a powerful point-of-care tool in clinical settings.

  17. Tracking plant, fungal, and bacterial DNA in honey specimens.

    Science.gov (United States)

    Olivieri, Cristina; Marota, Isolina; Rollo, Franco; Luciani, Stefania

    2012-01-01

    Consuming honey can result in adverse effects owing to poisoning by bacterial (botulism) or plant toxins. We have devised a method to extract polymerase chain reaction (PCR) amplifiable DNA of up to c. 400 bp in length based on dialysis of a 15-mL honey sample for 18 h against deionized water followed by sequential extraction using phenol, phenol/chloroform/isoamyl alcohol, chloroform/isoamyl alcohol, and ether. Sequence analysis of PCR products obtained using "universal" plant, fungal, and bacterial primers targeted to the ribosomal RNA genes has allowed us to identify six different orders of plants (Apiales, Fabales, Asterales, Solanales, Brassicales, and Sapindales), two orders of fungi (Entylomatales and Saccharomycetales), and six orders of bacteria (Sphingomonadales, Burkholderiales, Pseudomonadales, Enterobacteriales, Actinomycetales, and Bifidobacteriales) in a single honey specimen.

  18. Bioremediation Potential of Bacterial Isolates for Municipal Wastewater Treatment

    Directory of Open Access Journals (Sweden)

    Nilesh A. Sonune

    2015-08-01

    Full Text Available The potential of bacteria for the treatment of municipal wastewater was investigated in present study. Total eight bacterial isolates were used for this study that showed growth on wastewater agar medium. These isolates were identified on the basis of morphological and biochemical test and identified as Bacillus licheniformis NW16, Pseudomonas aeruginosa NS19, Pseudomonas sp. NS20, Planococcus salinarum NS23, Stenotrophomonas maltophilia NS21, Paenibacillus sp. NW9, Paenibacillus borealis NS3 and Aeromonas hydrophilia NS17. The B. licheniformis NW16 showed highest potential to reduce all parameter under study than other isolates except Ammonical nitrogen. B. licheniformis NW16 and Aeromonas hydrophilia NS17 showed maximum reduction (42.86% in BOD each. B. licheniformis NW16 and Paenibacillus sp. NW9 showed 82.76% and 81.61% reduction in COD respectively. B. licheniformis NW16, P. salinarum NS23 and Aeromonas hydrophilia NS17 showed reduction in nitrate ranging from 17.36%-63.64%. All the isolates have potential to reduced phosphate from 17.55% -72.3%. B. licheniformis NW16, Ps. aeruginosa NS19, Pseudomonas sp. NS20, Paenibacillus sp. NW9 and Aeromonas hydrophilia NS17 showed reduction in TSS ranging from 42.69%-79.94%. B. licheniformis NW16, Ps. aeruginosa NS19, Pseudomonas sp. NS20, S. maltophilia NS21 and Paenibacillus sp. NW9 showed reduction in TDS ranging from 14%-81.4%.

  19. VUV absorption spectroscopy of bacterial spores and DNA components

    Science.gov (United States)

    Fiebrandt, Marcel; Lackmann, Jan-Wilm; Raguse, Marina; Moeller, Ralf; Awakowicz, Peter; Stapelmann, Katharina

    2017-01-01

    Low-pressure plasmas can be used to inactivate bacterial spores and sterilize goods for medical and pharmaceutical applications. A crucial factor are damages induced by UV and VUV radiation emitted by the plasma. To analyze inactivation processes and protection strategies of spores, absorption spectra of two B. subtilis strains are measured. The results indicate, that the inner and outer coat of the spore significantly contribute to the absorption of UV-C and also of the VUV, protecting the spore against radiation based damages. As the sample preparation can significantly influence the absorption spectra due to salt residues, the cleaning procedure and sample deposition is tested for its reproducibility by measuring DNA oligomers and pUC18 plasmid DNA. The measurements are compared and discussed with results from the literature, showing a strong decrease of the salt content enabling the detection of absorption structures in the samples.

  20. Differential effect of three polyunsaturated aldehydes on marine bacterial isolates.

    Science.gov (United States)

    Ribalet, Francois; Intertaglia, Laurent; Lebaron, Philippe; Casotti, Raffaella

    2008-01-31

    Bioactive polyunsaturated aldehydes (PUAs) are produced by several marine phytoplankton (mainly diatoms) and have been shown to have a detrimental effect on a wide variety of organisms, including phytoplankton and invertebrates. However, their potential impact on marine bacteria has been largely neglected. We assess here the effect of three PUAs produced by marine diatoms: 2E,4E-decadienal, 2E,4E-octadienal and 2E,4E-heptadienal, on the growth of 33 marine bacterial strains, including 16 strains isolated during a bloom of the PUA-producing diatom Skeletonema marinoi in the Northern Adriatic Sea. A concentration-dependent growth reduction was observed for 19 bacterial strains at concentrations ranging from 3 to 145 micromolL(-1). Surprisingly, Eudora adriatica strain MOLA358 (Flavobacteriaceae) and Alteromonas hispanica strain MOLA151 (Alteromonadaceae) showed growth stimulation upon exposure to PUAs at concentrations between 13 and 18 micromolL(-1). The remaining 12 strains were unaffected by even very high PUA concentrations. Strains isolated during the diatom bloom showed remarkable resistance to PUA exposures, with only two out of 16 strains showing growth inhibition at PUA concentrations below 106, 130, and 145 micromolL(-1) for 2E,4E-decadienal, 2E,4E-octadienal and 2E,4E-heptadienal, respectively. No correlation between taxonomical position and sensitivity to PUA was observed. Considering that many bacteria thrive in close vicinity of diatom cells, it is likely that these compounds may shape the structure of associated bacterial communities by representing a selection force. This is even more relevant during the final stages of blooms, when senescence and nutrient limitation increase the potential production and release of aldehydes.

  1. Isolation and screening of azo dye decolorizing bacterial isolates from dye-contaminated textile wastewater

    Directory of Open Access Journals (Sweden)

    Shahid Mahmood

    2011-04-01

    Full Text Available Azo dyes are released into wastewater streams without any pretreatment and pollute water and soilenvironments. To prevent contamination of our vulnerable resources, removal of these dye pollutants is of greatimportance. For this purpose, wastewater samples were collected from dye-contaminated sites of Faisalabad. About200 bacterial isolates were isolated through enrichment and then tested for their potential to remove RemazolBlack-B azo dye in liquid medium. Five bacterial isolates capable of degrading Remazol Black-B azo dye efficientlywere screened through experimentation on modified mineral salt medium. Isolate SS1 (collected from wastewater ofSupreme Textile Industry was able to completely remove the Remazol Black-B dye from the liquid medium in 18 h.Further, the isolate showed the best performance at the dye concentration of 100 mg L-1 medium (pH 7 and attemperature 35oC. Similarly, yeast extract proved to be the best carbon source for decolorization purpose. Theresults imply that the isolate SS1 could be used for the removal of the reactive dyes from textile effluents.

  2. Isolation and Characterization of Bacteriophages Against Pseudomonas syringae pv. actinidiae Causing Bacterial Canker Disease in Kiwifruit.

    Science.gov (United States)

    Yu, Ji-Gang; Lim, Jeong-A; Song, Yu-Rim; Heu, Sunggi; Kim, Gyoung Hee; Koh, Young Jin; Oh, Chang-Sik

    2016-02-01

    Pseudomonas syringae pv. actinidiae causes bacterial canker disease in kiwifruit. Owing to the prohibition of agricultural antibiotic use in major kiwifruit-cultivating countries, alternative methods need to be developed to manage this disease. Bacteriophages are viruses that specifically infect target bacteria and have recently been reconsidered as potential biological control agents for bacterial pathogens owing to their specificity in terms of host range. In this study, we isolated bacteriophages against P. syringae pv. actinidiae from soils collected from kiwifruit orchards in Korea and selected seven bacteriophages for further characterization based on restriction enzyme digestion patterns of genomic DNA. Among the studied bacteriophages, two belong to the Myoviridae family and three belong to the Podoviridae family, based on morphology observed by transmission electron microscopy. The host range of the selected bacteriophages was confirmed using 18 strains of P. syringae pv. actinidiae, including the Psa2 and Psa3 groups, and some were also effective against other P. syringae pathovars. Lytic activity of the selected bacteriophages was sustained in vitro until 80 h, and their activity remained stable up to 50°C, at pH 11, and under UV-B light. These results indicate that the isolated bacteriophages are specific to P. syringae species and are resistant to various environmental factors, implying their potential use in control of bacterial canker disease in kiwifruits.

  3. [An efficient method for isolation of mitochondrial DNA in wheat].

    Science.gov (United States)

    Li, Wen-Qiang; Zhang, Gai-Sheng; Wang, Kui; Niu, Na; Pan, Dong-Liang

    2007-06-01

    An efficient method for isolation of mitochondrial DNA (mtDNA) from etiolated tissues of wheat was developed. The protocol consists of mitochondria isolation with differential centrifugation, Dnase I treatment, lysis with SDS and proteinase K, removing protein by TE-saturated phenol/chloroform extraction and a final RNase A treatment for obtaining mtDNA. The mtDNA samples were tested using spectrophotometry and agarose gel electrophoresis. It was proved that the mtDNA isolated by this method not only have the high yield but also structural complete, and contains no impurities, such as nuclear DNA, RNA and protein. The result showed that this high quality mtDNA can be successfully used in PCR and other genetic studies. In addition, it was found that adjusting the lysis temperature has a noticeable effect on the mtDNA yield.

  4. Analysis of endophytic bacterial communities of potato by plating and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA based PCR fragments

    NARCIS (Netherlands)

    Garbeva, P.; Overbeek, van L.S.; Vuurde, van J.W.L.; Elsas, van J.D.

    2001-01-01

    The diversity of endophytic bacterial populations of potato (Solanum tuberosum cv Desiree) was assessed using a combination of dilution plating of plant macerates followed by isolation and characterization of isolates, and direct PCR-DGGE on the basis of DNA extracted from plants. The culturable end

  5. Examination of bacterial inhibition using a catalytic DNA.

    Directory of Open Access Journals (Sweden)

    Long Qu

    Full Text Available Determination of accurate dosage of existing antibiotics and discovery of new antimicrobials or probiotics entail simple but effective methods that can conveniently track bacteria growth and inhibition. Here we explore the application of a previously reported fluorogenic E. coli-specific DNAzyme (catalytic DNA, RFD-EC1, as a molecular probe for monitoring bacterial inhibition exerted by antibiotics and for studying bacterial competition as a result of cohabitation. Because the DNAzyme method provides a convenient way to monitor the growth of E. coli, it is capable of determining the minimal inhibitory concentration (MIC of antibiotics much faster than the conventional optical density (OD method. In addition, since the target for RFD-EC1 is an extracellular protein molecule from E. coli, RFD-EC1 is able to identify pore-forming antibiotics or compounds that can cause membrane leakage. Finally, RFD-EC1 can be used to analyse the competition of cohabitating bacteria, specifically the inhibition of growth of E. coli by Bacillus subtilis. The current work represents the first exploration of a catalytic DNA for microbiological applications and showcases the utility of bacteria-sensing fluorogenic DNAzymes as simple molecular probes to facilitate antibiotic and probiotic research.

  6. Effects of different methods of DNA extraction for activated sludge on the subsequent analysis of bacterial community profiles.

    Science.gov (United States)

    Sun, Lianpeng; Ouyang, Xiong; Tang, Yueheng; Yang, Ying; Luo, Ying

    2012-02-01

    The effect of different DNA extraction protocols on activated sludge DNA yield and bacterial community composition was evaluated by temperature gradient gel electrophoresis (TGGE). Nine different procedures to extract DNA were compared-sonication (30s), sonication (40s), sonication (50s), freezing-thawing, bead milling, sodium dodecyl sulfate (SDS)-lysozyme, SDS-proteinase K, SDS-lysozyme-proteinase, and a commercial extraction kit. It was found that the TGGE profiles and the DNA band numbers made significant differences via various extraction methods. The yield and purity of DNA extracted by sonication and other physical methods were not satisfactory, while the DNA purity extracted by SDS and other chemical-biological methods were better. Crude DNA extracts isolated by sonication and other physical methods passed the polymerase chain reaction, despite the absence of purification and acquired affluent DNA bands in TGGE. The affluence of bands in TGGE was not consistent with the yield and purification of DNA, but was correlative with extraction protocols. To analyze the activated sludge bacterial community by TGGE fingerprint, it is necessary to make a synthesis of the TGGE fingerprint profiles of chemical and physical DNA extraction methods to overcome the representative bias.

  7. Antibiogram study of aerobic bacterial isolates from uropathogens

    Directory of Open Access Journals (Sweden)

    Mallikarjuna Reddy C, Himabindu M, Maity Soumendranath, Kanta RC, Kapur Indu

    2014-04-01

    Full Text Available Background: Bacteria are capable of invading and infecting humans, leading to disease and sometimes death. Systems and tissues in human body are vulnerable to different organisms. Infection pattern is likely to differ by geographical regions. Aim: This study was aimed to isolate and identify the type of aerobic bacteria causing Urinary Tract Infections (UTI in different age groups and sexes, and also in some predisposing conditions. Their antibiogram also was done. Materials and Methods: Midstream urine sample collected aseptically from 276 patients were subjected for isolation and identification of aerobic bacteria by standard technique and subsequently antibiogram was done by Kirby –Bayer Method. Both sexes of patients with an age range of 10-70 years and patients with diabetes (22, hypertension (8 and anemia (8 were also included in the study. Results: Escherichia coli was the predominant organism(50% among other isolates – Klebsiella species (27.3%, Proteus species(7.14%, Staphylococcus saprophyticus (5.95%, Staphylococcus aureus (3.57%, Enterococci (3.57%, Pseudomonas species(2.38%. UTI was more common among patients of 60 and more years of age; however, incidence was more in female patients (36.2 – 38.5% compared with male patients (25-30%. Anemia, Diabetes and Hypertension conditions were found to predispose UTI. Aminoglycosides and Quinolones were found to be more effective against the isolates. Conclusion: The present study reveals in spite of the topographical diversity, the infecting bacterial isolates from this area were found to be the same as from any other part of India.

  8. Isolation and characteristics of a novel biphenyl-degrading bacterial strain, Dyella ginsengisoli LA-4

    Institute of Scientific and Technical Information of China (English)

    LI Ang; QU Yuanyuan; ZHOU Jiti; GOU Min

    2009-01-01

    A novel biphenyl-degrading bacterial strain LA-4 was isolated from activated sludge. It was identified as Dyella ginsengisoli according to phylogenetic similarity of 16S rRNA gene sequence. This isolate could utilize biphenyl as sole source of carbon and energy, which degraded over 95 mg/L biphenyl within 36 h. The major metabolites formed from biphenyl, such as 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) and benzoic acid, were identified by LC-MS. The crude cell extract of strain LA-4 exhibited the activity of 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DHBD) and the kinetic parameters were Km= 26.48 μmol/L and Vmax= 8.12 μmol/mg protein. A conserved region of the biphenyl dioxygenase gene bphA1 of strain LA-4 was amplified by PCR and confirmed by DNA sequencing.

  9. Co-production of two new peptide antibiotics by a bacterial isolate Paenibacillus alvei NP75.

    Science.gov (United States)

    Anandaraj, Balaiah; Vellaichamy, Adaikkalam; Kachman, Maureen; Selvamanikandan, Athinarayanan; Pegu, Shyamanta; Murugan, Vadivel

    2009-02-06

    Two new peptide antibiotics were secreted by a Gram-positive bacterial strain isolated from fermented tomato fruit. Based on its 99% 16S rDNA sequence similarity with Paenibacillus alvei, the isolate was designated as P. alvei NP75. Among these two peptides, one is active against Gram-positive pathogens while the other against Gram-negative pathogens; thus these peptides were named as paenibacillin P and paenibacillin N, respectively. After the purification of those peptide antibiotics from the cell free culture supernatant by RP-HPLC, they were analyzed for their temperature sensitivity and susceptibility to proteases. Higher-temperature tolerant paenibacillin N was easily degraded by proteinase K, while the temperature sensitive paenibacillin P was not affected by any of the proteases used in this study other than a specific protease that was secreted by the same NP75 strain. Mass-spectrometry analysis of the above peptide antibiotics further confirmed their distinction among the known peptide antibiotics. We are reporting first of its kind the co-production of two different new peptide antibiotics from a single bacterial isolate of P. alvei strain.

  10. Isolation and Characterization of Nickel Uptake by Nickel Resistant Bacterial Isolate (NiRBI)

    Institute of Scientific and Technical Information of China (English)

    JAGDISH S PATEL; PRERNA C PATEL; KIRAN KALIA

    2006-01-01

    Bioremediation technology has gained importance because microbes could be the convenient source of bio-absorption/bioaccumulation of metals from effluent streams. Methods The nickel-resistant bacterial isolates (NiRBI)were selected from various bacterial isolates from industrial effluent and grown in nutrient broth containing different concentrations of nickel sulfate (0.3-3.0 mmol/L) and their capability of accumulating metal from the medium. Results Well-defined growth of NiRBI was observed in the medium containing up to 2.5 mmol/L of nickel. The isolate was identified using 16S rRNA and closely related to Pseudomonas fragi. Maximum accumulation of nickel (0.59 mg/g dry weight of bacterial cells) was observed when NiRBI was grown in media containing 2 mmol/L of nickel. The protein profile of the NiRBI cellular extract by SDS-PAGE showed two metal stress-induced proteins of molecular weight 48 KD and 18 KD with a simultaneous down regulation of four proteins of 46.7 KD, 42.2 KD, 19.7 KD, and 4.0 KD. Conclusion 48 KD and 18 KD proteins play a role in metal resistance mechanism by NiRBI.

  11. Aerobic cyanide degradation by bacterial isolates from cassava factory wastewater.

    Science.gov (United States)

    Kandasamy, Sujatha; Dananjeyan, Balachandar; Krishnamurthy, Kumar; Benckiser, Gero

    2015-01-01

    Ten bacterial strains that utilize cyanide (CN) as a nitrogen source were isolated from cassava factory wastewater after enrichment in a liquid media containing sodium cyanide (1 mM) and glucose (0.2% w/v). The strains could tolerate and grow in cyanide concentrations of up to 5 mM. Increased cyanide levels in the media caused an extension of lag phase in the bacterial growth indicating that they need some period of acclimatisation. The rate of cyanide removal by the strains depends on the initial cyanide and glucose concentrations. When initial cyanide and glucose concentrations were increased up to 5 mM, cyanide removal rate increased up to 63 and 61 per cent by Bacillus pumilus and Pseudomonas putida. Metabolic products such as ammonia and formate were detected in culture supernatants, suggesting a direct hydrolytic pathway without an intermediate formamide. The study clearly demonstrates the potential of aerobic treatment with cyanide degrading bacteria for cyanide removal in cassava factory wastewaters.

  12. Antimicrobial resistance of bacterial strains isolated from avian cellulitis

    Directory of Open Access Journals (Sweden)

    MM Santos

    2014-03-01

    Full Text Available Avian cellulitis is an inflammatory process in the subcutaneous tissue, mainly located in the abdomen and thighs. This problem is commonly observed in poultry at slaughter and it is considered one of the major causes of condemnation of carcasses in Brazil. The aim of this study was to perform the microbial isolation of lesions of avian cellulitis from a processing plant located in the State of Goiás in order to analyze antimicrobial resistance by antibiogram test and to detect resistance genes by polymerase chain reaction. A total of 25 samples of avian cellulitis lesions were analyzed, from which 30 bacterial strains were isolated. There were eleven (44% strains of Escherichia coli, nine (36% strains of Staphylococcus epidermidis, seven (28% strains of Proteus mirabilis and three (12% strains of Manheimiahaemolytica. The antibiogram test showed that all strains were resistant to at least one antimicrobial. The gene of antimicrobial resistance tetB was detected in E. coli, S. epidermidis and P. mirabilis strains, and was the most frequently observed gene. The gene of antimicrobial resistance Sul1 was detected in all bacterial species, while tetA was found in E. coli and S. epidermidis strains, SHV in E. coli strains, S. epidermidis and P. mirabilis,and cat1 in one P. mirabilis strain. The results suggest a potential public health hazard due to the ability of these microorganisms to transmit antimicrobial resistancegenes to other microorganisms present in the intestinal tract of humans and animals, which may affect clinical-medical usage of these drugs.

  13. Determination of the hydrocarbon-degrading metabolic capabilities of tropical bacterial isolates

    Energy Technology Data Exchange (ETDEWEB)

    Marquez-Rocha, F.J.; Olmos-Soto, J. [Centro de Investigacion Cientifica y de Educacion Superior de Ensenada, San Diego, CA (United States). Departamento de Biotecnologia Marina; Rosano-Hernandez, M.A.; Muriel-Garcia, M. [Instituto Mexicano del Petroleo, CD Carmen Camp (Mexico). Zona Marina/Tecnologia Ambiental

    2005-01-01

    Of more than 20 bacteria isolated from a tropical soil using minimal medium supplemented with hydrocarbons, 11 grew well on diesel as sole carbon source, and another 11 grew in the presence of polynuclear aromatic hydrocarbons (PAHs). Ten isolates were identified phenotypically as Pseudomonas sp. and eight as Bacillus sp. Gene sequences representing the catabolic genes (alkM, todM, ndoM, and xylM) and 16S rRNA gene sequences characteristic for Pseudomona and Bacillus were amplified by PCR, using DNA recovered from the supernatant of hydrocarbon-contaminated soil suspensions. Based on their rapid growth characteristics in the presence of hydrocarbons and the formation of PCR products for the catabolic genes alkM and ndoM six isolates were selected for biodegradation assays. After 30 days a mixed culture of two isolates achieved close to 70% hydrocarbon removal and apparent mineralization of 16% of the hydrocarbons present in the soil. Biodegradation rates varied from 275 to 387 mg hydrocarbon kg{sup -1} day{sup -1}. Several bacterial isolates obtained in this study have catabolic capabilities for the biodegradation of alkanes and aromatic hydrocarbons including PAHs. (author)

  14. Effect of isolate of ruminal fibrolytic bacterial culture supplementation on fibrolytic bacterial population and survivability of inoculated bacterial strain in lactating Murrah buffaloes

    Directory of Open Access Journals (Sweden)

    Brishketu Kumar

    2013-02-01

    Full Text Available Aim: The present study was conducted to evaluate the effect of bacterial culture supplementation on ruminal fibrolytic bacterial population as well as on survivability of inoculated bacterial strain in lactating Murrah buffaloes kept on high fibre diet. Materials and Methods: Fibrolytic bacterial strains were isolated from rumen liquor of fistulated Murrah buffaloes and live bacterial culture were supplemented orally in treatment group of lactating Murrah buffaloes fed on high fibre diet to see it's effect on ruminal fibrolytic bacterial population as well as to see the effect of survivability of the inoculated bacterial strain at three different time interval in comparison to control group. Results: It has been shown by real time quantification study that supplementation of bacterial culture orally increases the population of major fibre degrading bacteria i.e. Ruminococcus flavefaciens, Ruminococcus albus as well as Fibrobacter succinogenes whereas there was decrease in secondary fibre degrading bacterial population i.e. Butyrivibrio fibrisolvens over the different time periods. However, the inoculated strain of Ruminococcus flavefaciens survived significantly over the period of time, which was shown in stability of increased inoculated bacterial population. Conclusion: The isolates of fibrolytic bacterial strains are found to be useful in increasing the number of major ruminal fibre degrading bacteria in lactating buffaloes and may act as probiotic in large ruminants on fibre-based diets. [Vet World 2013; 6(1.000: 14-17

  15. A novel bacterial isolate Stenotrophomonas maltophilia as living factory for synthesis of gold nanoparticles

    Directory of Open Access Journals (Sweden)

    Shekhawat G

    2009-07-01

    Full Text Available Abstract Background The synthesis of gold nanoparticles (GNPs has received considerable attention with their potential applications in various life sciences related applications. Recently, there has been tremendous excitement in the study of nanoparticles synthesis by using some natural biological system, which has led to the development of various biomimetic approaches for the growth of advanced nanomaterials. In the present study, we have demonstrated the synthesis of gold nanoparticles by a novel bacterial strain isolated from a site near the famous gold mines in India. A promising mechanism for the biosynthesis of GNPs by this strain and their stabilization via charge capping was investigated. Results A bacterial isolate capable of gold nanoparticle synthesis was isolated and identified as a novel strain of Stenotrophomonas malophilia (AuRed02 based on its morphology and an analysis of its 16S rDNA gene sequence. After 8 hrs of incubation, monodisperse preparation of gold nanoparticles was obtained. Gold nanoparticles were characterized and found to be of ~40 nm size. Electrophoresis, Zeta potential and FTIR measurements confirmed that the particles are capped with negatively charged phosphate groups from NADP rendering them stable in aqueous medium. Conclusion The process of synthesis of well-dispersed nanoparticles using a novel microorganism isolated from the gold enriched soil sample has been reported in this study, leading to the development of an easy bioprocess for synthesis of GNPs. This is the first study in which an extensive characterization of the indigenous bacterium isolated from the actual gold enriched soil was conducted. Promising mechanism for the biosynthesis of GNPs by the strain and their stabilization via charge capping is suggested, which involves an NADPH-dependent reductase enzyme that reduces Au3+ to Au0 through electron shuttle enzymatic metal reduction process.

  16. Plutonium interaction with a bacterial strain isolated from the waste isolation pilot plant (WIPP) environment

    Energy Technology Data Exchange (ETDEWEB)

    Strietelmeier, B.A.; Kraus, S.M.; Leonard, P.A.; Triay, I.R. [Los Alamos National Lab., NM (United States)] [and others

    1996-12-31

    This work was conducted as part of a series of experiments to determine the association and interaction of various actinides with bacteria isolated from the WIPP site. The majority of bacteria that exist at the site are expected to be halophiles, or extreme halophiles, due to the high concentration of salt minerals at the location. Experiments were conducted to determine the toxicity of plutonium-n-239, neptunium-237 and americium-243 to several species of these halophiles and the results were reported elsewhere. As an extension of these experiments, we report an investigation of the type of association that occurs between {sup 239}Pu and the isolate WIPP-1A, isolated by staff at Brookhaven National Laboratory, when grown in a high-salt, defined medium. Using scanning electron microscopy (SEM) techniques, we demonstrate a surface association of the {sup 239}Pu with the bacterial cells.

  17. Erwinia teleogrylli sp. nov., a Bacterial Isolate Associated with a Chinese Cricket.

    Directory of Open Access Journals (Sweden)

    Bo Liu

    Full Text Available A bacterial isolate (SCU-B244T was obtained in China from crickets (Teleogryllus occipitalis living in cropland deserted for approximately 10 years. The isolated bacteria were Gram-negative, facultatively anaerobic, oxidase-negative rods. A preliminary analysis of the 16S rRNA gene sequence indicated that the strain belongs to either the genus Erwinia or Pantoea. Analysis of multilocus sequence typing based on concatenated partial atpD, gyrB and infB gene sequences and physiological and biochemical characteristics indicated that the strain belonged to the genus Erwinia, as member of a new species as it was distinct from other known Erwinia species. Further analysis of the 16S rRNA gene showed SCU-B244T to have 94.71% identity to the closest species of that genus, Erwinia oleae (DSM 23398T, which is below the threshold of 97% used to discriminate bacterial species. DNA-DNA hybridization results (5.78±2.52% between SCU-B244T and Erwinia oleae (DSM 23398T confirmed that SCU-B244T and Erwinia oleae (DSM 23398T represent different species combined with average nucleotide identity values which range from 72.42% to 74.41. The DNA G+C content of SCU-B244T was 55.32 mol%, which also differs from that of Erwinia oleae (54.7 to 54.9 mol%. The polyphasic taxonomic approach used here confirmed that the strain belongs to the Erwinia group and represents a novel species. The name Erwinia teleogrylli sp. nov. is proposed for this novel taxon, for which the type strain is SCU-B244T (= CGMCC 1.12772T = DSM 28222T = KCTC 42022T.

  18. Erwinia teleogrylli sp. nov., a Bacterial Isolate Associated with a Chinese Cricket.

    Science.gov (United States)

    Liu, Bo; Luo, Jin; Li, Wei; Long, Xiu-Feng; Zhang, Yu-Qin; Zeng, Zhi-Gang; Tian, Yong-Qiang

    2016-01-01

    A bacterial isolate (SCU-B244T) was obtained in China from crickets (Teleogryllus occipitalis) living in cropland deserted for approximately 10 years. The isolated bacteria were Gram-negative, facultatively anaerobic, oxidase-negative rods. A preliminary analysis of the 16S rRNA gene sequence indicated that the strain belongs to either the genus Erwinia or Pantoea. Analysis of multilocus sequence typing based on concatenated partial atpD, gyrB and infB gene sequences and physiological and biochemical characteristics indicated that the strain belonged to the genus Erwinia, as member of a new species as it was distinct from other known Erwinia species. Further analysis of the 16S rRNA gene showed SCU-B244T to have 94.71% identity to the closest species of that genus, Erwinia oleae (DSM 23398T), which is below the threshold of 97% used to discriminate bacterial species. DNA-DNA hybridization results (5.78±2.52%) between SCU-B244T and Erwinia oleae (DSM 23398T) confirmed that SCU-B244T and Erwinia oleae (DSM 23398T) represent different species combined with average nucleotide identity values which range from 72.42% to 74.41. The DNA G+C content of SCU-B244T was 55.32 mol%, which also differs from that of Erwinia oleae (54.7 to 54.9 mol%). The polyphasic taxonomic approach used here confirmed that the strain belongs to the Erwinia group and represents a novel species. The name Erwinia teleogrylli sp. nov. is proposed for this novel taxon, for which the type strain is SCU-B244T (= CGMCC 1.12772T = DSM 28222T = KCTC 42022T).

  19. Recycling Isolation of Plant DNA, A Novel Method

    Institute of Scientific and Technical Information of China (English)

    Lingling Zhang; Bo Wang; Lei Pan; Junhua Peng

    2013-01-01

    DNA is one of the most basic and essential genetic materials in the field of molecular biology.To date,isolation of sufficient and goodquality DNA is still a challenge for many plant species,though various DNA extraction methods have been published.In the present paper,a recycling DNA extraction method was proposed.The key step of this method was that a single plant tissue sample was recycled for DNA extraction for up to four times,and correspondingly four DNA precipitations (termed as the 1st,2nd,3rd and 4th DNA sample,respectively) were conducted.This recycling step was integrated into the conventional CTAB DNA extraction method to establish a recycling CTAB method.This modified CTAB method was tested in eight plant species,wheat,sorghum,barley,corn,rice,Brachypodium distachyon,Miscanthus sinensis and tung tree.The results showed that high-yield and good-quality DNA samples could be obtained by using this new method in all the eight plant species.The DNA samples were good templates for PCR amplification of both ISSR and SSR markers.The recycling method can be used in multiple plant species and can be integrated with multiple conventional DNA isolation methods,and thus is an effective and universal DNA isolation method.

  20. Detection and identification of bacterial DNA in serum from patients with acute pancreatitis

    Science.gov (United States)

    de Madaria, E; Martínez, J; Lozano, B; Sempere, L; Benlloch, S; Such, J; Uceda, F; Francés, R; Pérez-Mateo, M

    2005-01-01

    Background and aims: Bacterial infections are common complications in patients with acute pancreatitis, and translocation of bacteria from the intestinal lumen is probably the first step in the pathogenesis of these infections. As blood cultures in afebrile patients are usually negative, more sensitive methods to investigate this hypothesis in patients are needed. Our group has recently developed a method to detect the presence of bacterial DNA in biological fluids, and we aimed to detect bacterial DNA in patients with acute pancreatitis, as molecular evidences of bacterial translocation. Methods: Samples of blood were obtained on three consecutive days within the first six days after admission. Bacterial DNA was detected using a polymerase chain reaction based method, and an automated DNA nucleotide sequencing process allowed identification of bacteria species. Results: Thirty one consecutively admitted patients with acute pancreatitis were studied. Bacterial DNA was detected in six patients (19.3%), and the sequencing process allowed identification of Citrobacter freundii and Pseudomonas aeruginosa. In two patients the same bacteria detected at admission was detected 24 hours later (above 99.9% homology of nucleotide sequence). Basic clinical and biochemical characteristics were similar among patients with or without the presence of bacterial DNA. Conclusion: Detection of gram negative bacteria derived bacterial DNA in our series supports the contention that bacterial translocation is a systemic process in approximately 20% of patients with acute pancreatitis that does not seem to be related to the severity of the episode or immediate development of infection. PMID:16099797

  1. Characterization of corrosive bacterial consortia isolated from petroleum-product-transporting pipelines.

    Science.gov (United States)

    Rajasekar, Aruliah; Anandkumar, Balakrishnan; Maruthamuthu, Sundaram; Ting, Yen-Peng; Rahman, Pattanathu K S M

    2010-01-01

    Microbiologically influenced corrosion is a problem commonly encountered in facilities in the oil and gas industries. The present study describes bacterial enumeration and identification in diesel and naphtha pipelines located in the northwest and southwest region in India, using traditional cultivation technique and 16S rDNA gene sequencing. Phylogenetic analysis of 16S rRNA sequences of the isolates was carried out, and the samples obtained from the diesel and naphtha-transporting pipelines showed the occurrence of 11 bacterial species namely Serratia marcescens ACE2, Bacillus subtilis AR12, Bacillus cereus ACE4, Pseudomonas aeruginosa AI1, Klebsiella oxytoca ACP, Pseudomonas stutzeri AP2, Bacillus litoralis AN1, Bacillus sp., Bacillus pumilus AR2, Bacillus carboniphilus AR3, and Bacillus megaterium AR4. Sulfate-reducing bacteria were not detected in samples from both pipelines. The dominant bacterial species identified in the petroleum pipeline samples were B. cereus and S. marcescens in the diesel and naphtha pipelines, respectively. Therefore, several types of bacteria may be involved in biocorrosion arising from natural biofilms that develop in industrial facilities. In addition, localized (pitting) corrosion of the pipeline steel in the presence of the consortia was observed by scanning electron microscopy analysis. The potential role of each species in biofilm formation and steel corrosion is discussed.

  2. Characterization of corrosive bacterial consortia isolated from petroleum-product-transporting pipelines

    Energy Technology Data Exchange (ETDEWEB)

    Rajasekar, Aruliah; Ting, Yen-Peng [National Univ. of Singapore (Singapore). Dept. of Chemical and Biomolecular Engineering; Anandkumar, Balakrishnan [Sourashtra Coll., Madurai (India). Dept. of Biotechnology; Maruthamuthu, Sundaram [Central Electrochemical Research Inst., Karaikudi (India). Biocorrosion Group; Rahman, Pattanathu K.S.M. [Teesside Univ., Tees Valley (United Kingdom). Chemical and Bioprocess Engineering Group

    2010-01-15

    Microbiologically influenced corrosion is a problem commonly encountered in facilities in the oil and gas industries. The present study describes bacterial enumeration and identification in diesel and naphtha pipelines located in the northwest and southwest region in India, using traditional cultivation technique and 16S rDNA gene sequencing. Phylogenetic analysis of 16S rRNA sequences of the isolates was carried out, and the samples obtained from the diesel and naphtha-transporting pipelines showed the occurrence of 11 bacterial species namely Serratia marcescens ACE2, Bacillus subtilis AR12, Bacillus cereus ACE4, Pseudomonas aeruginosa AI1, Klebsiella oxytoca ACP, Pseudomonas stutzeri AP2, Bacillus litoralis AN1, Bacillus sp., Bacillus pumilus AR2, Bacillus carboniphilus AR3, and Bacillus megaterium AR4. Sulfate-reducing bacteria were not detected in samples from both pipelines. The dominant bacterial species identified in the petroleum pipeline samples were B. cereus and S. marcescens in the diesel and naphtha pipelines, respectively. Therefore, several types of bacteria may be involved in biocorrosion arising from natural biofilms that develop in industrial facilities. In addition, localized (pitting) corrosion of the pipeline steel in the presence of the consortia was observed by scanning electron microscopy analysis. The potential role of each species in biofilm formation and steel corrosion is discussed. (orig.)

  3. Characterization of Bacterial Strains Isolated from a Novel Seawater-based Retting Treatment of Hemp

    Institute of Scientific and Technical Information of China (English)

    ZHU Run-ye; CHEN Jian-yong; FENG Xin-xing; ZHANG Jian-chun

    2008-01-01

    Cultivable bacteria were isolated from seawater-based retting treatment of hemp, in which three of purified strains (SW - 1, SW - 2, and S - SW1) produced relatively high levels of pectinase activities, and also produced mannanases and xylanases.PCR - based entebacterial repetitive intergenic consensus primers (ERIC- PCR) were employed for fingerprinting DNA of the bacterial strains.The ERIC - PCR fingerprints of stains SW- 1, SW -1, and S -SW1 were found to be different, and should be further identified for each isolate.Strains SW - 1 and SW - 2 were identified as Stenotrophomnas maltophilia, while strain S - SW1 was assigned to Ochrobactrum anthropi by BIOLOG system.These two species represented rhizosphere bacterial genera, and possibly were introduced by the hemp plants.These organisms seemed potentially capable of producing pectinase and hemicellulase, and thus effectively degrading the gum substances in the seawater retting.This research could be helpful for improving a novel seawater-based retting treatment of hemp.

  4. Genetic and biochemical diversity of Gardnerella vaginalis strains isolated from women with bacterial vaginosis.

    Science.gov (United States)

    Pleckaityte, Milda; Janulaitiene, Migle; Lasickiene, Rita; Zvirbliene, Aurelija

    2012-06-01

    Gardnerella vaginalis is considered a substantial player in the progression of bacterial vaginosis (BV). We analysed 17 G. vaginalis strains isolated from the genital tract of women diagnosed with BV to establish a potential link between genotypes/biotypes and the expression of virulence factors, vaginolysin (VLY) and sialidase, which are assumed to play a substantial role in the pathogenesis of BV. Amplified ribosomal DNA restriction analysis revealed two G. vaginalis genotypes. Gardnerella vaginalis isolates of genotype 2 appeared more complex than genotype 1 and were subdivided into three subtypes. Biochemical typing allowed us to distinguish four different biotypes. A great diversity of the level of VLY production among the isolates of G. vaginalis may be related to a different cytotoxicity level of the strains. We did not find any correlation between VLY production level and G. vaginalis genotype/biotype. In contrast, a link between G. vaginalis genotype and sialidase production was established. Our findings on the diversity of VLY expression level in different clinical isolates and linking sialidase activity with the genotype of G. vaginalis could help to evaluate the pathogenic potential of different G. vaginalis strains.

  5. Diversity, Bacterial Symbionts and Antibacterial Potential of Gut-Associated Fungi Isolated from the Pantala flavescens Larvae in China.

    Science.gov (United States)

    Shao, Ming-Wei; Lu, Yi-Hui; Miao, Shuang; Zhang, Yun; Chen, Ting-Ting; Zhang, Ying-Lao

    2015-01-01

    The diversity of fungi associated with the gut of Pantala flavescens larvae was investigated using a culture-dependent method and molecular identification based on an analysis of the internally transcribed spacer sequence. In total, 48 fungal isolates were obtained from P. flavescens larvae. Based on phylogenetic analyses, the fungal isolates were grouped in 5 classes and 12 different genera. Fourteen bacterial 16S rDNA sequences derived from total genomic DNA extractions of fungal mycelia were obtained. The majority of the sequences were associated with Proteobacteria (13/14), and one Bacillaceae (1/14) was included. Leclercia sp., Oceanobacillus oncorhynchi and Methylobacterium extorquens, were reported for the first time as bacterial endosymbionts in fungi. High-performance liquid chromatography (HPLC) analysis indicated that bacterial symbionts produced specific metabolites and also exerted an inhibitory effect on fungal metabolites. The biological activity of the fungal culture extracts against the pathogenic bacteria Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633) and Escherichia coli (ATCC 8739) was investigated, and 20 extracts (42%) exhibited antibacterial activity against at least one of the tested bacterial strains. This study is the first report on the diversity and antibacterial activity of symbiotic fungi residing in the gut of P. flavescens larvae, and the results show that these fungi are highly diverse and could be exploited as a potential source of bioactive compounds.

  6. Kinetics of zinc toxicity to environmental bacterial isolates

    Directory of Open Access Journals (Sweden)

    Christian Okechukwu Nweke

    2009-12-01

    Full Text Available Toxicity of zinc to Pseudomonas, Escherichia, Proteus, Bacillus and Arthrobacter species isolated from a tropical river and petroleum refinery effluent was assessed using TTC-dehydrogenase activity (DHA inhibition test. At sufficient concentrations, zinc is toxic to these bacterial cells, and the exposure of the cells to zinc ion resulted in repression of dehydrogenase activity. The patterns of these toxic effects can be mathematically described with logistic dose-response models and in a manner similar to the non-competitive inhibition of enzymes. The threshold concentration above which toxic effect is observed ranged from 0.008 mM for Pseudomonas sp. DAF1 to 0.364 mM for Proteus sp. PLK2. The coefficients of inhibition Ki correlated with the IC50 and indicate that zinc toxicity is dependent on the organism. The Ki and toxicity threshold values predicted from the equations are comparable and are suitable indicators for kinetic analyses of zinc toxicity against bacteria.

  7. Decolorization of the textile dyes by newly isolated bacterial strains.

    Science.gov (United States)

    Chen, Kuo-Cheng; Wu, Jane-Yii; Liou, Dar-Jen; Hwang, Sz-Chwun John

    2003-02-27

    Six bacterial strains with the capability of degrading textile dyes were isolated from sludge samples and mud lakes. Aeromonas hydrophila was selected and identified because it exhibited the greatest color removal from various dyes. Although A. hydrophila displayed good growth in aerobic or agitation culture (AGI culture), color removal was the best in anoxic or anaerobic culture (ANA culture). For color removal, the most suitable pH and temperature were pH 5.5-10.0 and 20-35 degrees C under anoxic culture (ANO culture). More than 90% of RED RBN was reduced in color within 8 days at a dye concentration of 3,000 mg l(-1). This strain could also decolorize the media containing a mixture of dyes within 2 days of incubation. Nitrogen sources such as yeast extract or peptone could enhance strongly the decolorization efficiency. In contrast to a nitrogen source, glucose inhibited decolorization activity because the consumed glucose was converted to organic acids that might decrease the pH of the culture medium, thus inhibiting the cell growth and decolorization activity. Decolorization appeared to proceed primarily by biological degradation.

  8. Isolation of DNA from forensic evidence.

    Science.gov (United States)

    Bing, D H; Bieber, F R; Holland, M M; Huffine, E F

    2001-05-01

    This unit covers the many and varied methods for extracting DNA from such diverse specimens as blood, tissue, stamps and envelopes, and cigarette butts, among others. Modifications to the methods that allow the DNA to be used for either PCR or Southern blotbased analyses are also included.

  9. Influence of calcium in extracellular DNA mediated bacterial aggregation and biofilm formation.

    Directory of Open Access Journals (Sweden)

    Theerthankar Das

    Full Text Available Calcium (Ca(2+ has an important structural role in guaranteeing the integrity of the outer lipopolysaccharide layer and cell walls of bacterial cells. Extracellular DNA (eDNA being part of the slimy matrix produced by bacteria promotes biofilm formation through enhanced structural integrity of the matrix. Here, the concurrent role of Ca(2+ and eDNA in mediating bacterial aggregation and biofilm formation was studied for the first time using a variety of bacterial strains and the thermodynamics of DNA to Ca(2+ binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca(2+ had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca(2+ at biologically relevant concentrations was shown further increase in bacterial aggregation via cationic bridging. Negative Gibbs free energy (ΔG values in iTC data confirmed that the interaction between DNA and Ca(2+ is thermodynamically favourable and that the binding process is spontaneous and exothermic owing to its highly negative enthalpy. Removal of eDNA through DNase I treatment revealed that Ca(2+ alone did not enhance cell aggregation and biofilm formation. This discovery signifies the importance of eDNA and concludes that existence of eDNA on bacterial cell surfaces is a key facilitator in binding of Ca(2+ to eDNA thereby mediating bacterial aggregation and biofilm formation.

  10. Biotypes and virulence factors of Gardnerella vaginalis isolated from cases of bacterial vaginosis

    OpenAIRE

    Udayalaxmi, J.; Bhat, G. K.; S Kotigadde

    2011-01-01

    The present study was conducted to correlate the biotypes of Gardnerella vaginalis strains isolated from cases of bacterial vaginosis and their virulence factors. Thirty-two strains of G. vaginalis isolated from cases of bacterial vaginosis were biotyped. Adherence to vaginal epithelial cells, biofilm production, surface hydrophobicity, phospholipase C and protease activity were tested on these isolates. Biotype 1 was the most prevalent (8; 25%), followed by biotype 2 (7; 21.9%) and biotypes ...

  11. NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes.

    Science.gov (United States)

    Yoo, Dae-goon; Floyd, Madison; Winn, Matthew; Moskowitz, Samuel M; Rada, Balázs

    2014-08-01

    Cystic fibrosis (CF) airway disease is characterized by Pseudomonas aeruginosa infection and recruitment of neutrophil granulocytes. Neutrophil granule components (myeloperoxidase (MPO), human neutrophil elastase (HNE)), extracellular DNA and P. aeruginosa can all be found in the CF respiratory tract and have all been associated with worsening CF lung function. Pseudomonas-induced formation of neutrophil extracellular traps (NETs) offers a likely mechanism for release of MPO, HNE and DNA from neutrophils. NETs are composed of a DNA backbone decorated with granule proteins like MPO and HNE. Here we sought to examine whether CF clinical isolates of Pseudomonas are capable of inducing NET release from human neutrophil granulocytes. We used two methods to quantify NETs. We modified a previously employed ELISA that detects MPO-DNA complexes and established a new HNE-DNA ELISA. We show that these methods reliably quantify MPO-DNA and HNE-DNA complexes, measures of NET formation. We have found that CF isolates of P. aeruginosa stimulate robust respiratory burst and NET release in human neutrophils. By comparing paired "early" and "late" bacterial isolates obtained from the same CF patient we have found that early isolates induced significantly more NET release than late isolates. Our data support that Pseudomonas-induced NET release represents an important mechanism for release of neutrophil-derived CF inflammatory mediators, and confirm that decreased induction of NET formation is required for long-term adaptation of P. aeruginosa to CF airways.

  12. “Lachnoclostridium touaregense,” a new bacterial species isolated from the human gut microbiota

    OpenAIRE

    M. Tidjani Alou; S. Khelaifia; B. La Scola; Cassir, N.

    2016-01-01

    We report the main characteristics of “Lachnoclostridium touaregense” strain Marseille-P2415T (= CSUR P2415 = DSM 102219), a new bacterial species isolated from the gut microbiota of a healthy young girl from Niger.

  13. ‘Lachnoclostridium massiliosenegalense’, a new bacterial species isolated from the human gut microbiota

    OpenAIRE

    M. Tidjani Alou; J.-C. Lagier; B. La Scola; Cassir, N.

    2016-01-01

    We report the main characteristics of ‘Lachnoclostridium massiliosenegalense’ strain mt23T (=CSUR P299 =DSM 102084), a new bacterial species isolated from the gut microbiota of a healthy young girl from Senegal.

  14. Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute

    NARCIS (Netherlands)

    Swarts, Daan C.; Szczepaniak, Malwina; Sheng, Gang; Chandradoss, Stanley D.; Zhu, Yifan; Timmers, Elizabeth M.; Zhang, Yong; Zhao, Hongtu; Lou, Jizhong; Wang, Yanli; Joo, Chirlmin; Oost, van der John

    2017-01-01

    Several prokaryotic Argonaute proteins (pAgos) utilize small DNA guides to mediate host defense by targeting invading DNA complementary to the DNA guide. It is unknown how these DNA guides are being generated and loaded onto pAgo. Here, we demonstrate that guide-free Argonaute from Thermus thermophi

  15. Screening of bacterial strains isolated from uranium mill tailings porewaters for bioremediation purposes.

    Science.gov (United States)

    Sánchez-Castro, Iván; Amador-García, Ahinara; Moreno-Romero, Cristina; López-Fernández, Margarita; Phrommavanh, Vannapha; Nos, Jeremy; Descostes, Michael; Merroun, Mohamed L

    2017-01-01

    The present work characterizes at different levels a number of bacterial strains isolated from porewaters sampled in the vicinity of two French uranium tailing repositories. The 16S rRNA gene from 33 bacterial isolates, corresponding to the different morphotypes recovered, was almost fully sequenced. The resulting sequences belonged to 13 bacterial genera comprised in the phyla Firmicutes, Actinobacteria and Proteobacteria. Further characterization at physiological level and metals/metalloid tolerance provided evidences for an appropriate selection of bacterial strains potentially useful for immobilization of uranium and other common contaminants. By using High Resolution Transmission Electron Microscope (HRTEM), this potential ability to immobilize uranium as U phosphate mineral phases was confirmed for the bacterial strains Br3 and Br5 corresponding to Arthrobacter sp. and Microbacterium oxydans, respectively. Scanning Transmission Electron Microscope- High-Angle Annular Dark-Field (STEM-HAADF) analysis showed U accumulates on the surface and within bacterial cytoplasm, in addition to the extracellular space. Energy Dispersive X-ray (EDX) element-distribution maps demonstrated the presence of U and P within these accumulates. These results indicate the potential of certain bacterial strains isolated from porewaters of U mill tailings for immobilizing uranium, likely as uranium phosphates. Some of these bacterial isolates might be considered as promising candidates in the design of uranium bioremediation strategies.

  16. Mechanisms of ion-bombardment-induced DNA transfer into bacterial E. coli cells

    Energy Technology Data Exchange (ETDEWEB)

    Yu, L.D., E-mail: yuld@thep-center.org [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Sangwijit, K. [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Prakrajang, K. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Faculty of Science, Maejo University, Chiang Mai 50290 (Thailand); Phanchaisri, B. [Institute of Science and Technology Research, Chiang Mai University, Chiang Mai 50200 (Thailand); Thongkumkoon, P. [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thopan, P. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Singkarat, S. [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Anuntalabhochai, S. [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand)

    2014-05-01

    Highlights: • Ion bombardment could induce DNA transfer into E. coli cells. • The DNA transfer induction depended on ion energy and fluence. • The mechanism was associated with the bacterial cell envelope structure. • A mechanism phase diagram was proposed to summarize the mechanism. - Abstract: As a useful ion beam biotechnology, ion-bombardment-induced DNA transfer into bacterial Escherichia coli (E. coli) cells has been successfully operated using argon ions. In the process ion bombardment of the bacterial cells modifies the cell envelope materials to favor the exogenous DNA molecules to pass through the envelope to enter the cell. The occurrence of the DNA transfer induction was found ion energy and fluence dependent in a complex manner. At ion energy of a few keV and a few tens of keV to moderate fluences the DNA transfer could be induced by ion bombardment of the bacterial cells, while at the same ion energy but to high fluences DNA transfer could not be induced. On the other hand, when the ion energy was medium, about 10–20 keV, the DNA transfer could not be induced by ion bombardment of the cells. The complexity of the experimental results indicated a complex mechanism which should be related to the complex structure of the bacterial E. coli cell envelope. A phase diagram was proposed to interpret different mechanisms involved as functions of the ion energy and fluence.

  17. Isolation of the bacterial causes of tonsillitis in dogs

    Directory of Open Access Journals (Sweden)

    B. Al-Mufti

    2014-06-01

    Full Text Available The study was performed to identify the bacterial causes of tonsillitis in dogs. Twelve clinical cases of dogs (5 males and 7 females of different ages and breeds were observed. Tonsils swabs were taken from all the dogs, then cultured on different agars and bacterial smears prepared from all cultures and Gram stains were done. The study confirmed that the most bacterial causes of tonsillitis in dogs were Escherichia coli, Staphylococcus aureus, Staphylococcus intermedius, Staphylococcus albus, Streptococcus pyogenes, Klebsiella spp. and Pasteurella spp.

  18. Paramagnetic Cellulose DNA Isolation Improves DNA Yield and Quality Among Diverse Plant Taxa

    Directory of Open Access Journals (Sweden)

    Jackson R. Moeller

    2014-10-01

    Full Text Available Premise of the study: The chemical diversity of land plants ensures that no single DNA isolation method results in high yield and purity with little effort for all species. Here we evaluate a new technique originally developed for forensic science, based on MagnaCel paramagnetic cellulose particles (PMC, to determine its efficacy in extracting DNA from 25 plant species representing 21 families and 15 orders. Methods and Results: Yield and purity of DNA isolated by PMC, DNeasy Plant Mini Kit (silica column, and cetyltrimethylammonium bromide (CTAB methods were compared among four individuals for each of 25 plant species. PMC gave a two-fold advantage in average yield, and the relative advantage of the PMC method was greatest for samples with the lowest DNA yields. PMC also produced more consistent sample purity based on absorbance ratios at 260 : 280 and 260 : 230 nm. Conclusions: PMC technology is a promising alternative for plant DNA isolation.

  19. Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute.

    Science.gov (United States)

    Swarts, Daan C; Szczepaniak, Malwina; Sheng, Gang; Chandradoss, Stanley D; Zhu, Yifan; Timmers, Elizabeth M; Zhang, Yong; Zhao, Hongtu; Lou, Jizhong; Wang, Yanli; Joo, Chirlmin; van der Oost, John

    2017-03-16

    Several prokaryotic Argonaute proteins (pAgos) utilize small DNA guides to mediate host defense by targeting invading DNA complementary to the DNA guide. It is unknown how these DNA guides are being generated and loaded onto pAgo. Here, we demonstrate that guide-free Argonaute from Thermus thermophilus (TtAgo) can degrade double-stranded DNA (dsDNA), thereby generating small dsDNA fragments that subsequently are loaded onto TtAgo. Combining single-molecule fluorescence, molecular dynamic simulations, and structural studies, we show that TtAgo loads dsDNA molecules with a preference toward a deoxyguanosine on the passenger strand at the position opposite to the 5' end of the guide strand. This explains why in vivo TtAgo is preferentially loaded with guides with a 5' end deoxycytidine. Our data demonstrate that TtAgo can independently generate and selectively load functional DNA guides.

  20. Isolation of RNA and DNA from leukocytes and cDNA synthesis.

    NARCIS (Netherlands)

    Jansen, J.H.; Reijden, B.A. van der

    2006-01-01

    In this chapter, methods to isolate RNA and DNA from human leukocytes for the subsequent use in molecular diagnostic tests are described. In addition, protocols for cDNA synthesis are given, both for the use in conventional reverse transcription (RT)-polymerase chain reaction (PCR), and for the use

  1. Biodegradation of malathion, α- and β-endosulfan by bacterial strains isolated from agricultural soil in Veracruz, Mexico.

    Science.gov (United States)

    Jimenez-Torres, Catya; Ortiz, Irmene; San-Martin, Pablo; Hernandez-Herrera, R Idalia

    2016-12-01

    The objective of this study was to evaluate the capacity of two bacterial strains isolated, cultivated, and purified from agricultural soils of Veracruz, Mexico, for biodegradation and mineralisation of malathion (diethyl 2-(dimethoxyphosphorothioyl) succinate) and α- and β-endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6-9-methano-2,4,3-benzodioxathiepine-3-oxide). The isolated bacterial strains were identified using biochemical and morphological characterization and the analysis of their 16S rDNA gene, as Enterobacter cloacae strain PMM16 (E1) and E. amnigenus strain XGL214 (M1). The E1 strain was able to degrade endosulfan, whereas the M1 strain was capable of degrading both pesticides. The E1 strain degraded 71.32% of α-endosulfan and 100% of β-endosulfan within 24 days. The absence of metabolites, such as endosulfan sulfate, endosulfan lactone, or endosulfan diol, would suggest degradation of endosulfan isomers through non-oxidative pathways. Malathion was completely eliminated by the M1 strain. The major metabolite was butanedioic acid. There was a time-dependent increase in bacterial biomass, typical of bacterial growth, correlated with the decrease in pesticide concentration. The CO2 production also increased significantly with the addition of pesticides to the bacterial growth media, demonstrating that, under aerobic conditions, the bacteria utilized endosulfan and malathion as a carbon source. Here, two bacterial strains are shown to metabolize two toxic pesticides into non-toxic intermediates.

  2. In vivo compaction dynamics of bacterial DNA: A fingerprint of DNA/RNA demixing ?

    CERN Document Server

    Joyeux, Marc

    2016-01-01

    The volume occupied by unconstrained bacterial DNA in physiological solutions exceeds 1000 times the volume of the cell. Still, it is confined to a well defined region of the cell called the nucleoid, which occupies only a fraction of the cell volume. There is still no general agreement on the mechanism leading to the compaction of the DNA and the formation of the nucleoid. However, advances in in vivo sub-wavelength resolution microscopy techniques have recently allowed the observation of the nucleoid at an unprecedented level of detail. In particular, these observations show that the compaction of the nucleoid is not static but is instead a highly dynamic feature, which depends on several factors, like the richness of the nutrient, the cell cycle stage, temperature, the action of an osmotic shock or antibiotics, etc. After a short description of the electrolyte content of the cytosol and a brief overview of the different mechanisms that may lead to the formation of the nucleoid, this paper reviews some of t...

  3. Biotypes and virulence factors of Gardnerella vaginalis isolated from cases of bacterial vaginosis.

    Science.gov (United States)

    Udayalaxmi, J; Bhat, G K; Kotigadde, S

    2011-01-01

    The present study was conducted to correlate the biotypes of Gardnerella vaginalis strains isolated from cases of bacterial vaginosis and their virulence factors. Thirty-two strains of G. vaginalis isolated from cases of bacterial vaginosis were biotyped. Adherence to vaginal epithelial cells, biofilm production, surface hydrophobicity, phospholipase C and protease activity were tested on these isolates. Biotype 1 was the most prevalent (8; 25%), followed by biotype 2 (7; 21.9%) and biotypes 5 and 8 (5; 15.6%). We did not find any statistical correlation between G. vaginalis biotypes and its virulence factors. Virulence factors expressed by G. vaginalis were not associated with a single biotype.

  4. Biotypes and virulence factors of Gardnerella vaginalis isolated from cases of bacterial vaginosis

    Directory of Open Access Journals (Sweden)

    J Udayalaxmi

    2011-01-01

    Full Text Available The present study was conducted to correlate the biotypes of Gardnerella vaginalis strains isolated from cases of bacterial vaginosis and their virulence factors. Thirty-two strains of G. vaginalis isolated from cases of bacterial vaginosis were biotyped. Adherence to vaginal epithelial cells, biofilm production, surface hydrophobicity, phospholipase C and protease activity were tested on these isolates. Biotype 1 was the most prevalent (8; 25%, followed by biotype 2 (7; 21.9% and biotypes 5 and 8 (5; 15.6%. We did not find any statistical correlation between G. vaginalis biotypes and its virulence factors. Virulence factors expressed by G. vaginalis were not associated with a single biotype.

  5. Protocol for Evaluating the Permissiveness of Bacterial Communities Toward Conjugal Plasmids by Quantification and Isolation of Transconjugants

    DEFF Research Database (Denmark)

    Klümper, Uli; Dechesne, Arnaud; Smets, Barth F.

    2014-01-01

    The transfer of conjugal plasmids is the main bacterial process of horizontal gene transfer to potentially distantly related bacteria. These extrachromosomal, circular DNA molecules host genes that code for their own replication and transfer to other organisms. Because additional accessory genes...... transfer, the ability of a plasmid to invade a mixed community is crucial. The main parameter that controls the possible extent of horizontal plasmid transfer (HGT) in a bacterial community is the in situ community permissiveness for the considered plasmid. Permissiveness describes the fraction...... a gfp-tagged plasmid in a mCherry red fluorescently tagged donor strain repressing gfp expression. We take advantage of fluorescent marker genes to microscopically detect plasmid transfer events and use subsequent high-throughput fluorescence-activated cell sorting (FACS) to isolate...

  6. Rapid isolation of yeast genomic DNA: Bust n' Grab

    Directory of Open Access Journals (Sweden)

    Peterson Kenneth R

    2004-04-01

    Full Text Available Abstract Background Mutagenesis of yeast artificial chromosomes (YACs often requires analysis of large numbers of yeast clones to obtain correctly targeted mutants. Conventional ways to isolate yeast genomic DNA utilize either glass beads or enzymatic digestion to disrupt yeast cell wall. Using small glass beads is messy, whereas enzymatic digestion of the cells is expensive when many samples need to be analyzed. We sought to develop an easier and faster protocol than the existing methods for obtaining yeast genomic DNA from liquid cultures or colonies on plates. Results Repeated freeze-thawing of cells in a lysis buffer was used to disrupt the cells and release genomic DNA. Cell lysis was followed by extraction with chloroform and ethanol precipitation of DNA. Two hundred ng – 3 μg of genomic DNA could be isolated from a 1.5 ml overnight liquid culture or from a large colony. Samples were either resuspended directly in a restriction enzyme/RNase coctail mixture for Southern blot hybridization or used for several PCR reactions. We demonstrated the utility of this method by showing an analysis of yeast clones containing a mutagenized human β-globin locus YAC. Conclusion An efficient, inexpensive method for obtaining yeast genomic DNA from liquid cultures or directly from colonies was developed. This protocol circumvents the use of enzymes or glass beads, and therefore is cheaper and easier to perform when processing large numbers of samples.

  7. MALDI-TOF mass spectrometry proteomic based identification of clinical bacterial isolates

    Directory of Open Access Journals (Sweden)

    Ashutosh Panda

    2014-01-01

    Full Text Available Background & objectives: Pathogenic bacteria often cause life threatening infections especially in immunocompromised individuals. Therefore, rapid and reliable species identification is essential for a successful treatment and disease management. We evaluated a rapid, proteomic based technique for identification of clinical bacterial isolates by protein profiling using matrix-assisted laser desorption-ionization time - of - flight mass spectrometry (MALDI-TOF MS. Methods: Freshly grown bacterial isolates were selected from culture plates. Ethanol/formic acid extraction procedure was carried out, followed by charging of MALDI target plate with the extract and overlaying with α-cyano-4 hydroxy-cinnamic acid matrix solution. Identification was performed using the MALDI BioTyper 1.1, software for microbial identification (Bruker Daltonik GmbH, Bremen, Germany. Results: A comparative analysis of 82 clinical bacterial isolates using MALDI -TOF MS and conventional techniques was carried out. Amongst the clinical isolates, the accuracy at the species level for clinical isolates was 98.78%. One out of 82 isolates was not in accordance with the conventional assays because MALDI-TOF MS established it as Streptococcus pneumoniae and conventional methods as Streptococcus viridans. Interpretation & conclusions: MALDI - TOF MS was found to be an accurate, rapid, cost-effective and robust system for identification of clinical bacterial isolates. This innovative approach holds promise for earlier therapeutic intervention leading to better patient care.

  8. An easy and versatile 2-step protocol for targeted modification and subcloning of DNA from bacterial artificial chromosomes using non-commercial plasmids

    Directory of Open Access Journals (Sweden)

    Hartwich Heiner

    2012-03-01

    Full Text Available Abstract Background Promoter-specific expression of foreign DNA in transgenic organisms often relies on bacterial artificial chromosomes (BACs. This approach requires modification and subcloning of BAC-DNA by recombineering technologies in Escherichia coli. Most current protocols rely on commercial kits or isolation of BACs, their transfer between different host strains, and their restriction. Findings In this report we present a 2-step protocol for efficient modification and subcloning of DNA from bacterial artificial chromosomes using the non-commercial plasmids pKM208 and pTP223, distributed from addgene.com. A targeting cassette was successfully integrated into a BAC and 42 kb of this construct were subcloned. Both a plasmid-derived substrate with longer homology arms and a PCR-generated substrate with short homology arms (50 bp were used for recombination. pKM208 and pTP223 contain all required genes for recombineering, but differ in their antibiotic resistance genes. This makes the system independent of the selection markers on the DNA molecules targeted for recombination. Conclusions The time and cost saving protocol presented here compares favorably to currently used systems. Using non-commercial plasmids, it allows targeted modification and cloning of large DNA (> 40 kb fragments in vivo without restriction and ligation. Furthermore, both steps are performed in the same host eliminating the need to isolate BAC DNA and to use different bacterial strains.

  9. Identification and characterization of humic substances-degrading bacterial isolates from an estuarine environment.

    Science.gov (United States)

    Esham; Ye; Moran

    2000-12-01

    Bacterial isolates were obtained from enrichment cultures containing humic substances extracted from estuarine water using an XAD-8 resin. Eighteen isolates were chosen for phylogenetic and physiological characterization based on numerical importance in serial dilutions of the enrichment culture and unique colony morphology. Partial sequences of the 16S rRNA genes indicated that six of the isolates were associated with the alpha subclass of Proteobacteria, three with the gamma-Proteobacteria, and nine with the Gram-positive bacteria. Ten isolates degraded at least one (and up to six) selected aromatic single-ring compounds. Six isolates showed ability to degrade [(14)C]humic substances derived from the dominant salt marsh grass in the estuary from which they were isolated (Spartina alterniflora), mineralizing 0.4-1.1% of the humic substances over 4 weeks. A mixture of all 18 isolates did not degrade humic substances significantly faster than any of the individual strains, however, and no isolate degraded humic substances to the same extent as the natural marine bacterial community (3.0%). Similar studies with a radiolabeled synthetic lignin ([beta-(14)C]dehydropolymerisate) showed measurable levels of degradation by all 18 bacteria (3.0-8.8% in 4 weeks), but mineralization levels were again lower than that observed for the natural marine bacterial community (28.2%). Metabolic capabilities of the 18 isolates were highly variable and generally did not map to phylogenetic affiliation.

  10. Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing

    Directory of Open Access Journals (Sweden)

    Zdepski Anna

    2011-05-01

    Full Text Available Abstract Background High throughput sequencing (HTS technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination. Results We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR. We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment. Conclusions Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.

  11. Genome Calligrapher: A Web Tool for Refactoring Bacterial Genome Sequences for de Novo DNA Synthesis.

    Science.gov (United States)

    Christen, Matthias; Deutsch, Samuel; Christen, Beat

    2015-08-21

    Recent advances in synthetic biology have resulted in an increasing demand for the de novo synthesis of large-scale DNA constructs. Any process improvement that enables fast and cost-effective streamlining of digitized genetic information into fabricable DNA sequences holds great promise to study, mine, and engineer genomes. Here, we present Genome Calligrapher, a computer-aided design web tool intended for whole genome refactoring of bacterial chromosomes for de novo DNA synthesis. By applying a neutral recoding algorithm, Genome Calligrapher optimizes GC content and removes obstructive DNA features known to interfere with the synthesis of double-stranded DNA and the higher order assembly into large DNA constructs. Subsequent bioinformatics analysis revealed that synthesis constraints are prevalent among bacterial genomes. However, a low level of codon replacement is sufficient for refactoring bacterial genomes into easy-to-synthesize DNA sequences. To test the algorithm, 168 kb of synthetic DNA comprising approximately 20 percent of the synthetic essential genome of the cell-cycle bacterium Caulobacter crescentus was streamlined and then ordered from a commercial supplier of low-cost de novo DNA synthesis. The successful assembly into eight 20 kb segments indicates that Genome Calligrapher algorithm can be efficiently used to refactor difficult-to-synthesize DNA. Genome Calligrapher is broadly applicable to recode biosynthetic pathways, DNA sequences, and whole bacterial genomes, thus offering new opportunities to use synthetic biology tools to explore the functionality of microbial diversity. The Genome Calligrapher web tool can be accessed at https://christenlab.ethz.ch/GenomeCalligrapher  .

  12. Use of single-strand conformation polymorphism of amplified 16S rDNA for grouping of bacteria isolated from foods.

    Science.gov (United States)

    Takahashi, Hajime; Kimura, Bon; Tanaka, Yuichiro; Mori, Mayumi; Yokoi, Asami; Fujii, Tateo

    2008-04-01

    The grouping method for isolated strains from foods using single-strand conformation polymorphism (SSCP) after PCR amplification of a portion of 16S rDNA was developed. This method was able to group the strains from various food samples based on 16S rDNA sequence. As 97.8% of the isolated strains from various foods were grouped correctly, use of the PCR-SSCP method enables the prompt and labor-saving analysis of microbial population of food-derived bacterial strains. Advantages in speed and accuracy of bacterial population identification by the PCR-SSCP method have practical application for food suppliers and testing laboratories.

  13. Isolation and molecular characterisation of malathion-degrading bacterial strains from waste water in Egypt

    Directory of Open Access Journals (Sweden)

    Zeinat K. Mohamed

    2010-04-01

    Full Text Available Efficiencies of local bacterial isolates in malathion degradation were investigated. Five bacterial isolates obtained from agricultural waste water were selected due to their ability to grow in minimal salt media, supplied with 250 ppm malathion as sole source of carbon and phosphorus. The purified bacterial isolates (MOS-1, MOS-2, MOS-3, MOS-4 and MOS-5 were characterised and identified using a combination of cellular profile (SDS-PAGE, genetic make up profile (RAPD-PCR, and morphological and biochemical characteristics. Four bacterial isolates (MOS-1, MOS-2, MOS-3 and MOS-4 with identical genetic characteristics were identified as Enterobacter aerogenes, whereas isolate MOS-5 was identified as Bacillus thuringiensis. The degradation rate of malathion in liquid culture was estimated during 15 days of incubation for the isolate MOS-5 of B. thuringiensis. Slightly more than 50% of the initial malathion was decomposed within 3 days. The malathion concentration decreased to almost 17% in the inoculated medium after 10 days incubation, while more than 91% of the initial malathion was degraded after 15 days.

  14. REVIEW ARTICLE: DNA protein interactions and bacterial chromosome architecture

    Science.gov (United States)

    Stavans, Joel; Oppenheim, Amos

    2006-12-01

    Bacteria, like eukaryotic organisms, must compact the DNA molecule comprising their genome and form a functional chromosome. Yet, bacteria do it differently. A number of factors contribute to genome compaction and organization in bacteria, including entropic effects, supercoiling and DNA-protein interactions. A gamut of new experimental techniques have allowed new advances in the investigation of these factors, and spurred much interest in the dynamic response of the chromosome to environmental cues, segregation, and architecture, during both exponential and stationary phases. We review these recent developments with emphasis on the multifaceted roles that DNA-protein interactions play.

  15. A simple and efficient DNA isolation method for Salvia officinalis.

    Science.gov (United States)

    Aleksić, Jelena M; Stojanović, Danilo; Banović, Bojana; Jančić, Radiša

    2012-12-01

    We report an efficient, simple, and cost-effective protocol for the isolation of genomic DNA from an aromatic medicinal plant, common sage (Salvia officinalis L.). Our modification of the standard CTAB protocol includes two polyphenol adsorbents (PVP 10 and activated charcoal), high NaCl concentrations (4 M) for removing polysaccharides, and repeated Sevag treatment to remove proteins and other carbohydrate contaminants. The mean DNA yield obtained with our Protocol 2 was 330.6 μg DNA g(-1) of dry leaf tissue, and the absorbance ratios 260/280 and 260/230 nm averaged 1.909 and 1.894, respectively, revealing lack of contamination. PCR amplifications of one nuclear (26S rDNA) and one chloroplast (rps16-trnK) locus indicated that our DNA isolation protocol may be used in common sage and other aromatic and medicinal plants containing essential oil for molecular biologic and biotechnological studies and for population genetics, phylogeographic, and conservation surveys in which nuclear or chloroplast genomes would be studied in large numbers of individuals.

  16. A simple method for DNA isolation from Xanthomonas spp.

    Directory of Open Access Journals (Sweden)

    Gomes Luiz Humberto

    2000-01-01

    Full Text Available A simple DNA isolation method was developed with routine chemicals that yields high quality and integrity preparations when compared to some of the most well known protocols. The method described does not require the use of lysing enzymes, water bath and the DNA was obtained within 40 minutes The amount of nucleic acid extracted (measured in terms of absorbancy at 260 nm from strains of Xanthomonas spp., Pseudomonas spp. and Erwinia spp. was two to five times higher than that of the most commonly used method.

  17. Genome Sequences of Nine Gram-Negative Vaginal Bacterial Isolates

    Science.gov (United States)

    Deitzler, Grace E.; Ruiz, Maria J.; Lu, Wendy; Weimer, Cory; Park, SoEun; Robinson, Lloyd S.; Hallsworth-Pepin, Kymberlie; Wollam, Aye; Mitreva, Makedonka

    2016-01-01

    The vagina is home to a wide variety of bacteria that have great potential to impact human health. Here, we announce reference strains (now available through BEI Resources) and draft genome sequences for 9 Gram-negative vaginal isolates from the taxa Citrobacter, Klebsiella, Fusobacterium, Proteus, and Prevotella. PMID:27688330

  18. Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding surface.

    Science.gov (United States)

    Williamson, Adele; Rothweiler, Ulli; Leiros, Hanna Kirsti Schrøder

    2014-11-01

    DNA ligases are a structurally diverse class of enzymes which share a common catalytic core and seal breaks in the phosphodiester backbone of double-stranded DNA via an adenylated intermediate. Here, the structure and activity of a recombinantly produced ATP-dependent DNA ligase from the bacterium Psychromonas sp. strain SP041 is described. This minimal-type ligase, like its close homologues, is able to ligate singly nicked double-stranded DNA with high efficiency and to join cohesive-ended and blunt-ended substrates to a more limited extent. The 1.65 Å resolution crystal structure of the enzyme-adenylate complex reveals no unstructured loops or segments, and suggests that this enzyme binds the DNA without requiring full encirclement of the DNA duplex. This is in contrast to previously characterized minimal DNA ligases from viruses, which use flexible loop regions for DNA interaction. The Psychromonas sp. enzyme is the first structure available for the minimal type of bacterial DNA ligases and is the smallest DNA ligase to be crystallized to date.

  19. Bacterial-Feeding Nematode Growth and Preference for Biocontrol Isolates of the Bacterium Burkholderia cepacia.

    Science.gov (United States)

    Carta, L K

    2000-12-01

    The potential of different bacterial-feeding Rhabditida to consume isolates of Burkholderia cepacia with known agricultural biocontrol ability was examined. Caenorhabditis elegans, Diploscapter sp., Oscheius myriophila, Pelodera strongyloides, Pristionchus pacificus, Zeldia punctata, Panagrellus redivivus, and Distolabrellus veechi were tested for growth on and preference for Escherichia coli OP50 or B. cepacia maize soil isolates J82, BcF, M36, Bc2, and PHQM100. Considerable growth and preference variations occurred between nematode taxa on individual bacterial isolates, and between different bacterial isolates on a given nematode. Populations of Diploscapter sp. and P. redivivus were most strongly suppressed. Only Z. punctata and P. pacificus grew well on all isolates, though Z. punctata preferentially accumulated on all isolates and P. pacificus had no preference. Oscheius myriophila preferentially accumulated on growth-supportive Bc2 and M36, and avoided less supportive J82 and PHQM100. Isolates with plant-parasitic nematicidal properties and poor fungicidal properties supported the best growth of three members of the Rhabditidae, C. elegans, O. myriophila, and P. strongyloides. Distolabrellus veechi avoided commercial nematicide M36 more strongly than fungicide J82.

  20. Isolation, Characterization and Application of Bacterial Population From Agricultural Soil at Sohag Province, Egypt

    Directory of Open Access Journals (Sweden)

    Bahig, A. E.

    2008-01-01

    Full Text Available Forty soil samples of agriculture soil were collected from two different sites in Sohag province, Egypt, during hot and cold seasons. Twenty samples were from soil irrigated with canal water (site A and twenty samples were from soil irrigated with wastewater (site B. This study aimed to compare the incidence of plasmids in bacteria isolated from soil and to investigate the occurrence of metal and antibiotic resistance bacteria, and consequently to select the potential application of these bacteria in bioremediation. The total bacterial count (CFU/gm in site (B was higher than that in site (A. Moreover, the CFU values in summer were higher than those values in winter at both sites. A total of 771 bacterial isolates were characterized as Bacillus, Micrococcus, Staphylococcus, Pseudomonas, Eschershia, Shigella, Xanthomonas, Acetobacter, Citrobacter, Enterobacter, Moraxella and Methylococcus. Minimum inhibitory concentrations (MICs of Pb+2, Cu+2, Zn+2, Hg+2, Co+2, Cd+2, Cr+3, Te+2, As+2 and Ni+2 for plasmid-possessed bacteria were determined and the highest MICs were 1200 µg/mL for lead, 800 µg/mL for both Cobalt and Arsenate, 1200 µg/mL for Nickel, 1000 µg/ml for Copper and less than 600 µg/mL for other metals. Bacterial isolates from both sites A and B showed multiple heavy metal resistance. A total of 337 bacterial isolates contained plasmids and the incidence of plasmids was approximately 25-50% higher in bacteria isolated from site (B than that from site (A. These isolates were resistance to different antibiotics. Approximately, 61% of the bacterial isolates were able to assimilate insecticide, carbaryl, as a sole source of carbon and energy. However, the Citrobacter AA101 showed the best growth on carbaryl.

  1. Comparison of Hemagglutination and Hemolytic Activity of Various Bacterial Clinical Isolates Against Different Human Blood Groups

    Science.gov (United States)

    HRV, Rajkumar; Devaki, Ramakrishna

    2016-01-01

    Among the various pathogenic determinants shown by microorganisms hemagglutination and hemolysin production assume greater significance in terms of laboratory identification. This study evaluated the hemagglutination and hemolytic activity of various bacterial isolates against different blood groups. One hundred and fifty bacterial strains, isolated from clinical specimens like urine, pus, blood, and other body fluids were tested for their hemagglutinating and hemolytic activity against human A, B, AB, and O group red blood cells. Among the 150 isolates 81 were Escherichia coli, 18 were Klebsiella pneumoniae, 19 were Pseudomonas aeruginosa, 10 were Pseudomonas spp, six were Proteus mirabilis, and the rest 16 were Staphylococcus aureus. Nearly 85% of the isolates agglutinated A group cells followed by B and AB group (59.3% and 60.6% respectively). Least number of isolates agglutinated O group cells (38.0%). When the hemolytic activity was tested, out of these 150 isolates 79 (52.6%) hemolyzed A group cells, 61 (40.6%) hemolyzed AB group cells, 46 (30.6%) hemolyzed B group cells, and 57 (38.6%) isolates hemolyzed O group cells. Forty-six percent of the isolates exhibited both hemagglutinating and hemolytic property against A group cells, followed by B and AB group cells (28.6% and 21.3% respectively). Least number of isolates i.e., 32 (21.3%) showed both the properties against O group cells. The isolates showed wide variation in their hemagglutination and hemolytic properties against different combinations of human blood group cells. The study highlights the importance of selection of the type of cells especially when human RBCs are used for studying the hemagglutination and hemolytic activity of bacterial isolates because these two properties are considered as characteristic of pathogenic strains. PMID:27014523

  2. Importance of isolation and biotypization of Gardnerella vaginalis in diagnosis of bacterial vaginosis.

    Science.gov (United States)

    Numanović, Fatima; Hukić, Mirsada; Nurkić, Mahmud; Gegić, Merima; Delibegović, Zineta; Imamović, Alma; Pasić, Selma

    2008-08-01

    The natural habitat of Gardnerella vaginalis is a vagina since it could be located among 69% of women who have no signs of vaginal infection and in the vagina of as many as 13.5% girls. G. vaginalis is almost certainly identified among women diagnosed with bacterial vaginosis as well as in the urethra of their sexual partner. The increase in prevalence and concentration of G. vaginalis among patients diagnosed with this syndrome confirms that G. vaginalis plays a significant role in its pathogenesis. In our research, based on Amsel criteria for three or more clinical signs of bacterial vaginosis, it was diagnosed in 20.5% of women with subjective problems of vaginal infection, and in 48.80% of women with subjective symptoms characteristic of this disease. G. vaginalis was isolated from vaginal secretion of women without clinical signs characteristic of bacterial vaginosis. In 2.58% of cases it was solitary, while in 1.28% it was found in combination with other aerobic and anaerobic bacteria and, in 1.28% women combined with Candida albicans. The isolation of G. vaginalis was significantly increased (pbiotype of G. vaginalis, different from a source biotype or as a consequence of wrong treatment. Following Piot biotype scheme, biotypes 2., 3. and 7. G. vaginalis are significantly more often isolated from women who suffer from bacterial vaginosis. Biotype 7. G. vaginalis, isolated from the group of women without clinical signs of bacterial vaginosis, accounted for 2.58% cases. Following Benit biotype scheme, biotypes IVa, IVc and IIc were identified in 12.90% cases, while biotypes IIIa, IIa, Ia, IVb, IIb were found in 6.45% cases. Lipase-positive isolates of G. vaginalis were significantly more frequently accompanied by the syndrome of bacterial vaginosis.

  3. Pseudomonas granadensis sp. nov., a new bacterial species isolated from the Tejeda, Almijara and Alhama Natural Park, Granada, Spain.

    Science.gov (United States)

    Pascual, Javier; García-López, Marina; Bills, Gerald F; Genilloud, Olga

    2015-02-01

    During the course of screening bacterial isolates as sources of as-yet unknown bioactive compounds with pharmaceutical applications, a chemo-organotrophic, Gram-negative bacterium was isolated from a soil sample taken from the Tejeda, Almijara and Alhama Natural Park, Granada, Spain. Strain F-278,770(T) was oxidase- and catalase-positive, aerobic, with a respiratory type of metabolism with oxygen as the terminal electron acceptor, non-spore-forming and motile by one polar flagellum, although some cells had two polar flagella. Phylogenetic analysis of the 16S rRNA, gyrB, rpoB and rpoD genes revealed that strain F-278,770(T) belongs to the Pseudomonas koreensis subgroup (Pseudomonas fluorescens lineage), with Pseudomonas moraviensis, P. koreensis, P. baetica and P. helmanticensis as its closest relatives. Chemotaxonomic traits such as polar lipid and fatty acid compositions and G+C content of genomic DNA corroborated the placement of strain F-278,770(T) in the genus Pseudomonas. DNA-DNA hybridization assays and phenotypic traits confirmed that this strain represents a novel species of the genus Pseudomonas, for which the name Pseudomonas granadensis sp. nov. is proposed. The type strain is F-278,770(T) ( = DSM 28040(T) = LMG 27940(T)).

  4. UV Radiation Damage and Bacterial DNA Repair Systems

    Science.gov (United States)

    Zion, Michal; Guy, Daniel; Yarom, Ruth; Slesak, Michaela

    2006-01-01

    This paper reports on a simple hands-on laboratory procedure for high school students in studying both radiation damage and DNA repair systems in bacteria. The sensitivity to ultra-violet (UV) radiation of both "Escherichia coli" and "Serratia marcescens" is tested by radiating them for varying time periods. Two growth temperatures are used in…

  5. Fragment-based discovery of 6-azaindazoles as inhibitors of bacterial DNA ligase.

    Science.gov (United States)

    Howard, Steven; Amin, Nader; Benowitz, Andrew B; Chiarparin, Elisabetta; Cui, Haifeng; Deng, Xiaodong; Heightman, Tom D; Holmes, David J; Hopkins, Anna; Huang, Jianzhong; Jin, Qi; Kreatsoulas, Constantine; Martin, Agnes C L; Massey, Frances; McCloskey, Lynn; Mortenson, Paul N; Pathuri, Puja; Tisi, Dominic; Williams, Pamela A

    2013-12-12

    Herein we describe the application of fragment-based drug design to bacterial DNA ligase. X-ray crystallography was used to guide structure-based optimization of a fragment-screening hit to give novel, nanomolar, AMP-competitive inhibitors. The lead compound 13 showed antibacterial activity across a range of pathogens. Data to demonstrate mode of action was provided using a strain of S. aureus, engineered to overexpress DNA ligase.

  6. Selective Removal of DNA from Dead Cells of Mixed Bacterial Communities by Use of Ethidium Monoazide

    OpenAIRE

    Nocker, Andreas; Anne K. Camper

    2006-01-01

    The distinction between viable and dead bacterial cells poses a major challenge in microbial diagnostics. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based quantification methods overestimate the number of viable cells in mixed populations or even lead to false-positive results in the absence of viable cells. On the other hand, RNA-based diagnostic methods, which circumvent this problem, are technically demanding and suffer from some drawbacks. A prom...

  7. Bacterial DNA segregation by dynamic SopA polymers

    OpenAIRE

    2005-01-01

    Many bacterial plasmids and chromosomes rely on ParA ATPases for proper positioning within the cell and for efficient segregation to daughter cells. Here we demonstrate that the F-plasmid-partitioning protein SopA polymerizes into filaments in an ATP-dependent manner in vitro, and that the filaments elongate at a rate that is similar to that of plasmid separation in vivo. We show that SopA is a dynamic protein within the cell, undergoing cycles of polymerization and depolymerization, and shut...

  8. MICROFLUIDIC MODULES FOR ISOLATION OF RECOMBINANT CYTOKINE FROM BACTERIAL LYSATES

    Energy Technology Data Exchange (ETDEWEB)

    Retterer, Scott T [ORNL; Doktycz, Mitchel John [ORNL

    2014-01-01

    The portability and personalization of health-care diagnostics and treatments benefits from advancements and applications of micro and nanotechnology. Modularization and miniaturization of standardized biochemical processes and tests facilitates the advancement and customization of analyte detection and diagnosis on-chip. The goal of our work here is to develop modular platforms for on-chip biochemical processing of synthesized biologics for a range of on-demand applications. Our report focuses on the initial development, characterization and application of microfluidic size exclusion/gel filtration and ion exchange protein concentration modules for cytokine isolation from spiked cell extracts.

  9. Isolation of Biosurfactant–Producing Bacteria with Antimicrobial Activity against Bacterial Pathogens

    Directory of Open Access Journals (Sweden)

    Siripun Sarin

    2011-01-01

    Full Text Available The aims of this research were to study biosurfactant producing bacteria isolated from soil and to determine their property and efficiency as biosurfactants in order to inhibit bacterial pathogens. The result showed that there were 8 bacterial isolates out of 136 isolates of the total biosurfactant producing bacteria screened that exhibited the diameter of clear zone more than 1.5 cm. in the oil spreading test. The highest potential of emulsifying activity (%EA24 of 54.4 and the maximum additive concentration, (%MAC of 24.2 was obtained from the fermentation broth of the G7 isolate which the G7 isolate was later identified as Pseudomonas fluorescens. Escherichia coli, Staphylococcus aureus and Psuedomonas aeruginosa were the tested bacterial pathogens that were most sensitive to the acid precipitated biosurfactant obtained from P. fluorescens G7 with the lowest minimum inhibitory concentration (MIC of 41.6 mg/ml and minimum bactericidal concentration (MBC of 41.6 mg/ml compared with the acid precipitated bisurfactants of the other isolates used in the antimicrobial activity test. The type of the separated crude biosurfactant produced by P. fluorescens G7 analyzed later by using the rhamose test, TLC and FT-IR techniques was rhamnolipid.

  10. Gyramides prevent bacterial growth by inhibiting DNA gyrase and altering chromosome topology.

    Science.gov (United States)

    Rajendram, Manohary; Hurley, Katherine A; Foss, Marie H; Thornton, Kelsey M; Moore, Jared T; Shaw, Jared T; Weibel, Douglas B

    2014-06-20

    Antibiotics targeting DNA gyrase have been a clinical success story for the past half-century, and the emergence of bacterial resistance has fueled the search for new gyrase inhibitors. In this paper we demonstrate that a new class of gyrase inhibitors, the gyramides, are bacteriostatic agents that competitively inhibit the ATPase activity of Escherichia coli gyrase and produce supercoiled DNA in vivo. E. coli cells treated with gyramide A have abnormally localized, condensed chromosomes that blocks DNA replication and interrupts chromosome segregation. The resulting alterations in DNA topology inhibit cell division through a mechanism that involves the SOS pathway. Importantly, gyramide A is a specific inhibitor of gyrase and does not inhibit the closely related E. coli enzyme topoisomerase IV. E. coli mutants with reduced susceptibility to gyramide A do not display cross-resistance to ciprofloxacin and novobiocin. The results demonstrate that the gyramides prevent bacterial growth by a mechanism in which the topological state of chromosomes is altered and halts DNA replication and segregation. The specificity and activity of the gyramides for inhibiting gyrase makes these compounds important chemical tools for studying the mechanism of gyrase and the connection between DNA topology and bacterial cell division.

  11. Testing of DNA isolation for the identification of hemp

    Directory of Open Access Journals (Sweden)

    Tomáš Vyhnánek

    2015-12-01

    Full Text Available Hemp is diploid organism (2n = 2x = 20, genome size 534 Mb with nine pairs of autosomes plus XX (♀ or XY (♂ chromosomes. Cannabis sativa L. is an important economic plant for the production of food, fibre, oils, and intoxicants. Genotypes (varieties or chemovar of hemp with low Δ9-tetrahydrocannabinol content are used for industrial applications. Varieties with high Δ9-tetrahydrocannabinol or high cannabidiol content are used for medicinal applications. Biochemical and molecular methods can be used for identification and classification. An important step for molecular biology methods is to obtain the matrix of the native and sufficiently pure DNA. We tested two different experimental variant of samples (20 mg and 100 mg of seeds, oilcake and dried flowers for analysis of the Italian variety Carmagnola for analysis (harvested in 2014, Hempoint Ltd., Czech Republic. The DNeasy® Plant Mini Kit (Qiagen, GE was used to isolate the DNA. The DNA concentration and purity was assessed by agarose electrophoresis and via a spectrophotometer. Samples of lower weight yielded lower values of DNA concentration (average 16.30 - 38.90 ng.µL-1, but with better purity than samples of higher weight (ratio A260nm/A280nm for low-weight samples was near 1.80. To test the applicability of DNA analysis, we used two SSR markers (CAN1347 and CAN2913. PCR products were separated on 1% agarose and on 8% polyacrylamide electrophoresis. DNA samples obtained from samples of higher weight exhibited less PCR amplification than samples of lower weight. We found no effect of sample weight on the formation of non-specific amplification products during the PCR reaction. Based on our results we can be recommended for practical isolation procedure using DNeasy® Plant Mini Kit with lower of sample weight (20 mg. In future work the procedure for DNA isolating from wheat-cannabis products, e. g. breads, rolls or pasta, will be optimized.

  12. Isolation and characterization of novel bacterial strains exhibiting ligninolytic potential

    Directory of Open Access Journals (Sweden)

    Bandounas Luaine

    2011-10-01

    Full Text Available Abstract Background To expand on the range of products which can be obtained from lignocellulosic biomass, the lignin component should be utilized as feedstock for value-added chemicals such as substituted aromatics, instead of being incinerated for heat and energy. Enzymes could provide an effective means for lignin depolymerization into products of interest. In this study, soil bacteria were isolated by enrichment on Kraft lignin and evaluated for their ligninolytic potential as a source of novel enzymes for waste lignin valorization. Results Based on 16S rRNA gene sequencing and phenotypic characterization, the organisms were identified as Pandoraea norimbergensis LD001, Pseudomonas sp LD002 and Bacillus sp LD003. The ligninolytic capability of each of these isolates was assessed by growth on high-molecular weight and low-molecular weight lignin fractions, utilization of lignin-associated aromatic monomers and degradation of ligninolytic indicator dyes. Pandoraea norimbergensis LD001 and Pseudomonas sp. LD002 exhibited best growth on lignin fractions, but limited dye-decolourizing capacity. Bacillus sp. LD003, however, showed least efficient growth on lignin fractions but extensive dye-decolourizing capacity, with a particular preference for the recalcitrant phenothiazine dye class (Azure B, Methylene Blue and Toluidene Blue O. Conclusions Bacillus sp. LD003 was selected as a promising source of novel types of ligninolytic enzymes. Our observations suggested that lignin mineralization and depolymerization are separate events which place additional challenges on the screening of ligninolytic microorganisms for specific ligninolytic enzymes.

  13. Isolation and Identification of Active Compound Cause Light Emmitting of Bacterial Photobacterium phosphoreum Isolated from the Indonesia Jepara Marine Squid

    Directory of Open Access Journals (Sweden)

    Idam Arif

    2005-04-01

    Full Text Available This research carried out to study the bioluminescence process of bacterial Photobacterium phosphoreum isolated from Indonesia marine squid. The method used in the present study involved isolation, purification, electrophoresis, and the absorbance and light intensity measurement. This result show that the luciferace enzyme of bacterial Photobacterium phosphoreum or called LBPP catalyzes the emission of visible light from the reaction of reduced flavin mononucleotide (FMNH2, molecular oxygen (O2, and an aldehyde (RCOH. The electrophoresis data show that LBPP comprised of two different subunits α and βwith 41kD and 38 kD molecular weights. The absorb pattern showed that the bioluminescence process centered around 516 nm and are consistent with the fluorescence data. This result concluded that the excitation state formed after LBPP bind subtracts and the ground state formed after LBPP releases product and visible light.

  14. Isolation, identification and characterization of Bacillus amyloliquefaciens BZ-6, a bacterial isolate for enhancing oil recovery from oily sludge.

    Science.gov (United States)

    Liu, Wuxing; Wang, Xiaobing; Wu, Longhua; Chen, Mengfang; Tu, Chen; Luo, Yongming; Christie, Peter

    2012-06-01

    Over 100 biosurfactant-producing microorganisms were isolated from oily sludge and petroleum-contaminated soil from Shengli oil field in north China. Sixteen of the bacterial isolates produced biosurfactants and reduced the surface tension of the growth medium from 71 to BZ-6 was found to be the most efficient strain and the three phases (oil, water and sediment) were separated automatically after the sludge was treated with the culture medium of BZ-6. Based on morphological, physiological characteristics and molecular identification, isolate BZ-6 was identified as Bacillus amyloliquefaciens. The biosurfactant produced by isolate BZ-6 was purified and analyzed by high performance liquid chromatography-electrospray ionization tandem mass spectrometry. There were four ion peaks representing four different fengycin A homologues.

  15. Isolation and characterization of rice lesion mimic mutants from a T-DNA tagged population

    Institute of Scientific and Technical Information of China (English)

    LI Shutian; PEI Zhongyou; LUO Lijuan; TIAN Yingchuan; HE Chaozu

    2005-01-01

    A rice ( Oryza sativa L. ssp. japonica cv. Nipponbare) T-DNA tagged population consisting of about 7000 individual lines was generated and screened for rice lesion mimic mutants in the T1 generation. Ten lines were found to develop spontaneous lesions in the absence of pathogen infection and displayed distinct lesion phenotypes. These mutants were tentatively designated as lm1 -lm10 (for lesion mimic), respectively. Lesion formation of lm mutants was developmentally regulated, and all the mutants showed stunted growth and reduced fertility. Genetic analysis demonstrated that all the mutations were recessive, and five partially fertile mutants (lm4-lm8) were derived from different loci. Mimic lesions occurring on the leaves of lm mutants resulted from cell death as revealed by trypan blue staining. Six of them ( lm3 -lm8 ) exhibited enhanced resistance to five bacterial blight isolates, indicating their wide-spectrum resistance to this pathogen. These results imply that some lesion mimic mutations of rice might be involved in disease resistance signaling pathways,and that isolation of these mutated genes may be useful for elucidating molecular mechanisms of plant disease resistance. Among the mutants, only one mutant, lm6, was preliminarily shown to cosegregate with the inserted T-DNA in its T1 generation, making it feasible to isolate the gene responsible for the phenotype of this mutant.

  16. The Prevalence and Antimicrobial Susceptibility of Bacterial Uropathogens Isolated from Pediatric Patients

    Directory of Open Access Journals (Sweden)

    R Ranjbar

    2009-06-01

    Full Text Available "nBackground: Urinary tract infection (UTI is considered as the most common bacterial infectious disease seen among the pediatric patients. The aim of this study was to investigate the prevalence and antimicrobial susceptibility of bacterial uropathogens isolated from the pediatric patients with urinary tract infections."nMethods: This descriptive study was conducted in Children Medial Center, Tehran, Iran from March 2006 to Feb 2007. Clean-catch midstream urine specimens were obtained from the patients and cultured on the appropriate bacteriological media. Bacterial isolates were identified by standard biochemical and serological tests. Antimicrobial susceptibility testing was performed according to CLSI guidelines."nResults: From 14199 urine specimens, 16.2% had positive results for bacterial cultures. Nine hundred twenty one strains were identified as Escherichia coli; 412 as Klebsiella spp., 285 as Coagulase negative Staphylocococci, 202 as Enterococcus spp., 158 as Pseudomonas spp., and 83 as Staphylococcus aureus. E. coli isolates showed high resistance to carbenicillin (68%, ampicillin (96%, trimethoprim-sulfomethoxazol (70% and kanamycin (65%. More than 30% of isolates of Klebsiella spp., Pseudomonas spp. and Enterobacter spp. have shown high degree of resistance to commonly used antibiotics."nConclusion: Our findings reinforce the need for ongoing investigation to show trends in antibiotic resistance, which can help to prescribing of antibiotics in clinics.

  17. Antibiotic resistance among cultured bacterial isolates from bioethanol fermentation facilities across the United States.

    Science.gov (United States)

    Murphree, Colin A; Heist, E Patrick; Moe, Luke A

    2014-09-01

    Bacterial contamination of fuel ethanol fermentations by lactic acid bacteria (LAB) can have crippling effects on bioethanol production. Producers have had success controlling bacterial growth through prophylactic addition of antibiotics to fermentors, yet concerns have arisen about antibiotic resistance among the LAB. Here, we report on mechanisms used by 32 LAB isolates from eight different US bioethanol facilities to persist under conditions of antibiotic stress. Minimum inhibitory concentration assays with penicillin, erythromycin, and virginiamycin revealed broad resistance to each of the antibiotics as well as high levels of resistance to individual antibiotics. Phenotypic assays revealed that antibiotic inactivation mechanisms contributed to the high levels of individual resistances among the isolates, especially to erythromycin and virginiamycin, yet none of the isolates appeared to use a β-lactamase. Biofilm formation was noted among the majority of the isolates and may contribute to persistence under low levels of antibiotics. Nearly all of the isolates carried at least one canonical antibiotic resistance gene and many carried more than one. The erythromycin ribosomal methyltransferase (erm) gene class was found in 19 of 32 isolates, yet a number of these isolates exhibit little to no resistance to erythromycin. The erm genes were present in 15 isolates that encoded more than one antibiotic resistance mechanism, suggestive of potential genetic linkages.

  18. Bacterial DNA Sequence Compression Models Using Artificial Neural Networks

    Directory of Open Access Journals (Sweden)

    Armando J. Pinho

    2013-08-01

    Full Text Available It is widely accepted that the advances in DNA sequencing techniques have contributed to an unprecedented growth of genomic data. This fact has increased the interest in DNA compression, not only from the information theory and biology points of view, but also from a practical perspective, since such sequences require storage resources. Several compression methods exist, and particularly, those using finite-context models (FCMs have received increasing attention, as they have been proven to effectively compress DNA sequences with low bits-per-base, as well as low encoding/decoding time-per-base. However, the amount of run-time memory required to store high-order finite-context models may become impractical, since a context-order as low as 16 requires a maximum of 17.2 x 109 memory entries. This paper presents a method to reduce such a memory requirement by using a novel application of artificial neural networks (ANN to build such probabilistic models in a compact way and shows how to use them to estimate the probabilities. Such a system was implemented, and its performance compared against state-of-the art compressors, such as XM-DNA (expert model and FCM-Mx (mixture of finite-context models , as well as with general-purpose compressors. Using a combination of order-10 FCM and ANN, similar encoding results to those of FCM, up to order-16, are obtained using only 17 megabytes of memory, whereas the latter, even employing hash-tables, uses several hundreds of megabytes.

  19. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Science.gov (United States)

    2010-07-01

    ... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.” (a... killing or growth inhibition of repair deficient bacteria in a set of repair proficient and...

  20. Isolation and characterization of DNA probes for human chromosome 21.

    Science.gov (United States)

    Watkins, P C

    1990-01-01

    A coordinated effort to map and sequence the human genome has recently become a national priority. Chromosome 21, the smallest human chromosome accounting for less than 2% of the human genome, is an attractive model system for developing and evaluating genome mapping technology. Several strategies are currently being explored including the development of chromosome 21 libraries from somatic cell hybrids as reported here, the cloning of chromosome 21 in yeast artificial chromosomes (McCormick et al., 1989b), and the construction of chromosome 21 libraries using chromosome flow-sorting techniques (Fuscoe et al., 1989). This report describes the approaches used to identify DNA probes that are useful for mapping chromosome 21. Probes were successfully isolated from both phage and cosmid libraries made from two somatic cell hybrids that contain human chromosome 21 as the only human chromosome. The 15 cosmid clones from the WA17 library, reduced to cloned DNA sequences of an average size of 3 kb, total 525 kb of DNA which is approximately 1% of chromosome 21. From these clones, a set of polymorphic DNA markers that span the length of the long arm of chromosome 21 has been generated. All of the probes thus far analyzed from the WA17 libraries have been mapped to chromosome 21 both by physical and genetic mapping methods. It is therefore likely that the WA17 hybrid cell line contains human chromosome 21 as the only human component, in agreement with cytogenetic observation. The 153E7b cosmid libraries will provide an alternative source of cloned chromosome 21 DNA. Library screening techniques can be employed to obtain cloned DNA sequences from the same genetic loci of the two different chromosome 21s. Comparative analysis will allow direct estimation of DNA sequence variation for different regions of chromosome 21. Mapped DNA probes make possible the molecular analysis of chromosome 21 at a level of resolution not achievable by classical cytogenetic techniques (Graw et al

  1. Biological decolorization of the reactive dyes Reactive Black 5 by a novel isolated bacterial strain Enterobacter sp. EC3.

    Science.gov (United States)

    Wang, Hui; Zheng, Xiao-Wei; Su, Jian-Qiang; Tian, Yun; Xiong, Xiao-Jing; Zheng, Tian-Ling

    2009-11-15

    Studies were carried out on the decolorization of the reactive dye Reactive Black 5 by a newly isolated bacterium, EC3. Phenotypic characterization and phylogenetic analysis based on 16S rDNA sequence comparisons indicate that this strain belonged to the genus Enterobacter. The optimal conditions for the decolorizing activity of Enterobacter sp. EC3 were anaerobic conditions with glucose supplementation, at pH 7.0, and 37 degrees C. The maximum decolorization efficiency against Reactive Black 5 achieved in this study was 92.56%. Ultra-violet and visible (UV-vis) analyses before and after decolorization and the colorless bacterial biomass after decolorization suggested that decolorization was due to biodegradation, rather than inactive surface adsorption. The bacterial strain also showed a strong ability to decolorize various reactive textile dyes, including both azo and anthraquinone dyes. To our knowledge, it is the first time that a bacterial strain of Enterobacter sp. has been reported with decolorizing ability against both azo and anthraquinone dyes.

  2. [A comparative study of different methods of determining the Tm of bacterial DNA (AUTHOR'S TRANSL)].

    Science.gov (United States)

    Ferragut, C; Leclerc, H

    1976-01-01

    Melting point curves of DNA are used to calculate average GC content. Four methods of Tm determination are described and are used in the case of all DNA samples studied. The results presented confirme that thermal denaturation curves are asymetrical, and this limits the use of "normal probability paper" and "regression lines" methods. Best reproductibility is obtained by a study of the function (see article) followed by a linear interpolation of the median. This approach yields at the same time interesting information about bacterial DNA. The graphic determination of Tm appears to be the method for choice for routine analysis.

  3. Biodegradation of Ochratoxin A by Bacterial Strains Isolated from Vineyard Soils

    Directory of Open Access Journals (Sweden)

    Palmira De Bellis

    2015-11-01

    Full Text Available Ochratoxin A (OTA is a mycotoxin with a main nephrotoxic activity contaminating several foodstuffs. In the present report, five soil samples collected from OTA-contaminated vineyards were screened to isolate microorganisms able to biodegrade OTA. When cultivated in OTA-supplemented medium, OTA was converted in OTα by 225 bacterial isolates. To reveal clonal relationships between isolates, molecular typing by using an automated rep-PCR system was carried out, thus showing the presence of 27 different strains (rep-PCR profiles. The 16S-rRNA gene sequence analysis of an isolate representative of each rep-PCR profiles indicated that they belonged to five bacterial genera, namely Pseudomonas, Leclercia, Pantoea, Enterobacter, and Acinetobacter. However, further evaluation of OTA-degrading activity by the 27 strains revealed that only Acinetobacter calcoaceticus strain 396.1 and Acinetobacter sp. strain neg1, consistently conserved the above property; their further characterization showed that they were able to convert 82% and 91% OTA into OTα in six days at 24 °C, respectively. The presence of OTα, as the unique OTA-degradation product was confirmed by LC-HRMS. This is the first report on OTA biodegradation by bacterial strains isolated from agricultural soils and carried out under aerobic conditions and moderate temperatures. These microorganisms might be used to detoxify OTA-contaminated feed and could be a new source of gene(s for the development of a novel enzymatic detoxification system.

  4. Antibacterial synergy of curcumin with antibiotics against biofilm producing clinical bacterial isolates

    Science.gov (United States)

    Kali, Arunava; Bhuvaneshwar, Devaraj; Charles, Pravin M. V.; Seetha, Kunigal Srinivasaiah

    2016-01-01

    Introduction: The role of natural bioactive substances in treating infections has been rediscovered as bacterial resistance become common to most of the antibiotics. Curcumin is a bioactive substance from turmeric. Owing to antimicrobial properties, its prospect as an antibacterial agent is currently under focus. Materials and Methods: We have evaluated the in vitro synergy of curcumin with antibiotics against sixty biofilm producing bacterial isolates. Congo red agar method was used to identify the biofilm producing isolates. Curcumin minimum inhibitory concentration (MIC) was determined by agar dilution method. Its antibiotic synergy was identified by the increase in disc diffusion zone size on Mueller-Hinton agar with 32 mg/L curcumin. Results: The mean MICs of curcumin against Gram-positive and Gram-negative isolates were 126.9 mg/L and 117.4 mg/L, respectively. Maximum synergy was observed with ciprofloxacin among Gram-positive and amikacin, gentamicin, and cefepime among Gram-negative isolates. Conclusions: Curcumin per se as well as in combination with other antibiotics has a demonstrable antibacterial action against biofilm producing bacterial isolates. It may have a beneficial role in supplementing antibiotic therapy. PMID:27330262

  5. Isolation and identification of bacterial causes of clinical mastitis in cattle in Sulaimania region

    Directory of Open Access Journals (Sweden)

    S. A. Hussein

    2008-01-01

    Full Text Available A total of 51 cases of bovine clinical mastitis in Sulaimani district were investigated for their bacteriological causative agents; 76 milk samples were cultured on primary and selective media and the isolated bacteria were tested for their susceptibility to antimicrobial agents used in commercial intramammary infusion products. Eighty two bacterial isolates were obtained and further identified using biochemical tests. Escherichia coli was the most common bacteria followed by Staphylococcus aureus, Streptococcus agalactia and coagulase–negative staphylococci. Two other bacterial species (Pseudomonas aeruginosa and Streptococcucs uberis were also isolated but in a lower proportion. Antibacterial susceptibility testing showed that the use of florfenicol, cephalexin and gentamicin may be useful for the treatment of clinical mastitis cases in cows.

  6. Acceleration of the direct identification of Staphylococcus aureus versus coagulase-negative staphylococci from blood culture material: a comparison of six bacterial DNA extraction methods.

    Science.gov (United States)

    Loonen, A J M; Jansz, A R; Kreeftenberg, H; Bruggeman, C A; Wolffs, P F G; van den Brule, A J C

    2011-03-01

    To accelerate differentiation between Staphylococcus aureus and coagulase-negative staphylococci (CNS), this study aimed to compare six different DNA extraction methods from two commonly used blood culture materials, i.e. BACTEC and BacT/ALERT. Furthermore, we analysed the effect of reduced blood culture incubation for the detection of staphylococci directly from blood culture material. A real-time polymerase chain reaction (PCR) duplex assay was used to compare the six different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with methicillin-resistant S. aureus (MRSA). Bacterial DNA was isolated with automated extractor easyMAG (three protocols), automated extractor MagNA Pure LC (LC Microbiology Kit M(Grade)), a manual kit MolYsis Plus and a combination of MolYsis Plus and the easyMAG. The most optimal isolation method was used to evaluate reduced bacterial incubation times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the easyMAG resulted in the most sensitive detection of S. aureus, with a detection limit of 10 CFU/ml, in BacT/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S. aureus or CNS load of 1 CFU/ml blood can be detected after 5 h of incubation in BacT/ALERT 3D by combining the sensitive isolation method and the tuf LightCycler assay.

  7. A bacterial antirepressor with SH3 domain topology mimics operator DNA in sequestering the repressor DNA recognition helix

    OpenAIRE

    León, Esther; Navarro-Avilés, Gloria; Santiveri, Clara M.; Flores-Flores, Cesar; Rico, Manuel; González, Carlos; Murillo, Francisco J; Elías-Arnanz, Montserrat; Jiménez, María Angeles; Padmanabhan, S.

    2010-01-01

    Direct targeting of critical DNA-binding elements of a repressor by its cognate antirepressor is an effective means to sequester the repressor and remove a transcription initiation block. Structural descriptions for this, though often proposed for bacterial and phage repressor–antirepressor systems, are unavailable. Here, we describe the structural and functional basis of how the Myxococcus xanthus CarS antirepressor recognizes and neutralizes its cognate repressors to turn on a photo-inducib...

  8. Molecular detection of bacterial pathogens using microparticle enhanced double-stranded DNA probes.

    Science.gov (United States)

    Riahi, Reza; Mach, Kathleen E; Mohan, Ruchika; Liao, Joseph C; Wong, Pak Kin

    2011-08-15

    Rapid, specific, and sensitive detection of bacterial pathogens is essential toward clinical management of infectious diseases. Traditional approaches for pathogen detection, however, often require time-intensive bacterial culture and amplification procedures. Herein, a microparticle enhanced double-stranded DNA probe is demonstrated for rapid species-specific detection of bacterial 16S rRNA. In this molecular assay, the binding of the target sequence to the fluorophore conjugated probe thermodynamically displaces the quencher probe and allows the fluorophore to fluoresce. By incorporation of streptavidin-coated microparticles to localize the biotinylated probes, the sensitivity of the assay can be improved by 3 orders of magnitude. The limit of detection of the assay is as few as eight bacteria without target amplification and is highly specific against other common pathogens. Its applicability toward clinical diagnostics is demonstrated by directly identifying bacterial pathogens in urine samples from patients with urinary tract infections.

  9. A Novel AT-Rich DNA Recognition Mechanism for Bacterial Xenogeneic Silencer MvaT.

    Directory of Open Access Journals (Sweden)

    Pengfei Ding

    2015-06-01

    Full Text Available Bacterial xenogeneic silencing proteins selectively bind to and silence expression from many AT rich regions of the chromosome. They serve as master regulators of horizontally acquired DNA, including a large number of virulence genes. To date, three distinct families of xenogeneic silencers have been identified: H-NS of Proteobacteria, Lsr2 of the Actinomycetes, and MvaT of Pseudomonas sp. Although H-NS and Lsr2 family proteins are structurally different, they all recognize the AT-rich DNA minor groove through a common AT-hook-like motif, which is absent in the MvaT family. Thus, the DNA binding mechanism of MvaT has not been determined. Here, we report the characteristics of DNA sequences targeted by MvaT with protein binding microarrays, which indicates that MvaT prefers binding flexible DNA sequences with multiple TpA steps. We demonstrate that there are clear differences in sequence preferences between MvaT and the other two xenogeneic silencer families. We also determined the structure of the DNA-binding domain of MvaT in complex with a high affinity DNA dodecamer using solution NMR. This is the first experimental structure of a xenogeneic silencer in complex with DNA, which reveals that MvaT recognizes the AT-rich DNA both through base readout by an "AT-pincer" motif inserted into the minor groove and through shape readout by multiple lysine side chains interacting with the DNA sugar-phosphate backbone. Mutations of key MvaT residues for DNA binding confirm their importance with both in vitro and in vivo assays. This novel DNA binding mode enables MvaT to better tolerate GC-base pair interruptions in the binding site and less prefer A tract DNA when compared to H-NS and Lsr2. Comparison of MvaT with other bacterial xenogeneic silencers provides a clear picture that nature has evolved unique solutions for different bacterial genera to distinguish foreign from self DNA.

  10. DNA-based stable isotope probing enables the identification of active bacterial endophytes in potatoes.

    Science.gov (United States)

    Rasche, Frank; Lueders, Tillmann; Schloter, Michael; Schaefer, Sabine; Buegger, Franz; Gattinger, Andreas; Hood-Nowotny, Rebecca C; Sessitsch, Angela

    2009-03-01

    A (13)CO2 (99 atom-%, 350 ppm) incubation experiment was performed to identify active bacterial endophytes in two cultivars of Solanum tuberosum, cultivars Desirée and Merkur. We showed that after the assimilation and photosynthetic transformation of (13)CO2 into (13)C-labeled metabolites by the plant, the most directly active, cultivar specific heterotrophic endophytic bacteria that consume these labeled metabolite scan be identified by DNA stable isotope probing (DNA-SIP).Density-resolved DNA fractions obtained from SIP were subjected to 16S rRNA gene-based community analysis using terminal restriction fragment length polymorphism analysis and sequencing of generated gene libraries.Community profiling revealed community compositions that were dominated by plant chloroplast and mitochondrial 16S rRNA genes for the 'light' fractions of (13)CO2-incubated potato cultivars and of potato cultivars not incubated with (13)CO2. In the 'heavy' fractions of the (13)CO2-incubated endophyte DNA, a bacterial 492-bp terminal restriction fragment became abundant, which could be clearly identified as Acinetobacter and Acidovorax spp. in cultivars Merkur and Desirée,respectively, indicating cultivar-dependent distinctions in (13)C-label flow. These two species represent two common potato endophytes with known plant-beneficial activities.The approach demonstrated the successful detection of active bacterial endophytes in potato. DNA-SIP therefore offers new opportunities for exploring the complex nature of plant-microbe interactions and plant-dependent microbial metabolisms within the endosphere.

  11. Phenol-stacked carbon nanotubes: A new approach to genomic DNA isolation from plants

    OpenAIRE

    2014-01-01

    Extraction of intact quality DNA from plant tissues, especially those rich in secondary metabolites, is often challenging. Literally, hundreds of different DNA isolation protocols from various plant species have been published over the last decades. Although many commercial DNA isolation kits are convenient and designed to be safe, their cost and availability cause limitations in small molecular labs in many developing countries. In nearly all protocols and DNA isolation kits, phenol and chlo...

  12. Circulating bacterial-derived DNA fragment level is a strong predictor of cardiovascular disease in peritoneal dialysis patients.

    Directory of Open Access Journals (Sweden)

    Cheuk-Chun Szeto

    Full Text Available Circulating bacterial DNA fragment is related to systemic inflammatory state in peritoneal dialysis (PD patients. We hypothesize that plasma bacterial DNA level predicts cardiovascular events in new PD patients.We measured plasma bacterial DNA level in 191 new PD patients, who were then followed for at least a year for the development of cardiovascular event, hospitalization, and patient survival.The average age was 59.3 ± 11.8 years; plasma bacterial DNA level 34.9 ± 1.5 cycles; average follow up 23.2 ± 9.7 months. At 24 months, the event-free survival was 86.1%, 69.8%, 55.4% and 30.8% for plasma bacterial DNA level quartiles I, II, III and IV, respectively (p < 0.0001. After adjusting for confounders, plasma bacterial DNA level, baseline residual renal function and malnutrition-inflammation score were independent predictors of composite cardiovascular end-point; each doubling in plasma bacterial DNA level confers a 26.9% (95% confidence interval, 13.0 - 42.5% excess in risk. Plasma bacterial DNA also correlated with the number of hospital admission (r = -0.379, p < 0.0001 and duration of hospitalization for cardiovascular reasons (r = -0.386, p < 0.0001. Plasma bacterial DNA level did not correlate with baseline arterial pulse wave velocity (PWV, but with the change in carotid-radial PWV in one year (r = -0.238, p = 0.005.Circulating bacterial DNA fragment level is a strong predictor of cardiovascular event, need of hospitalization, as well as the progressive change in arterial stiffness in new PD patients.

  13. Super-Resolution Microscopy and Tracking of DNA-Binding Proteins in Bacterial Cells

    Science.gov (United States)

    Uphoff, Stephan

    2016-01-01

    Summary The ability to detect individual fluorescent molecules inside living cells has enabled a range of powerful microscopy techniques that resolve biological processes on the molecular scale. These methods have also transformed the study of bacterial cell biology, which was previously obstructed by the limited spatial resolution of conventional microscopy. In the case of DNA-binding proteins, super-resolution microscopy can visualize the detailed spatial organization of DNA replication, transcription, and repair processes by reconstructing a map of single-molecule localizations. Furthermore, DNA binding activities can be observed directly by tracking protein movement in real time. This allows identifying subpopulations of DNA-bound and diffusing proteins, and can be used to measure DNA-binding times in vivo. This chapter provides a detailed protocol for super-resolution microscopy and tracking of DNA-binding proteins in Escherichia coli cells. The protocol covers the construction of cell strains and describes data acquisition and analysis procedures, such as super-resolution image reconstruction, mapping single-molecule tracks, computing diffusion coefficients to identify molecular subpopulations with different mobility, and analysis of DNA-binding kinetics. While the focus is on the study of bacterial chromosome biology, these approaches are generally applicable to other molecular processes and cell types. PMID:27283312

  14. Microbial interactions chapter: binding and entry of DNA in bacterial transformation

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.A.

    1977-01-01

    Genetic transformation of bacteria by DNA released from cells of a related strain is discussed. The mechanism by which the giant information-bearing molecules of DNA are transported into the bacterial cell was investigated. It was concluded that the overall process of DNA uptake consists of two main steps, binding of donor DNA to the outside of the cell and entry of the bound DNA into the cell. Each step is discussed in detail. Inasmuch as these phenomena occur at the cell surface, they are related to structures and functions of the cell wall and membrane. In addition, the development of competence, that is the formation of cell surface structures allowing DNA uptake, is examined from both a physiological and evolutionary point of view. Genetic transfer mediated by free DNA is an obvious and important form of cellular interaction. The development of competence involves another, quite distinct system of interaction between bacterial cells. Streptococcus pneumoniae, Bacillus subtilis, and Hemophilus influenzae were used as the test organisms. 259 references.

  15. Comparison of DNA preservation methods for environmental bacterial community samples

    Science.gov (United States)

    Gray, Michael A.; Pratte, Zoe A.; Kellogg, Christina A.

    2013-01-01

    Field collections of environmental samples, for example corals, for molecular microbial analyses present distinct challenges. The lack of laboratory facilities in remote locations is common, and preservation of microbial community DNA for later study is critical. A particular challenge is keeping samples frozen in transit. Five nucleic acid preservation methods that do not require cold storage were compared for effectiveness over time and ease of use. Mixed microbial communities of known composition were created and preserved by DNAgard™, RNAlater®, DMSO–EDTA–salt (DESS), FTA® cards, and FTA Elute® cards. Automated ribosomal intergenic spacer analysis and clone libraries were used to detect specific changes in the faux communities over weeks and months of storage. A previously known bias in FTA® cards that results in lower recovery of pure cultures of Gram-positive bacteria was also detected in mixed community samples. There appears to be a uniform bias across all five preservation methods against microorganisms with high G + C DNA. Overall, the liquid-based preservatives (DNAgard™, RNAlater®, and DESS) outperformed the card-based methods. No single liquid method clearly outperformed the others, leaving method choice to be based on experimental design, field facilities, shipping constraints, and allowable cost.

  16. Low-energy plasma immersion ion implantation to induce DNA transfer into bacterial E. coli

    Energy Technology Data Exchange (ETDEWEB)

    Sangwijit, K. [Biotechnology Unit, University of Phayao, Muang, Phayao 56000 (Thailand); Yu, L.D., E-mail: yuld@thep-center.org [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Sarapirom, S. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Faculty of Science, Maejo University, Bang Khen, Chiang Mai 50290 (Thailand); Pitakrattananukool, S. [School of Science, University of Phayao, Muang, Phayao 56000 (Thailand); Anuntalabhochai, S. [Biotechnology Unit, University of Phayao, Muang, Phayao 56000 (Thailand)

    2015-12-15

    Plasma immersion ion implantation (PIII) at low energy was for the first time applied as a novel biotechnology to induce DNA transfer into bacterial cells. Argon or nitrogen PIII at low bias voltages of 2.5, 5 and 10 kV and fluences ranging from 1 × 10{sup 12} to 1 × 10{sup 17} ions/cm{sup 2} treated cells of Escherichia coli (E. coli). Subsequently, DNA transfer was operated by mixing the PIII-treated cells with DNA. Successes in PIII-induced DNA transfer were demonstrated by marker gene expressions. The induction of DNA transfer was ion-energy, fluence and DNA-size dependent. The DNA transferred in the cells was confirmed functioning. Mechanisms of the PIII-induced DNA transfer were investigated and discussed in terms of the E. coli cell envelope anatomy. Compared with conventional ion-beam-induced DNA transfer, PIII-induced DNA transfer was simpler with lower cost but higher efficiency.

  17. Bacterial Community Analysis, New Exoelectrogen Isolation and Enhanced Performance of Microbial Electrochemical Systems Using Nano-Decorated Anodes

    Science.gov (United States)

    Xu, Shoutao

    Microbial electrochemical systems (MESs) have attracted much research attention in recent years due to their promising applications in renewable energy generation, bioremediation, and wastewater treatment. In a MES, microorganisms interact with electrodes via electrons, catalyzing oxidation and reduction reactions at the anode and the cathode. The bacterial community of a high power mixed consortium MESs (maximum power density is 6.5W/m2) was analyzed by using denature gradient gel electrophoresis (DGGE) and 16S DNA clone library methods. The bacterial DGGE profiles were relatively complex (more than 10 bands) but only three brightly dominant bands in DGGE results. These results indicated there are three dominant bacterial species in mixed consortium MFCs. The 16S DNA clone library method results revealed that the predominant bacterial species in mixed culture is Geobacter sp (66%), Arcobacter sp and Citrobacter sp. These three bacterial species reached to 88% of total bacterial species. This result is consistent with the DGGE result which showed that three bright bands represented three dominant bacterial species. Exoelectrogenic bacterial strain SX-1 was isolated from a mediator-less microbial fuel cell by conventional plating techniques with ferric citrate as electron acceptor under anaerobic conditions. Phylogenetic analysis of the 16S rDNA sequence revealed that it was related to the members of Citrobacter genus with Citrobacter sp. sdy-48 being the most closely related species. The bacterial strain SX-1 produced electricity from citrate, acetate, glucose, sucrose, glycerol, and lactose in MFCs with the highest current density of 205 mA/m2 generated from citrate. Cyclic voltammetry analysis indicated that membrane associated proteins may play an important role in facilitating electron transfer from the bacteria to the electrode. This is the first study that demonstrates that Citrobacter species can transfer electrons to extracellular electron acceptors

  18. Inhibitory Effect of Lactobacillus reuteri on Some Pathogenic Bacteria Isolated From Women With Bacterial Vaginosis

    Directory of Open Access Journals (Sweden)

    Eslami

    2014-08-01

    Full Text Available Background Considering the high prevalence of bacterial vaginosis and its association with urinary tract infection in women and treatment of gynecologic problems occur when a high recurrence of bacterial vaginosis is often treated with antibiotics. Objectives The purpose of this study was to investigate the inhibitory effect of Lactobacillus reuteri on pathogenic bacteria isolated from women with bacterial vaginosis. Materials and Methods Ninety-six samples were obtained from vaginal discharge of women with bacterial vaginosis by a gynecologist with a Dacron swab and put in sterile tubes containing TSB broth and Thioglycollate broth. Then were immediately sent to the laboratory in cold chain for further assessment. Afterward, culture was transferred on blood agar, EMB, Palcam and differential diagnosis environments. Then cultures were incubated for 24 hours at 37 °C. Lactobacillus reuteri strains were cultured in MRS environment and transferred to laboratory. After purification of pathogenic bacteria, Lactobacillus reuteri inhibitory effect on pathogenic bacteria was evaluated by minimum inhibitory concentration (MIC and antibiogram. Statistical analysis was performed using SPSS software v.16. Results The results of this study demonstrated the inhibitory effect of Lactobacillus reuteri on some pathogenic bacteria that cause bacterial, including Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Enterococcus, Listeria monocytogenes and E. coli. Microscopic examination of stained smears of most Lactobacillus and pathogenic bacteria showed reduced. The prevalence of abnormal vaginal discharge, history of drug use, contraceptive methods and douching were 61%, 55%, 42% and 13%, respectively. Significant difference was observed between the use and non-use of IUD in women with bacterial. Conclusions Our findings indicated the inhibitory effect of Lactobacillus reuteri on pathogenic bacteria that

  19. In-vitro efficacy of antibacterials against bacterial isolates from corneal ulcers.

    Directory of Open Access Journals (Sweden)

    Bharathi Jayahar

    2002-01-01

    Full Text Available PURPOSE: To analyse the in-vitro efficacy of commonly used antibacterials against bacterial pathogens from corneal ulcers. METHODS: We evaluated 596 patients seen over 18 months, period, September 1999 through March 2001. Corneal scrapings were subjected to microscopy and cultures using standard protocols. Antibacterial susceptibility of isolated bacteria were determined by the Kirby-Bauer disc-diffusion method. RESULTS: 626 bacterial pathogens were isolated from 596 corneal ulcer cases. 411(65.65% were gram positive cocci Streptococcus pneumoniae (41.85% was the predominant bacterial species. The antibacterial susceptibility was: 451(72.04% to cefazolin, 471(75.24% to chloramphenicol; 321(51.28% to cephaloridine; 430(68.69% to vancomycin; 564(90.09% to ciprofloxacin; 429(68.53% to norfloxacin; 464(74.12% to gentamicin and 202(32.27% to co.trimoxazole. CONCLUSION: This study provides information on the efficacy of ocular antibacterials commonly used against bacterial pathogens of keratitis. It is hoped that this information will help decision-making in empiric initial treatment of bacterial keratitis.

  20. Pathogenicity testing of shellfish hatchery bacterial isolates on Pacific oyster Crassostrea gigas larvae.

    Science.gov (United States)

    Estes, Robyn M; Friedman, Carolyn S; Elston, Ralph A; Herwig, Russell P

    2004-03-10

    Bacterial diseases are a major cause of larval mortality in shellfish hatcheries. Even with proper sanitation measures, bacterial pathogens cannot be eliminated in all cases. The pathogenicity of bacteria isolated from Pacific Northwest shellfish hatcheries to Pacific oyster Crassostrea gigas larvae was investigated. We found 3 highly pathogenic strains and 1 mildly pathogenic strain among 33 isolates tested. These strains appear to be members of the genus Vibrio. Although there have been many studies of bivalve bacterial pathogens, a standard method to assess bacterial pathogenicity in bivalve larvae is needed. Thus, we developed 2 methods using either 15 ml conical tubes or tissue culture plates that were employed for rapidly screening bacterial strains for pathogenicity to Pacific oyster larvae. The tissue culture plates worked well for screening both mildly pathogenic strains and LD50 (lethal dose) assays. This method allowed for non-intrusive and non-destructive observation of the oyster larvae with a dissecting microscope. The LD50 for the 3 highly pathogenic strains ranged between 1.6 and 3.6 x 10(4) colony forming units (CFU) ml(-1) after 24 h and between 3.2 x 102 and 1.9 x 10(3) CFU ml(-1) after 48 h.

  1. Macrophage activation induced by Brucella DNA suppresses bacterial intracellular replication via enhancing NO production.

    Science.gov (United States)

    Liu, Ning; Wang, Lin; Sun, Changjiang; Yang, Li; Tang, Bin; Sun, Wanchun; Peng, Qisheng

    2015-12-01

    Brucella DNA can be sensed by TLR9 on endosomal membrane and by cytosolic AIM2-inflammasome to induce proinflammatory cytokine production that contributes to partially activate innate immunity. Additionally, Brucella DNA has been identified to be able to act as a major bacterial component to induce type I IFN. However, the role of Brucella DNA in Brucella intracellular growth remains unknown. Here, we showed that stimulation with Brucella DNA promote macrophage activation in TLR9-dependent manner. Activated macrophages can suppresses wild type Brucella intracellular replication at early stage of infection via enhancing NO production. We also reported that activated macrophage promotes bactericidal function of macrophages infected with VirB-deficient Brucella at the early or late stage of infection. This study uncovers a novel function of Brucella DNA, which can help us further elucidate the mechanism of Brucella intracellular survival.

  2. Reconstruction of a Bacterial Genome from DNA Cassettes

    Energy Technology Data Exchange (ETDEWEB)

    Christopher Dupont; John Glass; Laura Sheahan; Shibu Yooseph; Lisa Zeigler Allen; Mathangi Thiagarajan; Andrew Allen; Robert Friedman; J. Craig Venter

    2011-12-31

    This basic research program comprised two major areas: (1) acquisition and analysis of marine microbial metagenomic data and development of genomic analysis tools for broad, external community use; (2) development of a minimal bacterial genome. Our Marine Metagenomic Diversity effort generated and analyzed shotgun sequencing data from microbial communities sampled from over 250 sites around the world. About 40% of the 26 Gbp of sequence data has been made publicly available to date with a complete release anticipated in six months. Our results and those mining the deposited data have revealed a vast diversity of genes coding for critical metabolic processes whose phylogenetic and geographic distributions will enable a deeper understanding of carbon and nutrient cycling, microbial ecology, and rapid rate evolutionary processes such as horizontal gene transfer by viruses and plasmids. A global assembly of the generated dataset resulted in a massive set (5Gbp) of genome fragments that provide context to the majority of the generated data that originated from uncultivated organisms. Our Synthetic Biology team has made significant progress towards the goal of synthesizing a minimal mycoplasma genome that will have all of the machinery for independent life. This project, once completed, will provide fundamentally new knowledge about requirements for microbial life and help to lay a basic research foundation for developing microbiological approaches to bioenergy.

  3. Bacterial isolates from the bryozoan Membranipora membranacea: influence of culture media on isolation and antimicrobial activity.

    Science.gov (United States)

    Heindl, Herwig; Thiel, Vera; Wiese, Jutta; Imhoff, Johannes F

    2012-03-01

    From specimens of the bryozoan Membranipora membranacea collected in the Baltic Sea, bacteria were isolated on four different media, which significantly increased the diversity of the isolated groups. All isolates were classified according to 16S rRNA gene sequence analysis and tested for antimicrobial properties using a panel of five indicator strains and six different media. Each medium featured a unique set of isolated phylotypes, and a phylogenetically diverse collection of isolates was obtained. A total of 96 isolates were assigned to 49 phylotypes and 29 genera. Only one-third of the members of these genera had been isolated previously from comparable sources. The isolates were affiliated with Alpha- and Gammaproteobacteria, Bacilli, and Actinobacteria. A comparable large portion of up to 22 isolates, i.e., 15 phylotypes, probably represent new species. Likewise, 47 isolates (approximately 50%) displayed antibiotic activities, mostly against grampositive indicator strains. Of the active strains, 63.8 % had antibiotic traits only on one or two of the growth media, whereas only 12.7 % inhibited growth on five or all six media. The application of six different media for antimicrobial testing resulted in twice the number of positive hits as obtained with only a single medium. The use of different media for the isolation of bacteria as well as the variation of media considered suitable for the production of antibiotic substances significantly enhanced both the number of isolates obtained and the proportion of antibiotic active cultures. Thus the approach described herein offers an improved strategy in the search for new antibiotic compounds.

  4. Spatial pattern in Antarctica: what can we learn from Antarctic bacterial isolates?

    Science.gov (United States)

    Chong, Chun Wie; Goh, Yuh Shan; Convey, Peter; Pearce, David; Tan, Irene Kit Ping

    2013-09-01

    A range of small- to moderate-scale studies of patterns in bacterial biodiversity have been conducted in Antarctica over the last two decades, most suggesting strong correlations between the described bacterial communities and elements of local environmental heterogeneity. However, very few of these studies have advanced interpretations in terms of spatially associated patterns, despite increasing evidence of patterns in bacterial biogeography globally. This is likely to be a consequence of restricted sampling coverage, with most studies to date focusing only on a few localities within a specific Antarctic region. Clearly, there is now a need for synthesis over a much larger spatial to consolidate the available data. In this study, we collated Antarctic bacterial culture identities based on the 16S rRNA gene information available in the literature and the GenBank database (n > 2,000 sequences). In contrast to some recent evidence for a distinct Antarctic microbiome, our phylogenetic comparisons show that a majority (~75 %) of Antarctic bacterial isolates were highly similar (≥99 % sequence similarity) to those retrieved from tropical and temperate regions, suggesting widespread distribution of eurythermal mesophiles in Antarctic environments. However, across different Antarctic regions, the dominant bacterial genera exhibit some spatially distinct diversity patterns analogous to those recently proposed for Antarctic terrestrial macroorganisms. Taken together, our results highlight the threat of cross-regional homogenisation in Antarctic biodiversity, and the imperative to include microbiota within the framework of biosecurity measures for Antarctica.

  5. Trends of Bacterial Keratitis Culture Isolates in Jerusalem; a 13- Years Analysis

    Science.gov (United States)

    Politis, Michael; Wajnsztajn, Denise; Rosin, Boris; Block, Colin; Solomon, Abraham

    2016-01-01

    Purpose To describe the trends in pathogens and antibacterial resistance of corneal culture isolates in infectious keratitis during a period of 13 years at Hadassah-Hebrew University Medical Center. Methods A Retrospective analysis of bacterial corneal isolates was performed during the months of January 2002 to December 2014 at Hadassah Hebrew University Medical Center. Demographics, microbiological data and antibiotic resistance and sensitivity were collected. Results A total of 943 corneal isolates were analyzed during a 13 year period. A total of 415 positive bacterial cultures and 37 positive fungal cultures were recovered, representing 48% of the total cultures. The Annual incidence was 34.78 ± 6.54 cases. The most common isolate was coagulase-negative staphylococcus (32%), which had a significant decrease in trend throughout the study period (APC = -8.1, p = 0.002). Methicillin-resistant Staphylococcus aureus (MRSA) appears to have a decrease trend (APC = -31.2, P = 0.5). There was an increase in the resistance trend of coagulase-negative staphylococci to penicillin (APC = 5.0, P = keratitis. There was no significant change in the annual incidence of cases of bacterial keratitis seen over the past 13 years. Keratitis caused by MRSA appeared to decrease in contrast to the reported literature. PMID:27893743

  6. Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing

    Science.gov (United States)

    Riba, J.; Gleichmann, T.; Zimmermann, S.; Zengerle, R.; Koltay, P.

    2016-09-01

    The isolation and analysis of single prokaryotic cells down to 1 μm and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35 pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20 μm in size. Further, the magnification of the optical system used for cell detection was increased. Redesign of the optical path allows for collision-free addressing of any flat substrate since no compartment protrudes below the nozzle of the dispenser chip anymore. The improved system allows for deterministic isolation of individual bacterial cells. A single-cell printing efficiency of 93% was obtained as shown by printing fluorescent labeled E. coli. A 96-well plate filled with growth medium is inoculated with single bacteria cells on average within about 8 min. Finally, individual bacterial cells from a heterogeneous sample of E. coli and E. faecalis were isolated for clonal culturing directly on agar plates in user-defined array geometry.

  7. Comparison of different DNA isolation methods and use of dodecyle trimethyl ammonium bromide (DTAB for the isolation of DNA from meat products

    Directory of Open Access Journals (Sweden)

    Yusuf OZsENSOY

    2016-12-01

    Conclusion: DNA isolation kit, another best method, is recommended due to quality and quantity of DNA for researchers who do not want that phenol/chloroform method have toxic substances. This study is also the first study in which DTAB method is used for DNA extraction from meat products. [J Adv Vet Anim Res 2016; 3(4.000: 368-374

  8. In vitro Evaluation of the Effct of Bacterial Extract, Isolated From Infected Hydatid Cysts, on Protoscolex

    Directory of Open Access Journals (Sweden)

    Sardari M

    2017-03-01

    Full Text Available Introduction: To date, surgery has been the treatment of choice for hydatid cyst, with regard to danger of leakage of hydatid cyst contents into viscera and production of secondary cysts, aftr spread of protoscolices. Diffrent scolicidal agents get injected into cyst for preventing the secondary cyst production, which may cause diffrent side effcts in host, especially in the surrounding tissues. In this research, the scolicidal effcts of bacterial extract isolated from infected hydatid cyst was evaluated. Methods: In this experimental-laboratorial study, at fist, isolation and identifiation of the infecting bacteria of hydatid cyst were performed at the level of species. Thn, total the bacterial extract was prepared by sonication method, and serial dilutions 1.1, 1.2, 1.4, 1.8, 1.16, 1.32 and 1.64( were prepared using sterile saline as the solvent. Th obtained alive larvae at the times of 5, 10, 20, 40 and 60 minutes were placed in those dilutions and mean of dead protoscoleces were determined using eosin exclusive staining method. Results: Th identifid bacteria isolated from the infected hydatid cysts were as follows: E. coli, Staphylococcus aureus, S. saprophyticus, Proteus mirabilis and Pseudomonas aeruginosa. Th extract of isolated bacteria at the mentioned times had no considerable scolicidal effcts. For example, the whole extract of P. aeruginosa aftr 60 minutes of exposure showed a maximum of 13.17%. scolicidal effct. Conclusion: Th results of this study showed low scolicidal effct of bacterial extracts isolated from hydatid cyst. Degeneration of scolices in infected cysts can be due to other reasons than bacterial extract.

  9. Characterization of IS6110 insertions in the dnaA-dnaN intergenic region of Mycobacterium tuberculosis clinical isolates.

    Science.gov (United States)

    Turcios, L; Casart, Y; Florez, I; de Waard, J; Salazar, L

    2009-02-01

    Mycobacterium tuberculosis isolates with identical IS6110 restriction fragment length polymorphism (RFLP) patterns are considered to be clonally related. The presence of IS6110 in the dnaA-dnaN intergenic region, one preferential locus for the integration of IS6110, was evaluated in 125 M. tuberculosis isolates. Five isolates had IS6110 inserted in this region, and two consisted of a mix of isogenic strains that putatively have evolved during a single infection. Strains from the same isolate had identical spoligo and mycobacterial interspersed repetitive unit-variable-number tandem repeat profiles, but had slight variations in IS6110 RFLP patterns, due to the presence of IS6110 in the dnaA-dnaN intergenic region. Duplication of the dnaA-dnaN intergenic region was found in one isogenic strain.

  10. The effects of a low-intensity red laser on bacterial growth, filamentation and plasmid DNA

    Science.gov (United States)

    Roos, C.; Santos, J. N.; Guimarães, O. R.; Geller, M.; Paoli, F.; Fonseca, A. S.

    2013-07-01

    Exposure of nonphotosynthesizing microorganisms to light could increase cell division in cultures, a phenomenon denominated as biostimulation. However, data concerning the importance of the genetic characteristics of cells on this effect are as yet scarce. The aim of this work was to evaluate the effects of a low-intensity red laser on the growth, filamentation and plasmids in Escherichia coli cells proficient and deficient in DNA repair. E. coli cultures were exposed to a laser (658 nm, 10 mW, 1 and 8 J cm-2) to study bacterial growth and filamentation. Also, bacterial cultures hosting pBSK plasmids were exposed to the laser to study DNA topological forms from the electrophoretic profile in agarose gels. Data indicate the low-intensity red laser: (i) had no effect on the growth of E. coli wild type and exonuclease III deficient cells; (ii) induced bacterial filamentation, (iii) led to no alteration in the electrophoretic profile of plasmids from exonuclease III deficient cells, but plasmids from wild type cells were altered. A low-intensity red laser at the low fluences used in phototherapy has no effect on growth, but induces filamentation and alters the topological forms of plasmid DNA in E. coli cultures depending on the DNA repair mechanisms.

  11. Bacterial repetitive extragenic palindromic sequences are DNA targets for Insertion Sequence elements

    Directory of Open Access Journals (Sweden)

    Pareja Eduardo

    2006-03-01

    Full Text Available Abstract Background Mobile elements are involved in genomic rearrangements and virulence acquisition, and hence, are important elements in bacterial genome evolution. The insertion of some specific Insertion Sequences had been associated with repetitive extragenic palindromic (REP elements. Considering that there are a sufficient number of available genomes with described REPs, and exploiting the advantage of the traceability of transposition events in genomes, we decided to exhaustively analyze the relationship between REP sequences and mobile elements. Results This global multigenome study highlights the importance of repetitive extragenic palindromic elements as target sequences for transposases. The study is based on the analysis of the DNA regions surrounding the 981 instances of Insertion Sequence elements with respect to the positioning of REP sequences in the 19 available annotated microbial genomes corresponding to species of bacteria with reported REP sequences. This analysis has allowed the detection of the specific insertion into REP sequences for ISPsy8 in Pseudomonas syringae DC3000, ISPa11 in P. aeruginosa PA01, ISPpu9 and ISPpu10 in P. putida KT2440, and ISRm22 and ISRm19 in Sinorhizobium meliloti 1021 genome. Preference for insertion in extragenic spaces with REP sequences has also been detected for ISPsy7 in P. syringae DC3000, ISRm5 in S. meliloti and ISNm1106 in Neisseria meningitidis MC58 and Z2491 genomes. Probably, the association with REP elements that we have detected analyzing genomes is only the tip of the iceberg, and this association could be even more frequent in natural isolates. Conclusion Our findings characterize REP elements as hot spots for transposition and reinforce the relationship between REP sequences and genomic plasticity mediated by mobile elements. In addition, this study defines a subset of REP-recognizer transposases with high target selectivity that can be useful in the development of new tools for

  12. Effect of metals on a siderophore producing bacterial isolate and its implications on microbial assisted bioremediation of metal contaminated soils.

    Science.gov (United States)

    Gaonkar, Teja; Bhosle, Saroj

    2013-11-01

    A bacterial isolate producing siderophore under iron limiting conditions, was isolated from mangroves of Goa. Based on morphological, biochemical, chemotaxonomical and 16S rDNA studies, the isolate was identified as Bacillus amyloliquefaciens NAR38.1. Preliminary characterization of the siderophore indicated it to be catecholate type with dihydroxy benzoate as the core component. Optimum siderophore production was observed at pH 7 in mineral salts medium (MSM) without any added iron with glucose as the carbon source. Addition of NaCl in the growth medium showed considerable decrease in siderophore production above 2% NaCl. Fe(+2) and Fe(+3) below 2 μM and 40 μM concentrations respectively, induced siderophore production, above which the production was repressed. Binding studies of the siderophore with Fe(+2) and Fe(+3) indicated its high affinity towards Fe(+3). The siderophore concentration in the extracellular medium was enhanced when MSM was amended with essential metals Zn, Co, Mo and Mn, however, decreased with Cu, while the concentration was reduced with abiotic metals As, Pb, Al and Cd. Significant increase in extracellular siderophore production was observed with Pb and Al at concentrations of 50 μM and above. The effect of metals on siderophore production was completely mitigated in presence of Fe. The results implicate effect of metals on the efficiency of siderophore production by bacteria for potential application in bioremediation of metal contaminated iron deficient soils especially in the microbial assisted phytoremediation processes.

  13. Screening of bovine milk samples for sub-clinical mastitis and antibiogram of bacterial isolates

    Directory of Open Access Journals (Sweden)

    Harini H. and Sumathi B.R.

    Full Text Available The study was undertaken to find out the incidence of subclinical mastitis (SCM and to assess the antibiotic sensitivity pattern of the causative organisms in lactating cows in and around Kanakapura taluk, Ramanagara district of Karnataka state. The prevalence of subclinical mastitis was assessed by the results of 3 different screening tests and bacteriological evaluation was done for the milk samples that were found positive. The predominant bacterial isolates recovered were Staphylococcus aureus (58% and Escherichia coli (23.5% followed by Staphylococcus epidermidis (8%, Streptococcus sp. (5.5%, Klebsiella sp. (3% and Bacillus sp. (2%. The in vitro antibiogram studies of bacterial isolates revealed higher sensitivity for ciprofloxacin (89%, ofloxacin (85%, enrofloxacin (82%, gentamicin (80% and chloramphenicol (75%, resistant to colistin, neomycin, streptomycin, penicillin and tetracycline. [Vet. World 2011; 4(8.000: 358-359

  14. A model of H-NS mediated compaction of bacterial DNA

    CERN Document Server

    Joyeux, Marc; 10.1016/j.bpj.2013.02.043

    2013-01-01

    The Histone-like Nucleoid Structuring protein (H-NS) is a nucleoid-associated protein, which is involved in both gene regulation and DNA compaction. H-NS can bind to DNA in two different ways: in trans, by binding to two separate DNA duplexes, or in cis, by binding to different sites on the same duplex. Based on scanning force microscopy imaging and optical trap-driven unzipping assays, it has recently been suggested that DNA compaction may result from the antagonistic effects of H-NS binding to DNA in trans and cis configurations. In order to get more insight into the compaction mechanism, we constructed a coarse-grained model description of the compaction of bacterial DNA by H-NS. These simulations highlight the fact that DNA compaction indeed results from the subtle equilibrium between several competing factors, which include the deformation dynamics of the plasmid and the several binding modes of protein dimers to DNA, i.e. dangling configurations, cis- and trans-binding. In particular, the degree of comp...

  15. Structure of Bacterial LigD -phosphoesterase Unveils a DNA Repair Superfamily

    Energy Technology Data Exchange (ETDEWEB)

    Nair, P.; Smith, P; Shuman, S

    2010-01-01

    The DNA ligase D (LigD) 3{prime}-phosphoesterase (PE) module is a conserved component of the bacterial nonhomologous end-joining (NHEJ) apparatus that performs 3{prime} end-healing reactions at DNA double-strand breaks. Here we report the 1.9 {angstrom} crystal structure of Pseudomonas aeruginosa PE, which reveals that PE exemplifies a unique class of DNA repair enzyme. PE has a distinctive fold in which an eight stranded {beta} barrel with a hydrophobic interior supports a crescent-shaped hydrophilic active site on its outer surface. Six essential side chains coordinate manganese and a sulfate mimetic of the scissile phosphate. The PE active site and mechanism are unique vis a vis other end-healing enzymes. We find PE homologs in archaeal and eukaryal proteomes, signifying that PEs comprise a DNA repair superfamily.

  16. Bacterial isolates from burn wound infections and their antibiograms: A eight-year study

    Directory of Open Access Journals (Sweden)

    Mehta Manjula

    2007-01-01

    Full Text Available Background: Infection is an important cause of mortality in burns. Rapidly emerging nosocomial pathogens and the problem of multi-drug resistance necessitates periodic review of isolation patterns and antibiogram in the burn ward. Aim: Keeping this in mind, the present retrospective study from wounds of patients admitted to burns unit was undertaken to determine the bacteriological profile and the resistance pattern from the burn ward over a period of three years (June 2002 to May 2005 and was compared with the results obtained during the previous five years (June 1997-May 2002, to ascertain any change in the bacteriological profile and antimicrobial resistance pattern. Materials and Methods: Bacterial isolates from 268 wound swabs taken from burn patients were identified by conventional biochemical methods and antimicrobial susceptibility was performed. Statistical comparison of bacterial isolates and their resistance pattern with previous five years data was done using c2 test. Results and Conclusions: During the period from 2002 to 2005 Pseudomonas species was the commonest pathogen isolated (51.5% followed by Acinetobacter species (14.28%, Staph. aureus (11.15%, Klebsiella species (9.23% and Proteus species (2.3%. When compared with the results of the previous five years i.e., 1997 to 2002, Pseudomonas species was still the commonest pathogen in the burns unit. However, the isolation of this organism and other gram-negative organisms had decreased in comparison to previous years. Newer drugs were found to be effective.

  17. Identification of a nanovirus-like DNA molecule associated with Tobacco curly shoot virus isolates containing satellite DNA

    Institute of Scientific and Technical Information of China (English)

    XIE Yan; WU Peijun; TAO Xiaorong; ZHOU Xueping

    2004-01-01

    A circular single-stranded DNA molecule, designated DNA1, was identified from Tobacco curly shoot virus (TbCSV) isolates Y35 and Y115 containing satellite DNAβ using abutting primers based on the two reported DNA1 sequences of whitefly-transmitted geminiviruses, while DNA1 molecule was not found in TbCSV isolates Y1 and Y121 without DNAβ. The immunotrapping PCR test showed that DNA1 could be encapsidated in virus particles. Southern blot further confirmed that DNA1 molecules were only associated with TbCSV isolates (Y35 and Y115) containing DNAβ. Sequences of Y35 and Y115 DNA1 comprise 1367 and 1368 nucleotides, respectively, each having a conserved ORF encoding nanovirus-like replication-associated protein (Rep). A low nucleotide sequence identity was found between DNA1 molecules and their cognate DNA-As. Y35 and Y115 DNA1 shared 92% overall nucleotide sequence identity and 96% amino acid sequence identity for Rep, while 69%~79% overall nucleotide sequence identity and 87%~90% amino acid sequence identity were found when compared with two reported DNA1 molecules associated with Ageratum yellow vein virus and Cotton leaf curl Multon virus. Sequence analysis showed that DNA1 was less related to nanovirus DNA.

  18. Characterization of the DNA-Mediated Oxidation of Dps, A Bacterial Ferritin.

    Science.gov (United States)

    Arnold, Anna R; Zhou, Andy; Barton, Jacqueline K

    2016-09-07

    Dps proteins are bacterial ferritins that protect DNA from oxidative stress and have been implicated in bacterial survival and virulence. In addition to direct oxidation of the Dps iron sites by diffusing oxidants, oxidation from a distance via DNA charge transport (CT), where electrons and electron holes are rapidly transported through the base-pair π-stack, could represent an efficient DNA protection mechanism utilized by Dps. Here, we spectroscopically characterize the DNA-mediated oxidation of ferrous iron-loaded Dps. X-band EPR was used to monitor the oxidation of DNA-bound Dps after DNA photooxidation using an intercalating ruthenium photooxidant and the flash-quench technique. Upon irradiation with poly(dGdC)2, a signal arises with g = 4.3, consistent with the formation of mononuclear high-spin Fe(III) sites of low symmetry, the expected oxidation product of Dps with one iron bound at each ferroxidase site. When poly(dGdC)2 is substituted with poly(dAdT)2, the yield of Dps oxidation is decreased significantly, consistent with guanine radical intermediates facilitating Dps oxidation. We have also explored possible protein electron transfer (ET) intermediates in the DNA-mediated oxidation of ferrous iron-loaded Dps. Dps proteins contain a conserved tryptophan residue in close proximity to the iron-binding ferroxidase site (W52 in E. coli Dps). In EPR studies of the oxidation of ferrous iron-loaded Dps following DNA photooxidation, a W52A Dps mutant was significantly deficient compared to WT Dps in forming the characteristic EPR signal at g = 4.3, consistent with W52 acting as an ET hopping intermediate. This effect is mirrored in vivo in E. coli survival in response to hydrogen peroxide, where mutation of W52 leads to decreased survival under oxidative stress.

  19. Noninvasive method of DNA isolation from fecal epithelial tissue of dairy animals.

    Science.gov (United States)

    Chandra De, Bidhan; Patra, Mahesh Chandra; Kumar, Sushil; Brahma, Biswajit; Goutam, Devika; Jaiswal, Latika; Sharma, Ashutosh; De, Sachinandan

    2015-01-01

    A novel noninvasive genomic DNA isolation protocol from fecal tissue, by the proteinase K digestion and guanidine hydrochloride extraction method, was assessed for the genotyping of cattle and buffalo. The epithelial tissues present on the surface of the feces were used as source for isolation of genomic DNA. The DNA isolated from fecal tissue was found to be similar as those obtained from other body tissues such as skin, brain, liver, kidney, and muscle. The quality of DNA was checked by agarose gel electrophoresis and polymerase chain reaction (PCR). We successfully amplified a 320 bp MHC class II DRB gene and a 125 bp mt-DNA D-loop region from isolated genomic DNA of cattle. Thus, the DNA isolated using this method was suitable for common molecular biology methods, such as restriction enzyme digestion and genotyping of dairy animals through PCR.

  20. Bacterial chromosome segregation: structure and DNA binding of the Soj dimer — a conserved biological switch

    OpenAIRE

    Leonard, Thomas A.; Butler, P Jonathan; Löwe, Jan

    2005-01-01

    Soj and Spo0J of the Gram-negative hyperthermophile Thermus thermophilus belong to the conserved ParAB family of bacterial proteins implicated in plasmid and chromosome partitioning. Spo0J binds to DNA near the replication origin and localises at the poles following initiation of replication. Soj oscillates in the nucleoid region in an ATP- and Spo0J-dependent fashion. Here, we show that Soj undergoes ATP-dependent dimerisation in solution and forms nucleoprotein filaments with DNA. Crystal s...

  1. Optimized rapid amplification of cDNA ends (RACE) for mapping bacterial mRNA transcripts.

    Science.gov (United States)

    Tillett, D; Burns, B P; Neilan, B A

    2000-03-01

    A simple, efficient and sensitive RACE-based procedure was developed for the determination of unknown 5' regions from bacterial cDNA. A number of critical modifications were made to the standard RACE method, including the optimization of the RNA extraction, reverse transcription and PCR conditions. This procedure was used to accurately determine the site of transcript initiation and structure of the promoter region of the Helicobacter pylori aspartate carbamoyltransferase gene (pyrB). The technique avoids many of the difficulties associated with established bacterial transcript mapping protocols and can be performed in two days starting with less than 1 microgram of total RNA. The modifications described here have significant potential for the identification of transcript start sites of bacterial genes and non-polyadenylated eukaryotic RNA.

  2. The action of the bacterial toxin, microcin B17, on DNA gyrase.

    Science.gov (United States)

    Parks, William M; Bottrill, Andrew R; Pierrat, Olivier A; Durrant, Marcus C; Maxwell, Anthony

    2007-04-01

    Microcin B17 (MccB17) is a peptide-based bacterial toxin that targets DNA gyrase, the bacterial enzyme that introduces supercoils into DNA. The site and mode of action of MccB17 on gyrase are unclear. We review what is currently known about MccB17-gyrase interactions and summarise approaches to understanding its mode of action that involve modification of the toxin. We describe experiments in which treatment of the toxin at high pH leads to the deamidation of two asparagine residues to aspartates. The modified toxin was found to be inactive in vivo and in vitro, suggesting that the Asn residues are essential for activity. Following on from these studies we have used molecular modelling to suggest a 3D structure for microcin B17. We discuss the implications of this model for MccB17 action and investigate the possibility that it binds metal ions.

  3. Assessment of biofilm formation in device-associated clinical bacterial isolates in a tertiary level hospital

    Directory of Open Access Journals (Sweden)

    Summaiya A Mulla

    2011-01-01

    Full Text Available Background: Biofilm formation is a developmental process with intercellular signals that regulate growth. Biofilms contaminate catheters, ventilators, and medical implants; they act as a source of disease for humans, animals, and plants. Aim: In this study we have done quantitative assessment of biofilm formation in device-associated clinical bacterial isolates in response to various concentrations of glucose in tryptic soya broth and with different incubation time. Materials and Methods: The study was carried out on 100 positive bacteriological cultures of medical devices, which were inserted in hospitalized patients. The bacterial isolates were processed as per microtitre plate method with tryptic soya broth alone and with varying concentrations of glucose and were observed in response to time. Results: Majority of catheter cultures were positive. Out of the total 100 bacterial isolates tested, 88 of them were biofilm formers. Incubation period of 16-20 h was found to be optimum for biofilm development. Conclusions: Availability of nutrition in the form of glucose enhances the biofilm formation by bacteria. Biofilm formation depends on adherence of bacteria to various surfaces. Time and availability of glucose are important factors for assessment of biofilm progress.

  4. [Polyvalence of bacteriophages isolated from fruit trees, affected by bacterial fire blight].

    Science.gov (United States)

    Tovkach, F I; Moroz, S N; Korol', N A; Faĭdiuk, Iu V; Kushkina, A I

    2013-01-01

    Phage populations appearing as a result of a pathogenic process caused by Erwinia amylovora have been discovered and described. They accompany bacterial fire blight development in the process of quince, pear and apple trees vegetation in Zakarpattya region of Ukraine. Phage isolates of the affected pear and quince include polyvalent virulent phages able to develop on bacterial strains associated with plants--E. amylovora. E. "horticola" and Pantoea agglomerans. E. amylovora isolated from the plant tissues affected by the fire blight and detected at the same time as phages proved to be resistant to the viral infection. It is hard to explain now this characteristic however it was noticed that resistance to phages can change drastically in case of dissociation, lysogenization and mutagenesis of erwinia in laboratory conditions. Phage population study shows that they are heterogeneous and can obviously include not only polyvalent but also specific viruses. Further studies of biology and molecular genetics of pure lines of isolated phages will help to get closer to understanding the place and role of bacteriophages in the complicated network of relations between bacterial pathogens and plants.

  5. Characterization of CCN and IN activity of bacterial isolates collected in Atlanta, GA

    Science.gov (United States)

    Purdue, Sara; Waters, Samantha; Karthikeyan, Smruthi; Konstantinidis, Kostas; Nenes, Athanasios

    2016-04-01

    Characterization of CCN activity of bacteria, other than a few select types such as Pseudomonas syringae, is limited, especially when looked at in conjunction with corresponding IN activity. The link between these two points is especially important for bacteria as those that have high CCN activity are likely to form an aqueous phase required for immersion freezing. Given the high ice nucleation temperature of bacterial cells, especially in immersion mode, it is important to characterize the CCN and IN activity of many different bacterial strains. To this effect, we developed a droplet freezing assay (DFA) which consists of an aluminum cold plate, cooled by a continuous flow of an ethylene glycol-water mixture, in order to observe immersion freezing of the collected bacteria. Here, we present the initial results on the CCN and IN activities of bacterial samples we have collected in Atlanta, GA. Bacterial strains were collected and isolated from rainwater samples taken from different storms throughout the year. We then characterized the CCN activity of each strain using a DMT Continuous Flow Streamwise Thermal Gradient CCN Counter by exposing the aerosolized bacteria to supersaturations ranging from 0.05% to 0.6%. Additionally, using our new DFA, we characterized the IN activity of each bacterial strain at temperatures ranging from -20oC to 0oC. The combined CCN and IN activity gives us valuable information on how some uncharacterized bacteria contribute to warm and mixed-phase cloud formation in the atmosphere.

  6. Surface physicochemistry and ionic strength affects eDNA's role in bacterial adhesion to abiotic surfaces.

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    Viduthalai R Regina

    Full Text Available Extracellular DNA (eDNA is an important structural component of biofilms formed by many bacteria, but few reports have focused on its role in initial cell adhesion. The aim of this study was to investigate the role of eDNA in bacterial adhesion to abiotic surfaces, and determine to which extent eDNA-mediated adhesion depends on the physicochemical properties of the surface and surrounding liquid. We investigated eDNA alteration of cell surface hydrophobicity and zeta potential, and subsequently quantified the effect of eDNA on the adhesion of Staphylococcus xylosus to glass surfaces functionalised with different chemistries resulting in variable hydrophobicity and charge. Cell adhesion experiments were carried out at three different ionic strengths. Removal of eDNA from S. xylosus cells by DNase treatment did not alter the zeta potential, but rendered the cells more hydrophilic. DNase treatment impaired adhesion of cells to glass surfaces, but the adhesive properties of S. xylosus were regained within 30 minutes if DNase was not continuously present, implying a continuous release of eDNA in the culture. Removal of eDNA lowered the adhesion of S. xylosus to all surfaces chemistries tested, but not at all ionic strengths. No effect was seen on glass surfaces and carboxyl-functionalised surfaces at high ionic strength, and a reverse effect occurred on amine-functionalised surfaces at low ionic strength. However, eDNA promoted adhesion of cells to hydrophobic surfaces irrespective of the ionic strength. The adhesive properties of eDNA in mediating initial adhesion of S. xylosus is thus highly versatile, but also dependent on the physicochemical properties of the surface and ionic strength of the surrounding medium.

  7. Bacterial DNA induces the complement system activation in serum and ascitic fluid from patients with advanced cirrhosis.

    Science.gov (United States)

    Francés, Rubén; González-Navajas, José M; Zapater, Pedro; Muñoz, Carlos; Caño, Rocío; Pascual, Sonia; Márquez, Dorkas; Santana, Francia; Pérez-Mateo, Miguel; Such, José

    2007-07-01

    Translocation of intestinal bacteria to ascitic fluid is, probably, the first step in the development of spontaneous bacterial peritonitis in patients with cirrhosis. Proteins of the complement system are soluble mediators implicated in the host immune response to bacterial infections and its activation has been traditionally considered to be an endotoxin-induced phenomenon. The aim of this study was to compare the modulation of these proteins in response to the presence of bacterial DNA and/or endotoxin in patients with advanced cirrhosis and ascites in different clinical conditions. Groups I and II consisted of patients without/with bacterial DNA. Group III included patients with spontaneous bacterial peritonitis and Group IV with patients receiving norfloxacin as secondary long-term prophylaxis of spontaneous bacterial peritonitis. Serum and ascitic fluid levels of endotoxin and truncated residues of the complement system were measured by ELISA. The complement system is triggered in response to bacterial DNA, as evidenced by significantly increased levels of C3b, membrane attack complex, and C5a in patients from Groups II and III compared with patients without bacterial DNA (Group I) and those receiving norfloxacin (Group IV). Gram classification did not further differentiate the immune response between patients within groups II and III, even though endotoxin levels were, as expected, significantly higher in patients with bacterial DNA from gram-negative microorganisms. The complement protein activation observed in patients with bacterial DNA in blood and ascitic fluid is indistinguishable from that observed in patients with spontaneous bacterial peritonitis and may occur in an endotoxin-independent manner.

  8. The role of enzymology in a structure-based drug discovery program: bacterial DNA gyrase.

    Science.gov (United States)

    Cunningham, Mark L

    2012-01-01

    The capability to accurately, rapidly, and reproducibly determine the affinity of a ligand for a target protein or enzyme is a vital component for a successful structure-based drug design effort. In order to successfully drive a structure-based drug design (SBDD) project forward, multiple distinct assays, each with particular strengths and weaknesses, need to be employed. Using bacterial DNA gyrase as an example, a range of assays are described that will fully support an SBDD program.

  9. Autoclave method for rapid preparation of bacterial PCR-template DNA.

    Science.gov (United States)

    Simmon, Keith E; Steadman, Dewey D; Durkin, Sarah; Baldwin, Amy; Jeffrey, Wade H; Sheridan, Peter; Horton, Rene; Shields, Malcolm S

    2004-02-01

    An autoclave method for preparing bacterial DNA for PCR template is presented, it eliminates the use of detergents, organic solvents, and mechanical cellular disruption approaches, thereby significantly reducing processing time and costs while increasing reproducibility. Bacteria are lysed by rapid heating and depressurization in an autoclave. The lysate, cleared by microcentrifugation, was either used directly in the PCR reaction, or concentrated by ultrafiltration. This approach was compared with seven established methods of DNA template preparation from four bacterial sources which included boiling Triton X-100 and SDS, bead beating, lysozyme/proteinase K, and CTAB lysis method components. Bacteria examined were Enterococcus and Escherichia coli, a natural marine bacterial community and an Antarctic cyanobacterial-mat. DNAs were tested for their suitability as PCR templates by repetitive element random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE) analysis. The autoclave method produced PCR amplifiable template comparable or superior to the other methods, with greater reproducibility, much shorter processing time, and at a significantly lower cost.

  10. Phenol-stacked carbon nanotubes: A new approach to genomic DNA isolation from plants

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    Farhad Nazarian-Firouzabadi

    2014-09-01

    Full Text Available Extraction of intact quality DNA from plant tissues, especially those rich in secondary metabolites, is often challenging. Literally, hundreds of different DNA isolation protocols from various plant species have been published over the last decades. Although many commercial DNA isolation kits are convenient and designed to be safe, their cost and availability cause limitations in small molecular labs in many developing countries. In nearly all protocols and DNA isolation kits, phenol and chloroform are used to precipitate various classes of impurities. However, phenol is partially soluble in water, resulting in the co-existence of proteins in upper (aqueous phases. This phenomenon results in the contamination of the nucleic acids and low quality DNA. Nanotechnology advances have helped many areas of molecular biology such as the development of new diagnosis and purification kits. In this study, for the first time, we report a different approach to isolate DNA from plants based on carbon nanotubes (CNTs. The results show that the phenol reagent stack on CNTs can effectively remove proteins, polysaccharides and other polyphenol constituents. The A260/A280nm absorbance ratios of isolated DNA samples were 1.9 and 1.8 for chamomile and opium plants, respectively, indicating the high purity of the isolated DNA. DNA yield was more than two times the standard Doyle and Doyle method. Furthermore, the isolated DNA proved amenable to PCR amplification, using Random Amplified Polymorphic DNA (RAPD analysis.

  11. Aerobic De-Epoxydation of Trichothecene Mycotoxins by a Soil Bacterial Consortium Isolated Using In Situ Soil Enrichment

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    Wei-Jie He

    2016-09-01

    Full Text Available Globally, the trichothecene mycotoxins deoxynivalenol (DON and nivalenol (NIV are among the most widely distributed mycotoxins that contaminate small grain cereals. In this study, a bacterial consortium, PGC-3, with de-epoxydation activity was isolated from soil by an in situ soil enrichment method. Screening of 14 soil samples that were sprayed with DON revealed that 4 samples were able to biotransform DON into de-epoxydized DON (dE-DON. Among these, the PGC-3 consortium showed the highest and most stable activity to biotransform DON into dE-DON and NIV into dE-NIV. PGC-3 exhibited de-epoxydation activity at a wide range of pH (5–10 and temperatures (20–37 °C values under aerobic conditions. Sequential subculturing with a continued exposure to DON substantially reduced the microbial population diversity of this consortium. Analyses of the 16S rDNA sequences indicated that PGC-3 comprised 10 bacterial genera. Among these, one species, Desulfitobacterium, showed a steady increase in relative abundance, from 0.03% to 1.55% (a 52-fold increase, as higher concentrations of DON were used in the subculture media, from 0 to 500 μg/mL. This study establishes the foundation to further develop bioactive agents that can detoxify trichothecene mycotoxins in cereals and enables for the characterization of detoxifying genes and their regulation.

  12. Aerobic De-Epoxydation of Trichothecene Mycotoxins by a Soil Bacterial Consortium Isolated Using In Situ Soil Enrichment

    Science.gov (United States)

    He, Wei-Jie; Yuan, Qing-Song; Zhang, You-Bing; Guo, Mao-Wei; Gong, An-Dong; Zhang, Jing-Bo; Wu, Ai-Bo; Huang, Tao; Qu, Bo; Li, He-Ping; Liao, Yu-Cai

    2016-01-01

    Globally, the trichothecene mycotoxins deoxynivalenol (DON) and nivalenol (NIV) are among the most widely distributed mycotoxins that contaminate small grain cereals. In this study, a bacterial consortium, PGC-3, with de-epoxydation activity was isolated from soil by an in situ soil enrichment method. Screening of 14 soil samples that were sprayed with DON revealed that 4 samples were able to biotransform DON into de-epoxydized DON (dE-DON). Among these, the PGC-3 consortium showed the highest and most stable activity to biotransform DON into dE-DON and NIV into dE-NIV. PGC-3 exhibited de-epoxydation activity at a wide range of pH (5–10) and temperatures (20–37 °C) values under aerobic conditions. Sequential subculturing with a continued exposure to DON substantially reduced the microbial population diversity of this consortium. Analyses of the 16S rDNA sequences indicated that PGC-3 comprised 10 bacterial genera. Among these, one species, Desulfitobacterium, showed a steady increase in relative abundance, from 0.03% to 1.55% (a 52-fold increase), as higher concentrations of DON were used in the subculture media, from 0 to 500 μg/mL. This study establishes the foundation to further develop bioactive agents that can detoxify trichothecene mycotoxins in cereals and enables for the characterization of detoxifying genes and their regulation. PMID:27669304

  13. A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs

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    Gong Shiaochin

    2009-03-01

    Full Text Available Abstract Background Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available. Results We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1 subclone genes of interest into BAC linking vectors, (2 insert desired reporter genes into respective genes and (3 link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts. Conclusion The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

  14. Survival and transfer ability of phylogenetically diverse bacterial endosymbionts in environmental Acanthamoeba isolates.

    Science.gov (United States)

    Matsuo, Junji; Kawaguchi, Kouhei; Nakamura, Shinji; Hayashi, Yasuhiro; Yoshida, Mitsutaka; Takahashi, Kaori; Mizutani, Yoshihiko; Yao, Takashi; Yamaguchi, Hiroyuki

    2010-08-01

    Obligate intracellular bacteria are commonly found as endosymbionts of acanthamoebae; however, their survival in and ability to transfer to amoebae are currently uncharacterized. In this study, six bacterial endosymbionts, found in five environmental Acanthamoeba isolates (S13, R18, S23, S31, S40) from different locations of Sapporo city, Japan, were characterized. Phylogenetic analysis revealed that three bacterial endosymbionts (eS23, eS31, eS40a) belonged to α- and β-Proteobacteria phyla and the remaining endosymbionts (eS13, eR18, eS40b) belonged to the order Chlamydiales. The Acanthamoeba isolate (S40) contained two phylogenetically different bacterial endosymbionts (eS40a, eS40b). Fluorescent in situ hybridization analysis showed that all bacterial endosymbionts were diffusely localized within amoebae. Transmission electron microscopy also showed that the endosymbionts were rod-shaped (eS23, eS31, eS40a) or sphere- or crescent-shaped (eS13, eR18, eS40b). No successful culture of these bacteria was achieved using conventional culture methods, but the viability of endosymbionts was confirmed by live/dead staining and RT-PCR methods. However, endosymbionts (except eR18) derived from original host cells lost the ability to be transferred to another Acanthamoebae strains [ATCC strain (C3), environmental strains (S14, R23, S24)]. Thus, our data demonstrate that phylogenetically diverse bacterial endosymbionts found in amoebae maintain a stable interaction with amoebae, but the transferability is limited.

  15. Proteolysis produced within biofilms of bacterial isolates from raw milk tankers.

    Science.gov (United States)

    Teh, Koon Hoong; Flint, Steve; Palmer, Jon; Andrewes, Paul; Bremer, Phil; Lindsay, Denise

    2012-06-15

    In this study, six bacterial isolates that produced thermo-resistant enzymes isolated from the internal surfaces of raw milk tankers were evaluated for their ability to produce proteolysis within either single culture biofilms or co-culture biofilms. Biofilms were formed in an in vitro model system that simulated the upper internal surface of a raw milk tanker during a typical summer's day of milk collection in New Zealand. The bacterial isolates were further evaluated for their ability to form biofilms at 25, 30 and 37°C. Mutual and competitive effects were observed in some of the co-culture biofilms, with all isolates being able to form biofilms in either single culture or co-culture at the three temperatures. The proteolysis was also evaluated in both biofilms and corresponding planktonic cultures. The proteolysis per cell decreased as the temperature of incubation (20-37°C) increased. Furthermore, mutualistic interactions in terms of proteolysis were observed when cultures were grown as co-culture biofilms. This is the first study to show that proteolytic enzymes can be produced in biofilms on the internal surfaces of raw milk tankers. This has important implications for the cleaning and the temperature control of raw milk transport tankers.

  16. Quantitative analysis of resistance in cotton to three new isolates of the bacterial blight pathogen.

    Science.gov (United States)

    Wallace, T P; El-Zik, K M

    1990-04-01

    Genetic variability for virulence of the bacterial blight pathogen [Xanthomonas campestris pv malvacearum (Smith) Dye] on cotton (Gossypium hirsutum L.) has been shown by the identification of 19 races of the pathogen based on disease reactions of a set of ten host differentials. This study was conducted to determine the inheritance of host resistance to three recently identified isolates of X. campestris pv malvacearum, which are virulent on the entire set of differentials. True leaves of Tamcot CAMD-E, LEBOCAS-3-80, Stoneville 825, and their f1, F2, and backcross progenies were wound-inoculated in the field with separate bacterial suspensions of the virulent HV3, HV7, and Sudan isolates of the pathogen. LEBOCAS-3-80 was replaced with S295, a new immune cultivar, for a greenhouse study in which both cotyledons and true leaves were inoculated. Disease reactions were rated on a scale of 1-10, and genetic models were proposed utilizing generation means analysis. Dominance, when significant, was in the direction of resistance in all but one cross-isolate combination. Digenic interaction components indicated a duplicate type. Narrow-sense heritability for resistance ranged from 0.59 to 0.68; therefore, primarily additive-genetic variability among the selected cutlivars was detected, indicating that breeding for improved resistance to these isolates is a practical goal.

  17. Bacterial Agents Isolated from Wards’ Environment and Staff’s Hands in Yahyanejad Hospital, Babol

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    Sadighian, F. (BSc

    2013-01-01

    Full Text Available Background and Objective: Nowadays, nosocomial infection is one of the greatest problems in hospitals. Normal flora of staff’s hands and the bacterial agents on the surface of medical equipment can become progressively colonized with potential pathogens during patient care. This study was carried out to determine the bacterial agents existed on staff’s hands and in the wards of hospital to step in to control nosocomial infection. Material and Methods: In this descriptive study, during 17 months (22.mar.2010- 30.aug.2011, 403 samples, using sterile swab , were randomly obtained from the staff’s hands and medical equipment of emergency departments , ICU, male operation room and female surgical unit . The samples were cultured on Blood agar (BA and Eosin methylene blue (EMB. Then, identification of isolated bacteria was done with diagnostic tests. Results: Of 430 samples, 530 bacteria were isolated from staff’s hands (N= 291 and medical equipment (N= 234. The most common bacterium from personnel’s hands (144; 49.5% and medical equipment (24; 10% is Staphylococcus aureus. Also, three isolates of pseudomonas aeruginosa from staff’s hands of male surgical ward and medical equipment of ICU, and two isolates of Acinetobacter.spp from ICU’s medical equipment were identified. Conclusion: With regard to the findings, it seems that applying the appropriate disinfectant agents by using standard procedures is necessary. Keywords: Medical Equipment; Staff’s Hand; Nosocomial Infection; Staphylococcus Aureus

  18. Linguistic isolates in Portugal: insights from the mitochondrial DNA pattern.

    Science.gov (United States)

    Mairal, Quim; Santos, Cristina; Silva, Marina; Marques, Sofia L; Ramos, Amanda; Aluja, Maria Pilar; Amorim, Antonio; Prata, Maria João; Alvarez, Luis

    2013-12-01

    Miranda do Douro, located in the northeastern region of Portugal, has notable characteristics not only from a geographic or naturalistic point of view, but also from a cultural perspective. A remarkable one is the coexistence of two different languages: Portuguese and Mirandese, the second being an Astur-Leonese dialect. The current persistence of the Astur-Leonese dialect in this population falls on the singularity of the region: relative isolation, implying difficulties to communicate with other Portuguese regions, while the same location facilitated the establishment of social and commercial relationships with adjacent Spanish territories, origin of the Astur-Leonese language. The objective of this study was to characterize the population from Miranda through the analysis of maternal lineages in order to evaluate whether its mitochondrial DNA diversity fitted the patterns previously reported for other populations from the Iberian Peninsula. Viewing that, the entire control region of mitochondrial DNA from 121 individuals was examined. Miranda showed a haplogroup composition usual for a Western European population, in the sense that as high as 63.6% of sequences belonged to macro-haplogroup R0. Lineages ascribed to have an African (L2a and L1b) origin, were detected, but reaching an amount commonly found in Portugal. Miranda also presented a few haplogroups typically found in Jewish populations, while rarely observed in other Iberian populations. The finding can be explained by gene flow with crypto-Jew communities that since long are known to be established in the region where Miranda is located. In Miranda, both genetic and nucleotide diversities presented low values (0.9292 ± 0.0180 and 0.01101 ± 0.00614 respectively) when compared to populations from its micro-geographical framework, which constitute a sign of population isolation that certainly provided conditions for the survival of the Astur-Leonese dialect in the region.

  19. A mitomycin-N6-deoxyadenosine adduct isolated from DNA.

    Science.gov (United States)

    Palom, Y; Lipman, R; Musser, S M; Tomasz, M

    1998-03-01

    A minor N6-deoxyadenosine adduct of mitomycin C (MC) was isolated from synthetic oligonucleotides and calf thymus DNA, representing the first adduct of MC and a DNA base other than guanine. The structure of the adduct (8) was elucidated using submilligram quantities of total available material. UV difference spectroscopy, circular dichroism, and electrospray mass spectroscopy as well as chemical transformations were utilized in deriving the structure of 8. A series of synthetic oligonucleotides was designed to probe the specificities of the alkylation of adenine by MC. The nature and frequency of the oligonucleotide-MC adducts formed under conditions of reductive activation of MC were determined by their enzymatic digestion to the nucleoside level followed by quantitative analysis of the products by HPLC. The analyses indicated the following: (i) (A)n sequence is favored over (AT)n for adduct formation; (ii) the alkylation favors the duplex structure; (iii) at adenine sites only monofunctional alkylation occurs; (iv) the adenine-to-alkylation frequency in the model oligonucleotides was 0.3-0.6 relative to guanine alkylation at the 5'-ApG sequence but only 0.02-0.1 relative to guanine alkylation at 5'-CpG. The 5'-phosphodiester linkage of the MC-adenine adduct is resistant to snake venom diesterase. The overall ratio of adenine to guanine alkylation in calf thymus DNA was 0.03, indicating that 8 is a minor MC-DNA adduct relative to MC-DNA adducts at guanine residues in the present experimental residues in the present experimental system. However, the HPLC elution time of 8 coincides with that of a major, unknown MC adduct detected previously in mouse mammary tumor cells treated with radiolabeled MC [Bizanek, R., Chowdary, D., Arai, H., Kasai, M., Hughes, C. S., Sartorelli, A. C., Rockwell, S., and Tomasz, M. (1993) Cancer Res. 53, 5127-5134]. Thus, 8 may be identical or closely related to this major adduct formed in vivo. This possibility can now be tested by

  20. Therapeutic efficacy of Lactobacillus acidophilus against bacterial isolates from burn wounds

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    Mohammed Sh. Jebur

    2010-12-01

    Full Text Available Background: Probiotics are live microorganisms which are mainly strains of Lactobacillus spp., Bifidobacterium spp. When administered in adequate amounts, these microorganisms offer a health benefit for the host. Probiotic organisms are also available commercially in milk, sour milk, ice cream and other foods. Aims: To identify bacterial species isolated from burn wounds, and also to evaluate (In-vitro the therapeutic efficacy of Lacto. acidophilus against these bacterial isolates. To compare this activity to other antibacterial agents which are used medically in the treatment of burn wound cases. Materials and Methods: Burn wound swabs were obtained from 50 patients who had been admitted to hospitals in Baghdad during August to November 2009. These swabs were inoculated onto enriched and differential culture media. Subcultures were performed on selective media. The necessary biochemical tests were conducted and the organisms identified using standard procedures. Susceptibility of isolated pathogens to local isolates Lacto. Acidophilus (with 1х108 cells/mL and 10 commonly used burn wounds antibiotics was examined using standard susceptibility testing. Results: Ninety different organisms were isolated. Gram-positive cocci accounted for 16 (17.7% and gram-negative bacilli for 74 (82.2% bacterial isolates. Pseudomonas aeruginosa 30(33.3% were the most commonly isolated organisms, followed by Escherichia coli, Enterobacter spp., Klebsiella spp., Proteus spp.(22.2,20,4.4,2.2%, respectively. Staphylococcus aureus isolates were performed in 8(8.8%. However, the incidence of Staphylococcus epidermidis was 2 (2.2%, while ß-haemolytic Streptococci was 4(4.4%. In susceptibility testing, Lacto. acidophilus had coverage against 90 (100% of 74 gram-negative and 16 of gram-positive bacteria tested. The coverage of the remaining 10 antibacterial agents used was different in their activity (resistance or sensitivity, which ranged between 50-100%. Conclusion

  1. Isolation of culturable endophytic bacteria from Moso bamboo (Phyllostachys edulis and 16S rDNA diversity analysis

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    Yuan Zong-Sheng

    2015-01-01

    Full Text Available We analyzed culturable endophytic bacteria from Moso bamboo (Phyllostachys edulis using traditional bacterial isolation and culture methods and then studied the colony characteristics and diversity with a 16S rDNA sequence analysis. We isolated 82 endophytic bacteria strains belonging to 47 species in 26 genera from the root, rhizome, stem and leaves of Moso bamboo species from populations on Wuyi Mountain, and in the Jiangle and Changting regions. There were significant differences in the composition of the culturable endophytic bacteria isolated from the different areas and from different tissues. The dominant bacteria strains from the Wuyi Mountain samples were Arthrobacter, Staphylococcus, Bacillus and Enterobacter, while the dominant bacteria from the Jiangle samples were Bacillus, Staphylococcus and Curtobacterium, and the dominant bacteria in the Changting samples were Alcaligenes, Pseudomonas, Staphylococcus and Bacillus. Our results demonstrate the abundant diversity of endophytic bacteria in Moso bamboo.

  2. DNA Barcoding Green Microalgae Isolated from Neotropical Inland Waters.

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    Sámed I I A Hadi

    Full Text Available This study evaluated the feasibility of using the Ribulose Bisphosphate Carboxylase Large subunit gene (rbcL and the Internal Transcribed Spacers 1 and 2 of the nuclear rDNA (nuITS1 and nuITS2 markers for identifying a very diverse, albeit poorly known group, of green microalgae from neotropical inland waters. Fifty-one freshwater green microalgae strains isolated from Brazil, the largest biodiversity reservoir in the neotropics, were submitted to DNA barcoding. Currently available universal primers for ITS1-5.8S-ITS2 region amplification were sufficient to successfully amplify and sequence 47 (92% of the samples. On the other hand, new sets of primers had to be designed for rbcL, which allowed 96% of the samples to be sequenced. Thirty-five percent of the strains could be unambiguously identified to the species level based either on nuITS1 or nuITS2 sequences' using barcode gap calculations. nuITS2 Compensatory Base Change (CBC and ITS1-5.8S-ITS2 region phylogenetic analysis, together with morphological inspection, confirmed the identification accuracy. In contrast, only 6% of the strains could be assigned to the correct species based solely on rbcL sequences. In conclusion, the data presented here indicates that either nuITS1 or nuITS2 are useful markers for DNA barcoding of freshwater green microalgae, with advantage for nuITS2 due to the larger availability of analytical tools and reference barcodes deposited at databases for this marker.

  3. Inhibitory effect of Lactobacillus rhamnosus on pathogenic bacteria isolated from women with bacterial vaginosis

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    Gita Eslami

    2014-06-01

    Full Text Available Background: Considering the high prevalence of bacterial vaginosis and its association with urinary tract infection in women and treatment of gynecologic problems occur when a high recurrence of bacterial vaginosis is often treated with antibiotics. The purpose of this study is to investigate the inhibitory effect of Lactobacillus rhamnosus on pathogenic bacteria isolated from women with bacterial vaginosis, respectively.Materials and Methods: 96 samples from women with bacterial vaginosis discharge referred to health centers dependent Shahid Beheshti University in 91-92 were taken by a gynecologist with a dacron swab and put in sterile tubes containing TSB broth and Thioglycollate broth and were immediately sent to the lab location in cold chain for the next stages of investigation. From Thioglycollate and TSB medium was cultured on blood agar and EMB and Palkam and Differential diagnosis environments, and then incubated for 24 h at 37°C. Strains of Lactobacillus rhamnosus were cultured in MRSA environment and were transfered to the lab. After purification of pathogenic bacteria, MIC methods and antibiogram, Lactobacillus rhamnosus inhibitory effect on pathogenic bacteria is checked. Statistical analysis was done by SPSS software v.16.Results: The results of this study show the inhibitory effect of Lactobacillus rhamnosus on some pathogenic bacteria that cause bacterial vaginosis, including Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Entrococcus, Listeria monocytogenes and E.Coli. Microscopic examination of stained smears of the large number of Lactobacillus and pathogenic bacteria showed reduced. The prevalence of abnormal vaginal discharge, history of drug use means of preventing pregnancy and douching, respectively, 61%, 55%, 42% and 13% respectively. Significant difference was observed between the use and non-use of IUD in women with bacterial vaginosis infection

  4. Integrative bacterial artificial chromosomes for DNA integration into the Bacillus subtilis chromosome.

    Science.gov (United States)

    Juhas, Mario; Ajioka, James W

    2016-06-01

    Bacillus subtilis is a well-characterized model bacterium frequently used for a number of biotechnology and synthetic biology applications. Novel strategies combining the advantages of B. subtilis with the DNA assembly and editing tools of Escherichia coli are crucial for B. subtilis engineering efforts. We combined Gibson Assembly and λ red recombineering in E. coli with RecA-mediated homologous recombination in B. subtilis for bacterial artificial chromosome-mediated DNA integration into the well-characterized amyE target locus of the B. subtilis chromosome. The engineered integrative bacterial artificial chromosome iBAC(cav) can accept any DNA fragment for integration into B. subtilis chromosome and allows rapid selection of transformants by B. subtilis-specific antibiotic resistance and the yellow fluorescent protein (mVenus) expression. We used the developed iBAC(cav)-mediated system to integrate 10kb DNA fragment from E. coli K12 MG1655 into B. subtilis chromosome. iBAC(cav)-mediated chromosomal integration approach will facilitate rational design of synthetic biology applications in B. subtilis.

  5. Gardnerella vaginalis isolated from patients with bacterial vaginosis and from patients with healthy vaginal ecosystems.

    Science.gov (United States)

    Aroutcheva, A A; Simoes, J A; Behbakht, K; Faro, S

    2001-10-01

    The differences in the phenotype and genotype of Gardnerella vaginalis isolates from patients with bacterial vaginosis (BV) and from patients without BV are unknown. In our study, 43 isolates of G. vaginalis were examined for biotype (hippurate hydrolysis, lipase, and beta-galactosidase activity), sensitivity to metronidazole, and genotype. Of the 117 women visiting the gynecology clinic at Rush-Presbyterian-St. Luke's Medical Center who were included in the study, 27.4% were found to have BV. G. vaginalis was found in samples from 87.5% of women with BV, from 34.0% of women with intermediate BV, and from 26.4% of women with healthy vaginal ecosystems. Among patients with G. vaginalis, biotypes 7 and 8 were isolated from 32% and 20% of patients, respectively. Biotype 5 was predominantly associated with a healthy vaginal ecosystem (P=.0004). Biotypes 5 and 7 were the most resistant to metronidazole. No specific phenotype or genotype of G. vaginalis causes BV.

  6. Antioxidant treatments counteract the non-culturability of bacterial endophytes isolated from legume nodules.

    Science.gov (United States)

    Muresu, Rosella; Tondello, Alessandra; Polone, Elisa; Sulas, Leonardo; Baldan, Barbara; Squartini, Andrea

    2013-06-01

    In many wild legumes, attempts to cultivate nodule bacteria fail. We hypothesized that the limited culturability could be related to injury from oxidative stress caused by disruption of plant tissues during isolation. To test that, we isolated bacteria from nodules of Hedysarum spinosissimum and Tetragonolobus purpureus using buffers supplemented with scavenging systems to prevent damage from reactive oxygen species (ROS). Treatments included the following: antioxidants (glutathione, ascorbate, EDTA) or enzymes (catalase, peroxidase, superoxide dismutase), tested either as modified squashing buffers or added in plates. Some combinations yielded dramatic increases of culturability. Different endophytes were found, including additional Rhizobiaceae that were not the primary symbiont and were unable to nodulate. Their H2O2 tolerance in broth culture showed differences consistent with the unequal culturability observed. In wild legumes species, ROS generation during extraction appears to be a major factor limiting microbiota isolation, and protocols presented here significantly improve the recovery of culturable bacterial endophytes from plants.

  7. Genome Sequences of 15 Gardnerella vaginalis Strains Isolated from the Vaginas of Women with and without Bacterial Vaginosis

    Science.gov (United States)

    Robinson, Lloyd S.; Perry, Justin; Lek, Sai; Wollam, Aye; Sodergren, Erica; Weinstock, George

    2016-01-01

    Gardnerella vaginalis is a predominant species in bacterial vaginosis, a dysbiosis of the vagina that is associated with adverse health outcomes, including preterm birth. Here, we present the draft genome sequences of 15 Gardnerella vaginalis strains (now available through BEI Resources) isolated from women with and without bacterial vaginosis. PMID:27688326

  8. Making ends meet: repairing breaks in bacterial DNA by non-homologous end-joining.

    Directory of Open Access Journals (Sweden)

    Richard Bowater

    2006-02-01

    Full Text Available DNA double-strand breaks (DSBs are one of the most dangerous forms of DNA lesion that can result in genomic instability and cell death. Therefore cells have developed elaborate DSB-repair pathways to maintain the integrity of genomic DNA. There are two major pathways for the repair of DSBs in eukaryotes: homologous recombination and non-homologous end-joining (NHEJ. Until very recently, the NHEJ pathway had been thought to be restricted to the eukarya. However, an evolutionarily related NHEJ apparatus has now been identified and characterized in the prokarya. Here we review the recent discoveries concerning bacterial NHEJ and discuss the possible origins of this repair system. We also examine the insights gained from the recent cellular and biochemical studies of this DSB-repair process and discuss the possible cellular roles of an NHEJ pathway in the life-cycle of prokaryotes and phages.

  9. Isolation and characterization of culturable seed-associated bacterial endophytes from gnotobiotically grown Marama bean seedlings.

    Science.gov (United States)

    Chimwamurombe, Percy Maruwa; Grönemeyer, Jann Lasse; Reinhold-Hurek, Barbara

    2016-06-01

    Marama bean (Tylosema esculentum) is an indigenous non-nodulating legume to the arid agro-ecological parts of Southern Africa. It is a staple food for the Khoisan and Bantu people from these areas. It is intriguing how it is able to synthesize the high-protein content in the seeds since its natural habitat is nitrogen deficient. The aim of the study was to determine the presence of seed transmittable bacterial endophytes that may have growth promoting effects, which may be particularly important for the harsh conditions. Marama bean seeds were surface sterilized and gnotobiotically grown to 2 weeks old seedlings. From surface-sterilized shoots and roots, 123 distinct bacterial isolates were cultured using three media, and identified by BOX-PCR fingerprinting and sequence analyses of the 16S rRNA and nifH genes. Phylogenetic analyses of 73 putative endophytes assigned them to bacterial species from 14 genera including Proteobacteria (Rhizobium, Massilia, Kosakonia, Pseudorhodoferax, Caulobacter, Pantoea, Sphingomonas, Burkholderia, Methylobacterium), Firmicutes (Bacillus), Actinobacteria (Curtobacterium, Microbacterium) and Bacteroidetes (Mucilaginibacter, Chitinophaga). Screening for plant growth-promoting activities revealed that the isolates showed production of IAA, ACC deaminase, siderophores, endoglucanase, protease, AHLs and capacities to solubilize phosphate and fix nitrogen. This is the first report that marama bean seeds may harbor endophytes that can be cultivated from seedlings; in this community of bacteria, physiological characteristics that are potentially plant growth promoting are widespread.

  10. Frequency of β-lactamase enzyme and antibiogram pattern in bacterial flora isolated from staffs hands

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    Shilla Jalalpoor

    2011-12-01

    Full Text Available Background: β-lactamase is an enzyme that can inactivate β–Lactam family antibiotics. High prevalence of β-lactamase producer bacteria on the staff hands, due to antibiotic resistance and nosocomial infection in hospitalized patients. The objective of this study was to assess the frequency of β-lactamase positive bacteria and antibiogram pattern in bacterial flora isolated from staff hands of the Al-Zahra hospital in Isfahan.Materials and Method: This laboratory research was performed during of 2005-2007 in Al-Zahra hospital in Isfahan. According to statistical formula, we randomly selected 80 samples from staff hands. Staff hand samples collected with finger print method. Bacterial identification was performed with microbiological methods and β–lactamase production was performed with Acidometric method and antibiogram pattern was performed with Kirby Bauer method.Results: According to the acidometric test results of 80 isolated staff hands, 61.85% of strains produce β–lactamase. Staphylococcus spp., Bacillus spp. and Enterobacteriaceae were the most important producers respectively (70.83%, 64.72% and 50%. According to antibiogram test results, penicillin and vancomycin had the highest and lowest resistance. Conclusion: High frequency of β–lactamase in bacterial survey represents colonization of bacteria in staff hands; may be due to facility transmission β–lactamase plasmid genes in bacteria. We suggest better hand washing in hospitals and prescription of β–lactame antibiotics was based only on antibiogram results

  11. Characterization of Bacterial Strains Isolated Through Microbial Profiling of Urine Samples

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    Poulomi Nandy

    2007-01-01

    Full Text Available The present study was conducted to determine the microbial profile in urine samples. Differential and selective chromogenic culture media were used for the rapid detection, identification and enumeration of urinary tract pathogens namely, E. coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Proteus mirabilis. Urine samples of normal healthy individuals as well as patients with Urinary Tract Infection (UTI were screened on hicrome agar plates. The cultivable bacteria present in urine were isolated based on chromogenic detection. Antibiotic sensitivity assay, morphological characterization and biochemical tests, namely protease, oxidase, catalase, lipase, DNase and lecithinase assay were performed with the 15 isolates obtained from urine samples. The molecular analyses of the isolates were done through partial sequencing of the 16SrDNA gene; six of them were found to be novel and submitted in GenBank under the accession numbers EF644491-96. Phylogenetic tree of the isolates were constructed by neighbour joining method.

  12. Tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives can specifically target bacterial DNA ligases and can distinguish them from human DNA ligase I.

    Science.gov (United States)

    Yadav, Nisha; Khanam, Taran; Shukla, Ankita; Rai, Niyati; Hajela, Kanchan; Ramachandran, Ravishankar

    2015-05-21

    DNA ligases are critical components for DNA metabolism in all organisms. NAD(+)-dependent DNA ligases (LigA) found exclusively in bacteria and certain entomopoxviruses are drawing increasing attention as therapeutic targets as they differ in their cofactor requirement from ATP-dependent eukaryotic homologs. Due to the similarities in the cofactor binding sites of the two classes of DNA ligases, it is necessary to find determinants that can distinguish between them for the exploitation of LigA as an anti-bacterial target. In the present endeavour, we have synthesized and evaluated a series of tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives for their ability to distinguish between bacterial and human DNA ligases. The in vivo inhibition assays that employed LigA deficient E. coli GR501 and S. typhimurium LT2 bacterial strains, rescued by ATP-dependent T4 DNA ligase or Mycobacterium tuberculosis NAD(+)-dependent DNA ligase (Mtb LigA), respectively, showed that the compounds can specifically inhibit bacterial LigA. The in vitro enzyme inhibition assays using purified MtbLigA, human DNA ligase I & T4 DNA ligase showed specific inhibition of MtbLigA at low micromolar range. Our results demonstrate that tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives can distinguish between bacterial and human DNA ligases by ∼5-folds. In silico docking and enzyme inhibition assays identified that the compounds bind to the cofactor binding site and compete with the cofactor. Ethidium bromide displacement and gel-shift assays showed that the inhibitors do not exhibit any unwanted general interactions with the substrate DNA. These results set the stage for the detailed exploration of this compound class for development as antibacterials.

  13. Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: Effective recovery of bacterial and archaeal DNA using mechanical cell lysis

    NARCIS (Netherlands)

    Salonen, A.; Nikkilä, J.; Jalanka-Tuovinen, J.; Immonen, O.; Rajilic-Stojanovic, M.; Kekkonen, R.A.; Palva, A.; Vos, de W.M.

    2010-01-01

    Several different protocols are used for fecal DNA extraction, which is an integral step in all phylogenetic and metagenomic approaches to characterize the highly diverse intestinal ecosystem. We compared four widely used methods, and found their DNA yields to vary up to 35-fold. Bacterial, archaeal

  14. Xanthomonas oryzae pv oryzae the Causal Agent of Bacterial Leaf Blight of rice: Isolation, Characterization, and Study of Transposon Mutagenesis

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    Abdjad Asih Nawangsih

    2011-04-01

    Full Text Available Xanthomonas oryzae pv oryzae the Causal Agent of Bacterial Leaf Blight of rice: Isolation, Characterization, and Study of Transposon Mutagenesis. X. oryzae pv. oryzae (Xoo causes bacterial leaf blight (BLB of rice (Oryza sativa L., a major disease that constrains production of the staple crop in many countries of the world. Identification of X. oryzae pv. oryzae (Xoo was conducted based on the disease symptoms, pathogenicity, morphological, physiological, and genetic characteristics of bacterial cultures isolated from the infected plants. Fifty bacterial isolates predicted as Xoo have been successfully isolated. They are aerobic, rod shaped, and Gram negative bacteria. The isolates were evaluated for their hypersensitivity in tobacco and pathogenicity in rice plant. Fifty isolates induced hypersensitive reaction in tobacco and showed pathogenicity symptom in rice in different length. Based on physiological test, hypersensitivity and pathogenicity reactions, three bacterial isolates strongly predicted as Xoo, i.e. STG21, STG42, and STG46, were non indole formation, non pigment fluorescent, hydrolyzed casein, catalase activity positive, but negative oxidase. Partial sequencing of 16S rRNA genes of STG21 and STG42 showed 80% and 82% homology with X. oryzae, respectively, while STG46 showed 84% homology with X. campestris. Mini-Tn5 transposon mutagenesis of STG21 generated one of the mutants (M5 lossed it’s ability to induce hypersensitive reaction in tobacco plant and deficient in pathogenicity on rice. The lesion length of rice leaf caused by the mutant M5 decreased up to 80%.

  15. Isolation of bacteria causing secondary bacterial infection in the lesions of cutaneous leishmaniasis

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    Ziaie Hengameh

    2008-01-01

    Full Text Available Background: Cutaneous Leishmaniasis (CL is a parasitic disease characterized by single or multiple ulcerations. Secondary bacterial infection is one of the complications of the disease that can increase the tissue destruction and the resulting scar. Objective: To effectively determine the incidence of real secondary bacteria infection in cutaneous leishmaniasis, we designed the current study. Methods and Materials: This was a cross-sectional study performed in Skin Diseases and Leishmaniasis Research Centre, Isfahan, Iran. In this study, 854 patients with confirmed CL were enrolled. Samples were taken from all the patients. Sterile swaps were achieved for the ulcer exudates and scraping was used for nonulcerated lesions. All the samples were transferred to tryptic soy broth medium. After 24 h of incubation in 37°C, they were transferred to eosin methylene blue agar (EBM and blood agar. Laboratory tests were used to determine the species of bacteria. All of the collected data were analyzed by SPSS software and chi-square. Results: Among 854 patients with confirmed cutaneous leishmaniasis, 177 patients (20.7% had positive cultures for secondary bacterial infection. Bacteria isolated from the lesions were as follows: Staphylococcus aureus - 123 cases (69.4%, coagulase negative Staphylococcus - 41 cases (23.1%, E. coil - 7 cases (3.9%, Proteus - 3 cases (1.7% and Klebsiella - 3 cases (1.7%. Conclusions: The incidence of secondary bacterial infection in lesions of CL was 20.7%. The most common isolated pathogen was Staphylococcus aureus . The incidence of secondary bacterial infection was significantly more in the ulcerated lesions as compared with nonulcerated lesions ( P = 0.00001.

  16. Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide.

    Science.gov (United States)

    Nocker, Andreas; Camper, Anne K

    2006-03-01

    The distinction between viable and dead bacterial cells poses a major challenge in microbial diagnostics. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based quantification methods overestimate the number of viable cells in mixed populations or even lead to false-positive results in the absence of viable cells. On the other hand, RNA-based diagnostic methods, which circumvent this problem, are technically demanding and suffer from some drawbacks. A promising and easy-to-use alternative utilizing the DNA-intercalating dye ethidium monoazide bromide (EMA) was published recently. This chemical is known to penetrate only into "dead" cells with compromised cell membrane integrity. Subsequent photoinduced cross-linking was reported to inhibit PCR amplification of DNA from dead cells. We provide evidence here that in addition to inhibition of amplification, most of the DNA from dead cells is actually lost during the DNA extraction procedure, probably together with cell debris which goes into the pellet fraction. Exposure of bacteria to increasing stress and higher proportions of dead cells in defined populations led to increasing loss of genomic DNA. Experiments were performed using Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium as model pathogens and using real-time PCR for their quantification. Results showed that EMA treatment of mixed populations of these two species provides a valuable tool for selective removal of DNA of nonviable cells by using conventional extraction protocols. Furthermore, we provide evidence that prior to denaturing gradient gel electrophoresis, EMA treatment of a mature mixed-population drinking-water biofilm containing a substantial proportion of dead cells can result in community fingerprints dramatically different from those for an untreated biofilm. The interpretation of such fingerprints can have important implications in the field of microbial ecology.

  17. Assessment of viable bacteria and bacterial DNA in blood and bloodstain specimens stored under various conditions.

    Science.gov (United States)

    Hosokawa-Muto, Junji; Fujinami, Yoshihito; Mizuno, Natsuko

    2013-11-01

    Microbial forensic specimens that are collected at biocrime and bioterrorism scenes include blood, tissue, cloths containing biological fluids, swabs, water, soil, and aerosols. It is preferable that pathogens in such specimens are alive and kept in a steady state. Specimens may be stored for a prolonged period before analysis; therefore, it is important to understand the effect of the storage conditions on the pathogens contained within the specimens. In this study, we prepared blood and bloodstain specimens containing Gram-negative or -positive bacteria, stored the samples for 482 days under various conditions, and measured viable bacterial counts and total bacterial contents in the samples. Viable bacteria were preserved well in the samples stored at -30 and -80 °C, but were diminished or undetectable in the samples stored at 4 °C and room temperature. The total bacterial content was maintained in the blood samples stored at -30 and -80 °C and in the bloodstain samples stored under all temperature conditions, but decreased in the blood samples stored at 4 °C and room temperature. This study showed that the storage conditions affected viable bacteria and bacterial DNA and that freezing and drying were significant for their long-term storage. We provide important information for the storage of microbial forensic specimens.

  18. Bacterial delivery of large intact genomic-DNA-containing BACs into mammalian cells.

    Science.gov (United States)

    Cheung, Wing; Kotzamanis, George; Abdulrazzak, Hassan; Goussard, Sylvie; Kaname, Tadashi; Kotsinas, Athanassios; Gorgoulis, Vassilis G; Grillot-Courvalin, Catherine; Huxley, Clare

    2012-01-01

    Efficient delivery of large intact vectors into mammalian cells remains problematical. Here we evaluate delivery by bacterial invasion of two large BACs of more than 150 kb in size into various cells. First, we determined the effect of several drugs on bacterial delivery of a small plasmid into different cell lines. Most drugs tested resulted in a marginal increase of the overall efficiency of delivery in only some cell lines, except the lysosomotropic drug chloroquine, which was found to increase the efficiency of delivery by 6-fold in B16F10 cells. Bacterial invasion was found to be significantly advantageous compared with lipofection in delivering large intact BACs into mouse cells, resulting in 100% of clones containing intact DNA. Furthermore, evaluation of expression of the human hypoxanthine phosphoribosyltransferase (HPRT) gene from its genomic locus, which was present in one of the BACs, showed that single copy integrations of the HPRT-containing BAC had occurred in mouse B16F10 cells and that expression of HPRT from each human copy was 0.33 times as much as from each endogenous mouse copy. These data provide new evidence that bacterial delivery is a convenient and efficient method to transfer large intact therapeutic genes into mammalian cells.

  19. Prevalence and antibiogram of bacterial isolates from urinary tract infections at Dessie Health Research Laboratory, Ethiopia

    Institute of Scientific and Technical Information of China (English)

    Mulugeta Kibret; Bayeh Abera

    2014-01-01

    Objective: To determine the prevalence and antimicrobial susceptibility of bacteria from suspected urinary tract infections.Methods:A retrospective analysis of bacterial pathogens and their antimicrobial susceptibility was done on urine samples at Dessie Regional Laboratory in the period 2003 to 2010. Antimicrobial susceptibility tests were done using disc diffusion technique as per the standard of Kirby-Bauer method.Results:The male to female ratio of the patients was 1:1.96. Of the total 1404 samples, 319 (22.7%) were culture positive. Escherichia coli was the dominant isolate (63.6%) followed by Klebsiella spp. (8.5%) and Proteus spp. (8.2%). The overall resistance rates to erythromycin, amoxycillin, and tetracycline were 85.6%, 88.9% and 76.7%, respectively. The three most frequently isolated bacteria had resistance rates of 80.1%-90.0% to, amoxycillin, and tetracycline and sensitivity rates of 0 to 25% to nitrofurantoin, ciprofloxacin and gentamicin. Antibiogram of isolates showed that 152 (47.85%) isolates were resistance to two and more antimicrobials.Conclusions:In the study area resistance rates to erythromycin, amoxycillin and tetracycline were high. Since most isolates were sensitive to nitrofurantoin and gentamicin, they are considered as appropriate antimicrobials for empirical treatment urinary tract infections.

  20. Highly effective bacterial agents against Cimbex quadrimaculatus (Hymenoptera: Cimbicidae): isolation of bacteria and their insecticidal activities.

    Science.gov (United States)

    Cakici, Filiz Ozkan; Ozgen, İnanc; Bolu, Halil; Erbas, Zeynep; Demirbağ, Zihni; Demir, İsmail

    2015-01-01

    Cimbex quadrimaculatus (Hymenoptera: Cimbicidae) is one of the serious pests of almonds in Turkey and worldwide. Since there is no effective control application against this pest, it has been a serious problem up to now. Therefore, we aimed to find an effective bacterium that can be utilized as a biocontrol agent against C. quadrimaculatus in pest management. We isolated seven bacteria from dead and live C. quadrimaculatus larvae, and evaluated the larvicidal potency of all isolates on the respective pest. Based on the morphological, physiological, biochemical and molecular properties (partial sequence of 16S rRNA gene), the isolates were identified to be Bacillus safensis (CQ1), Bacillus subtilis (CQ2), Bacillus tequilensis (CQ3), Enterobacter sp. (CQ4), Kurthia gibsonii (CQ5), Staphylococcus sp. (CQ6) and Staphylococcus sciuri (CQ7). The results of the larvicidal activities of these isolates indicated that the mortality value obtained from all treatments changed from 58 to 100 %, and reached 100 % with B. safensis (CQ1) and B. subtilis (CQ2) on the 3rd instar larvae within 10 days of application of 1.89 × 10(9) cfu/mL bacterial concentration at 25 °C under laboratory conditions. Findings from this study indicate that these isolates appear to be a promising biocontrol agent for C. quadrimaculatus.

  1. SLiCE: a novel bacterial cell extract-based DNA cloning method.

    Science.gov (United States)

    Zhang, Yongwei; Werling, Uwe; Edelmann, Winfried

    2012-04-01

    We describe a novel cloning method termed SLiCE (Seamless Ligation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from Bacteria Artificial Chromosomes (BACs) or other sources. SLiCE is highly cost effective as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. In addition, the cloning efficiencies and capabilities of these strains can be greatly improved by simple genetic modifications. As an example, we modified the DH10B Escherichia coli strain to express an optimized λ prophage Red recombination system. This strain, termed PPY, facilitates SLiCE with very high efficiencies and demonstrates the versatility of the method.

  2. Monitoring of oil pollution at Gemsa Bay and bioremediation capacity of bacterial isolates with biosurfactants and nanoparticles.

    Science.gov (United States)

    El-Sheshtawy, H S; Khalil, N M; Ahmed, W; Abdallah, R I

    2014-10-15

    Fifteen crude oil-degrading bacterial isolates were isolated from an oil-polluted area in Gemsa Bay, Red Sea, Egypt. Two bacterial species showed the highest growth rate on crude oil hydrocarbons. From an analysis of 16S rRNA sequences, these isolates were identified as Pseudomonas xanthomarina KMM 1447 and Pseudomonas stutzeri ATCC 17588. Gas Chromatographic (GC) analysis of the crude oil remaining in the culture medium after one week at 30°C showed that the optimum biodegradation of crude petroleum oil was demonstrated at 50% in medium containing biosurfactant with two types of nanoparticles separately and two bacterial species. The complete degradation of some different members of polyaromatics and the percentage biodegradation of other polyaromatics increased in microcosm containing two different types of nanoparticles with biosurfactant after 7 days. In conclusion, these bacterial strains may be useful for the bioremediation process in the Gemsa Bay, Red Sea decreasing oil pollution in this marine ecosystem.

  3. A case report of acute pediatric bacterial meningitis due to the rare isolate, Pseudomonas putida

    Institute of Scientific and Technical Information of China (English)

    Grishma V. Kulkarni

    2016-01-01

    Acute bacterial meningitis (ABM) is the medical emergency which warrants an early diagnosis and an aggressive therapy. Despite the availability of the potent newer antibiotics, the mortality caused by ABM and its complications remain high in India, ranging from 16% to 32%. The aim of this case report is to present the rare isolation ofPseudomonas putida from cerebrospinal lfuid sample. Besides this, the author also emphasizes the importance of correctly identifying the organism and thus the selection of the most accurate antibiotic from the susceptibility proifle to allow for early recovery and to improve the patient outcome and survival.

  4. The Extraction and Partial Purification of Bacterial DNA as a Practical Exercise for GCE Advanced Level Students.

    Science.gov (United States)

    Falconer, A. C.; Hayes, L. J.

    1986-01-01

    Describes a relatively simple method of extraction and purification of bacterial DNA. This technique permits advanced secondary-level science students to obtain adequate amounts of DNA from very small pellets of bacteria and to observe some of its polymer properties. (ML)

  5. Comparative DNA isolation behaviours of silica and polymer based sorbents in batch fashion: monodisperse silica microspheres with bimodal pore size distribution as a new sorbent for DNA isolation.

    Science.gov (United States)

    Günal, Gülçin; Kip, Çiğdem; Eda Öğüt, S; İlhan, Hasan; Kibar, Güneş; Tuncel, Ali

    2017-03-22

    Monodisperse silica microspheres with bimodal pore-size distribution were proposed as a high performance sorbent for DNA isolation in batch fashion under equilibrium conditions. The proposed sorbent including both macroporous and mesoporous compartments was synthesized 5.1 μm in-size, by a "staged shape templated hydrolysis and condensation method". Hydrophilic polymer based sorbents were also obtained in the form of monodisperse-macroporous microspheres ca 5.5 μm in size, with different functionalities, by a developed "multi-stage microsuspension copolymerization" technique. The batch DNA isolation performance of proposed material was comparatively investigated using polymer based sorbents with similar morphologies. Among all sorbents tried, the best DNA isolation performance was achieved with the monodisperse silica microspheres with bimodal pore size distribution. The collocation of interconnected mesoporous and macroporous compartments within the monodisperse silica microspheres provided a high surface area and reduced the intraparticular mass transfer resistance and made easier both the adsorption and desorption of DNA. Among the polymer based sorbents, higher DNA isolation yields were achieved with the monodisperse-macroporous polymer microspheres carrying trimethoxysilyl and quaternary ammonium functionalities. However, batch DNA isolation performances of polymer based sorbents were significantly lower with respect to the silica microspheres.

  6. Isolation and Partial Characterization of Bacterial Strains on Low Organic Carbon Medium from Soils Fertilized with Different Organic Amendments

    NARCIS (Netherlands)

    Senechkin, I.V.; Speksnijder, A.G.C.L.; Semenov, A.M.; Bruggen, van A.H.C.; Overbeek, van L.S.

    2010-01-01

    A total of 720 bacterial strains were isolated from soils with four different organic amendment regimes on a low organic carbon (low-C) agar medium (10 mu g C ml(-1)) traditionally used for isolation of oligotrophs. Organic amendments in combination with field history resulted in differences in diss

  7. Comparison of bacterial DNA profiles of footwear insoles and soles of feet for the forensic discrimination of footwear owners.

    Science.gov (United States)

    Goga, Haruhisa

    2012-09-01

    It is crucial to identify the owner of unattended footwear left at a crime scene. However, retrieving enough DNA for DNA profiling from the owner's foot skin (plantar skin) cells from inside the footwear is often unsuccessful. This is sometimes because footwear that is used on a daily basis contains an abundance of bacteria that degrade DNA. Further, numerous other factors related to the inside of the shoe, such as high humidity and temperature, can encourage bacterial growth inside the footwear and enhance DNA degradation. This project sought to determine if bacteria from inside footwear could be used for footwear trace evidence. The plantar skins and insoles of shoes of volunteers were swabbed for bacteria, and their bacterial community profiles were compared using bacterial 16S rRNA terminal restriction fragment length polymorphism analysis. Sufficient bacteria were recovered from both footwear insoles and the plantar skins of the volunteers. The profiling identified that each volunteer's plantar skins harbored unique bacterial communities, as did the individuals' footwear insoles. In most cases, a significant similarity in the bacterial community was identified for the matched foot/insole swabs from each volunteer, as compared with those profiles from different volunteers. These observations indicate the probability to discriminate the owner of footwear by comparing the microbial DNA fingerprint from inside footwear with that of the skin from the soles of the feet of the suspected owner. This novel strategy will offer auxiliary forensic footwear evidence for human DNA identification, although further investigations into this technique are required.

  8. In vitro antimicrobial activity of marbofloxacin and enrofloxacin against bacterial strains isolated from companion animals.

    Science.gov (United States)

    Farca, A M; Cavana, P; Robino, P; Nebbia, P

    2007-06-01

    Fluoroquinolones were originally developed for the Gram-negative aerobic spectrum, but the newer generation agents are also highly effective against some Gram-positive pathogens and cause few adverse effects. Owing to these characteristics, fluoroquinolones are often used in first line therapy in small animal practice. However, their widespread use has raised concern over emerging bacterial resistance. In this study we evaluated the in vitro efficacy of two fluoroquinolones, marbofloxacin and enrofloxacin, on field strains isolated from clinical infections between 2002 and 2005. Our data show that most of the isolates are still sensitive to both antimicrobials and marbofloxacin was more effective than enrofloxacin, especially against P. aeruginosa and beta-Streptococci (P < 0.01). beta-Streptococci demonstrated the greatest resistance to the two study drugs.

  9. Detection of processed genetically modified food using CIM monolithic columns for DNA isolation.

    Science.gov (United States)

    Jerman, Sergej; Podgornik, Ales; Cankar, Katarina; Cadet, Neza; Skrt, Mihaela; Zel, Jana; Raspor, Peter

    2005-02-11

    The availability of sufficient quantities of DNA of adequate quality is crucial in polymerase chain reaction (PCR)-based methods for genetically modified food detection. In this work, the suitability of anion-exchange CIM (Convective Interaction Media; BIA Separations, Ljubljana, Slovenia) monolithic columns for isolation of DNA from food was studied. Maize and its derivates corn meal and thermally pretreated corn meal were chosen as model food. Two commercially available CIM disk columns were tested: DEAE (diethylaminoethyl) and QA (quaternary amine). Preliminary separations were performed with standard solution of salmon DNA at different pH values and different NaCl concentrations in mobile phase. DEAE groups and pH 8 were chosen for further isolations of DNA from a complex matrix-food extract. The quality and quantity of isolated DNA were tested on agarose gel electrophoresis, with UV-scanning spectrophotometry, and by amplification with real-time PCR. DNA isolated in this way was of suitable quality for further PCR analyses. The described method is also applicable for DNA isolation from processed foods with decreased DNA content. Furthermore, it is more effective and less time-consuming in comparison with the existing proposed methods for isolation of DNA from plant-derived foods.

  10. Isolation and Identification of a New Tetrodotoxin-Producing Bacterial Species, Raoultella terrigena, from Hong Kong Marine Puffer Fish Takifugu niphobles

    Directory of Open Access Journals (Sweden)

    Fred Wang-Fat Lee

    2011-11-01

    Full Text Available Puffer fish, Takifugu niphobles, collected from the Hong Kong coastal waters were screened for tetrodotoxin-producing bacteria. A Gram-negative, non-acid-fast, non-sporing and rod shaped bacterial strain (designated as gutB01 was isolated from the intestine of the puffer fish and was shown to produce tetrodotoxin (TTX. Based on the Microbial Identification (MIDI and 16S-23S rDNA internal transcribed spacer (ITS phylogenetic analysis, the strain was identified as Raoultella terrigena. The TTX production ability of the strain was confirmed by mouse bioassay, ELISA and mass spectrometry (MALDI-TOF. Our results reiterate that the TTX found in puffer fish was likely produced by the associated bacteria and TTX are widely produced amongst a diversity of bacterial species.

  11. Isolation and identification of a new tetrodotoxin-producing bacterial species, Raoultella terrigena, from Hong Kong marine puffer fish Takifugu niphobles.

    Science.gov (United States)

    Yu, Vincent Chung-Him; Yu, Peter Hoi-Fu; Ho, Kin-Chung; Lee, Fred Wang-Fat

    2011-01-01

    Puffer fish, Takifugu niphobles, collected from the Hong Kong coastal waters were screened for tetrodotoxin-producing bacteria. A Gram-negative, non-acid-fast, non-sporing and rod shaped bacterial strain (designated as gutB01) was isolated from the intestine of the puffer fish and was shown to produce tetrodotoxin (TTX). Based on the Microbial Identification (MIDI) and 16S-23S rDNA internal transcribed spacer (ITS) phylogenetic analysis, the strain was identified as Raoultella terrigena. The TTX production ability of the strain was confirmed by mouse bioassay, ELISA and mass spectrometry (MALDI-TOF). Our results reiterate that the TTX found in puffer fish was likely produced by the associated bacteria and TTX are widely produced amongst a diversity of bacterial species.

  12. Susceptibility of different bacterial species isolated from food animals to copper sulphate, zinc chloride and antimicrobial substances used for disinfection

    DEFF Research Database (Denmark)

    Aarestrup, Frank Møller; Hasman, Henrik

    2004-01-01

    that Danish bacterial isolates from livestock so far have not or have only to a limited degree developed resistance to antimicrobial compounds commonly used for disinfection. Acquired copper resistance was only found in enterococci. There were large differences in the intrinsic susceptibility of the different......A total of 569 different bacterial isolates (156 Salmonella, 202 E. coli, 43 S. aureus, 38 S. hyicus, 52 E. faecalis, 78 E faecium) were tested for susceptibility to copper sulphate, benzalkonium chloride, hydrogen peroxide and chlorhexidine using MIC determinations. A total of 442 isolates were...... of susceptibilities to the different antimicrobial agents. Large variations were observed in the susceptibility of the different bacterial species to the different compounds. Staphylococci were in general very susceptible to all antimicrobial compounds tested. The Salmonella isolates were in general less susceptible...

  13. Bioremediation of petroleum based contaminants with biosurfactant produced by a newly isolated petroleum oil degrading bacterial strain

    Directory of Open Access Journals (Sweden)

    Debajit Borah

    2017-03-01

    Full Text Available Petroleum based hydrocarbon degrading and biosurfactant producing bacterial strain was isolated from an automobile engine. The strain was identified as Bacillus cereus DRDU1 on the basis of 16S rDNA sequencing analysis. The strain was found to be efficiently degrading 96% of kerosene making it a potential tool for bioremediation of petroleum based contaminants. Production and optimization of the biosurfactant produced by the isolate were also carried out. Surface hydrophobicity trait of isolate was found to be 60.67 ± 1.53% and foaming percentage of the crude biosurfactant was found to be 31.33 ± 0.58%. The presence of amino acids and sugar moieties in the biosurfactant was confirmed by biochemical tests and were further validated by FTIR (the Fourier transform infrared spectrometric analysis revealing the presence of υOH, υCOO, υCOOH, υCH (stretching, υNH, υCH2, υCH3, and υCH (bending, and υCO (ester in the surfactant. The decrease in contact angle of hydrocarbon oil from (30.67 ± 1.15° to (21.3 ± 1.53° respectively after 3 and 6 days of incubation reveals its potential to emulsify petroleum oil. Further, emulsification index (E24 of biosurfactant against kerosene, crude oil, and used engine oil were determined to be 55.33 ± 1.53%, 29.67 ± 1.53%, and 20 ± 1% respectively which attracts its future application in MEOR (microbial enhanced oil recovery process.

  14. Isolation, cultivation, purification and identification of bacterial species from microfauna of soil

    Directory of Open Access Journals (Sweden)

    Amna Ali

    2011-03-01

    Full Text Available

     

    Abstract:
    Soil is an excellent source of unknown microorganisms since bacteria, algae, protozoans, yeasts, moulds, and microscopic worms are routinely found in this environment. Therefore, soil is a medium in which life is sustained in a fragile biological balance. Bacteria play an important role in nutritional chains that are an important part of biological balance. In the present study, four different soil samples were collected from the rhizosphere of i Sapota zapotilla, ii Eucalyptus species, iii Ficus religiosa from Lahore and iv soil from Changa manga, Pakistan. A Total of 28 bacterial species were isolated and classified in the period between November 2008 and December 2009. All species were cultured on recommended media for verification of biochemical characteristics. The results showed that at least fifteen Gram-positive bacterial species were present in samples and these were considered as the major group constituting the bacterial population strains

  15. The interaction of DNA gyrase with the bacterial toxin CcdB

    DEFF Research Database (Denmark)

    Kampranis, S C; Howells, A J; Maxwell, A

    1999-01-01

    CcdB is a bacterial toxin that targets DNA gyrase. Analysis of the interaction of CcdB with gyrase reveals two distinct complexes. An initial complex (alpha) is formed by direct interaction between GyrA and CcdB; this complex can be detected by affinity column and gel-shift analysis, and has...... of this initial complex with ATP in the presence of GyrB and DNA slowly converts it to a second complex (beta), which has a lower rate of ATP hydrolysis and is unable to catalyse supercoiling. The efficiency of formation of this inactive complex is dependent on the concentrations of ATP and CcdB. We suggest...

  16. DNA isolation by Chelex-100:an efficient approach to consider in leptospirosis early stages

    Institute of Scientific and Technical Information of China (English)

    Angel Alberto Noda; Islay Rodríguez

    2014-01-01

    Objective:To compare the value of leptospiral DNA extraction procedures from clinical samples for the early diagnosis of leptospirosis. Methods:Three DNA extraction procedures were applied for microbiological analysis, results of QIAmp DNA mini kit (QIAGEN, Germany), CLART HPV kit (GENOMICA, Spain) and Chelex-100 assay were compared concerning extraction efficiency, DNA purity and DNA suitability for amplification by specific polymerase chain reaction for pathogenic leptospires from blood, plasma and serum artificially infected. Results: The comparison of extraction methods highlighted the efficiency of Chelex-100 and QIAmp DNA mini kit. Chelex-100 achieved the isolation of the highest concentration of leptospiral DNA from the culture and the spiked samples, with acceptable purities and without inhibitors to PCR. Conclusions: Chelex-100 assay is a rapid and effective approach for DNA isolation in clinical samples having pathogenic leptospires and it could be useful in the early diagnosis of leptospirosis.

  17. DNA isolation by Chelex-100: an efficient approach to consider in leptospirosis early stages

    Directory of Open Access Journals (Sweden)

    Angel Alberto Noda

    2014-06-01

    Full Text Available Objective: To compare the value of leptospiral DNA extraction procedures from clinical samples for the early diagnosis of leptospirosis. Methods: Three DNA extraction procedures were applied for microbiological analysis, results of QIAmp DNA mini kit (QIAGEN, Germany, CLART HPV kit (GENOMICA, Spain and Chelex-100 assay were compared concerning extraction efficiency, DNA purity and DNA suitability for amplification by specific polymerase chain reaction for pathogenic leptospires from blood, plasma and serum artificially infected. Results: The comparison of extraction methods highlighted the efficiency of Chelex-100 and QIAmp DNA mini kit. Chelex-100 achieved the isolation of the highest concentration of leptospiral DNA from the culture and the spiked samples, with acceptable purities and without inhibitors to PCR. Conclusions: Chelex-100 assay is a rapid and effective approach for DNA isolation in clinical samples having pathogenic leptospires and it could be useful in the early diagnosis of leptospirosis.

  18. Genomic DNA restriction endonuclease from Pasteurella multocida isolated from Indonesia, katha strain and reference strains and analysed by PFGE

    Directory of Open Access Journals (Sweden)

    Supar

    2003-10-01

    Full Text Available Pasteurella multocida strains are the causative disease agents of wide range of domestic and wild animals in Indonesia. The most important serotypes are associated with Hemorrhagic septicaemic (HS diseases in cattle and buffaloes, cholera in ducks and chickens. The HS disease associated with P. multocia in large ruminants in Indonesia is controled by killed whole cell vaccines produced by the use of P. multocida Katha strains. There is no discriminatory data of the molecular biology technique has been applied to investigate P. multocida isolates from different geographic locations in Indonesia. The purpose of this studies were to observe the genetic diversity among P. multocida isolated from various geograpic locations and compared with Katha vaccine strain and other reference strains. A total samples of 38 isolates and strains of P. multocida were analysed by means of pulsed-field gel electrophoresis (PFGE. Each sample was grown in nutrient broth, cells were separeted by centrifugation. Whole cell pellet was mixed with agarose and then prepared agarose plugs. The genomic DNA of each sample was digested in situ (plug with either restriction endonuclease of ApaI and/or BamHI. The digested genomic DNA of each sample was analysed by PFGE, the genomic DNA restricted profile of each sample was compared with others. The use of ApaI restriction endonuclease digestion and analysed by PFGE, demonstrated that 34 out of 38 P. multocia samples could be differentiated into 16 ApaI types, whereas based on the BamHI digestion of these samples were differentiated into 20 BamHI types. Genomic DNA restriction pattern of Indonesian P. multocida isolates originated from cattle and buffaloes associated with haemorrhagic septicaemic diseases demonstrated different pattern to those of vaccine Katha strain, poultry strains as well as the reference strains currenly kept at Balitvet Culture Collection (BCC unit. Two P. multocida isolates derived from ducks with cholera

  19. Enzymatic activity of endophytic bacterial isolates of Jacaranda decurrens Cham. (Carobinha-do-campo

    Directory of Open Access Journals (Sweden)

    Aysha Jussara Ivonilde Carrim

    2006-05-01

    Full Text Available The objectives of this work was to isolate endophytic bacteria from Jacaranda decurrens Cham. and screening of some enzymes of biotechnological interest. Ten (10 bacterial species were isolated and identified from the leaves and steams. All the isolates presented enzymatic activity, which was ranked as follows: proteolytic (60% and amilolytic activity (60%, lipolytic (40%, esterasic (40%. However, cellulolytic and pectinolytic activities were not detected. This is the first report on the isolation and identification of endophytic bacteria from Jacaranda decurrens Cham.O objetivo deste trabalho foi isolar bactérias endofíticas de Jacaranda decurrens Cham. e selecionar algumas enzimas de interesse biotecnológico. Dez espécies de bactérias foram isoladas e identificadas de caule e folhas. Todos os isolados de apresentaram atividade enzimática, e a maior predominância foi de atividade proteolítica (60% e atividade amilolítica (60%, seguidas atividades lipolítica (40% e esterásica (40%. Contudo, as atividades celulolítica e pectinolítica não foram detectadas. Este é o primeiro relato de isolamento e identificação de bactérias presentes em Jacaranda decurrens Cham.

  20. In vitro evaluation of Pseudomonas bacterial isolates from rice phylloplane for biocontrol of Rhizoctonia solani and plant growth promoting traits.

    Science.gov (United States)

    Akter, Shamima; Kadir, Jugah; Juraimi, Abdul Shukor; Saud, Halimi Mohd

    2016-07-01

    The ability for biocontrol and plant growth promotion of three Pseudomonas bacterial isolates namely Pseudomonas fluorescens (UMB20), Pseudomonas aeruginosa (KMB25) and Pseudomonas asplenii (BMB42) obtained from rice plants was investigated. Fungal growth inhibition by the isolates ranged from 86.85 to 93.15% in volatile and 100% in diffusible metabolites test. Among the isolates, BMB42 showed fungal growth inhibition significantly in the volatile metabolite test. Isolates UMB20 and BMB42 were able to synthesis chitinase with chitinolytic indices of 13.66 and 13.50, respectively. In case of -1,3-glucanase, all the isolates showed activity to produce this enzyme at varied levels and isolate KMB25 showed significantly highest activity (53.53 ppm). Among the three isolates, KMB25 showed positive response to protease production and all of them were negative to pectinase and lipase and positive to the production of siderophore, and HCN, and were able to solubilize tricalcium phosphate. All the three bacterial isolates were capable of forming biofilm at different levels. Above results suggest that phylloplane Pseudomonas bacterial isolates have potential for antifungal activities and plant growth promotion.

  1. Isolation of T—DNA flanking plant DNA from T—DNA insertional embryo—lethal mutants of Arabidopsis thaliana by plasmid rescue technique

    Institute of Scientific and Technical Information of China (English)

    YAOXIAOLI; JIANGESUN; 等

    1996-01-01

    Three T-DNA insertional embryonic lethal mutants from NASC(The Nottingham Arabidopsis Stock Center) were first checked with their segregation ratio of abortive and normal seeds and the copy number of T-DNA insertion.The N4081 mutant has a segregation ratio of 1:3.04 in average and one T-DNA insertion site according to our assay.It was therefore chosen for further analysis.To isolate the joint fragment of T-DNA and plant DNA,the plasmid rescue technique was used.pEL-7,one of plasmids from left border of T-DNA,which contained pBR322 was selected from ampicillin plate.The T-DNA fragment of pEL-7 was checked by restriction enzyme analysis and Southern Blot.Restriction analysis confirmed the presence of known sites of EcoRI,PstI and PvuII on it.For confirming the presence of flanking plant DNA in this plasmid,pEL-7 DNA was labeled and hybridized with wild type and mutant plant DNA.The Southern Blot indicated the hybridization band in both of them.Furthermore,the junction of T-DNA/plant DNA was subcloned into bluescript SK+ and sequenced by Applied Biosystem 373A sequencer.The results showed the 822 bp fragment contained a 274 bp sequence,which is 99.6%homolog(273bp/274bp) to Ti plasmid pTi 15955,DNA.The bp of left 25 bp border repeat were also found in the juction of T-DNA and Plant DNA. Taken together,pEL-7 should coutain a joint fragment of T-DNA and flanking plant DNA.This plasmid DNA could be used for the isolation of plant gene,which will be helpful to elucidate the relationship between gene function and plant embryo development.

  2. An improved method with a wider applicability to isolate plant mitochondria for mtDNA extraction

    OpenAIRE

    2015-01-01

    Background Mitochondria perform a principal role in eukaryotic cells. Mutations in mtDNA can cause mitochondrial dysfunction and are frequently associated with various abnormalities during plant development. Extraction of plant mitochondria and mtDNA is the basic requirement for the characterization of mtDNA mutations and other molecular studies. However, currently available methods for mitochondria isolation are either tissue specific or species specific. Extracted mtDNA may contain substant...

  3. Epidemiological Aspect and common Bacterial and Fungal isolates from Suppurative Corneal Ulcer in Mymensingh Region.

    Science.gov (United States)

    Moid, M A; Akhanda, A H; Islam, S; Halder, S K; Islam, R

    2015-04-01

    This prospective study was done to find out the epidemiological factors of suppurative corneal ulcer and the common causative bacterial and fungal isolates from the, patients with suppurative corneal ulcer in secondary and tertiary level hospital at Mymensingh region. A total 100 samples of corneal scrapings were collected purposively from clinically diagnosed suppurative corneal ulcer patients from March 18, 2012 to March 17, 2013. Out of the total 100 samples, bacterial species were 29(29%) cases and the fungal spacies were 71(71%) identified by the culture in blood agar, chocolate agar and sabouraud's agar media and also by microscopic examination. The bacterial species were streptococcus pneumonae 12 cases (12%), Staphylococcus aureus 9 cases (9%), pseudomonas in 6 cases (6%), and Streptococcus pyoganes 2 cases (2%). Fungal species were aspergillus fumigatus 61 cases (61%), aspergillus niger 10 cases (10%). Out of the study populations, most of the populations were from the age group of 41 to 60 years (39 %), followed 21 to 40 years (34%) age group. Considering the sex, male were 67%, female were 33%. The majority of patients came from the rural area of Mymensingh region; occupationally they were farmers (44%). Ocular trauma due to agricultural materials was the most common associated factor (71%). The etiological and epidemiological pattern of suppurative corneal ulcer varies significantly with geographical region, patient population and health of the cornea. The present study was carried out to explore the epidemiological pattern, causative bacterial and fungal specie by laboratory procedure from corneal scraping and to invent a prospective guide line for the management of corneal ulcer in the community.

  4. Comparison of DNA extraction methods in analysis of salivary bacterial communities.

    Science.gov (United States)

    Lazarevic, Vladimir; Gaïa, Nadia; Girard, Myriam; François, Patrice; Schrenzel, Jacques

    2013-01-01

    Culture-independent high-throughput sequencing-based methods are widely used to study bacterial communities. Although these approaches are superior to traditional culture-based methods, they introduce bias at the experimental and bioinformatics levels. We assessed the diversity of the human salivary microbiome by pyrosequencing of the 16S rDNA V1-3 amplicons using metagenomic DNA extracted by two different protocols: a simple proteinase K digestion without a subsequent DNA clean-up step, and a bead-beating mechanical lysis protocol followed by column DNA purification. A high degree of congruence was found between the two extraction methods, most notably in regard to the microbial community composition. The results showed that for a given bioinformatics pipeline, all the taxa with an average proportion >0.12% in samples processed using one extraction method were also detected in samples extracted using the other method. The same taxa tended to be abundant and frequent for both extraction methods. The relative abundance of sequence reads assigned to the phyla Actinobacteria, Spirochaetes, TM7, Synergistetes, and Tenericutes was significantly higher in the mechanically-treated samples than in the enzymatically-treated samples, whereas the phylum Firmicutes showed the opposite pattern. No significant differences in diversity indices were found between the extraction methods, although the mechanical lysis method revealed higher operational taxonomic unit richness. Differences between the extraction procedures outweighed the variations due to the bioinformatics analysis pipelines used.

  5. Comparison of DNA extraction methods in analysis of salivary bacterial communities.

    Directory of Open Access Journals (Sweden)

    Vladimir Lazarevic

    Full Text Available Culture-independent high-throughput sequencing-based methods are widely used to study bacterial communities. Although these approaches are superior to traditional culture-based methods, they introduce bias at the experimental and bioinformatics levels. We assessed the diversity of the human salivary microbiome by pyrosequencing of the 16S rDNA V1-3 amplicons using metagenomic DNA extracted by two different protocols: a simple proteinase K digestion without a subsequent DNA clean-up step, and a bead-beating mechanical lysis protocol followed by column DNA purification. A high degree of congruence was found between the two extraction methods, most notably in regard to the microbial community composition. The results showed that for a given bioinformatics pipeline, all the taxa with an average proportion >0.12% in samples processed using one extraction method were also detected in samples extracted using the other method. The same taxa tended to be abundant and frequent for both extraction methods. The relative abundance of sequence reads assigned to the phyla Actinobacteria, Spirochaetes, TM7, Synergistetes, and Tenericutes was significantly higher in the mechanically-treated samples than in the enzymatically-treated samples, whereas the phylum Firmicutes showed the opposite pattern. No significant differences in diversity indices were found between the extraction methods, although the mechanical lysis method revealed higher operational taxonomic unit richness. Differences between the extraction procedures outweighed the variations due to the bioinformatics analysis pipelines used.

  6. Isolation and 16S DNA characterization of soil microorganisms from tropical soils capable of utilizing the herbicides hexazinone and tebuthiuron.

    Science.gov (United States)

    Mostafa, Fadwa I Y; Helling, Charles S

    2003-11-01

    Six non-fermentative bacteria were isolated from Colombian (South America) and Hawaiian (USA) soils after enrichment with minimal medium supplemented with two herbicides, hexazinone (Hex) and tebuthiuron (Teb). Microscopic examination and physiological tests were followed by partial 16S DNA sequence analysis, using the first 527 bp of the 16S rRNA gene for bacterial identification. The isolated microorganisms (and in brackets, the herbicide that each degraded) were identified as: from Colombia. Methylobacterium organophilum [Teb], Paenibacillus pabuli [Teb], and Micrmbacterium foliorum [Hex]; and from Hawaii, Methylobacterium radiotolerans [Teb], Paenibacillus illinoisensis [Hex], and Rhodococcus equi [Hex]. The findings further explain how these herbicides, which have potential for illicit coca (Erythroxylum sp.) control, dissipate following their application to tropical soils.

  7. An improved method of mitochondrial DNA isolation for XL-PCR

    Institute of Scientific and Technical Information of China (English)

    SHI Duo; ZHU Ke-jun; WANG Xue-min; WANG Zhen-cheng; ZHENG Jian-ming; MIAO Ming-yong; JIAO Bing-hua

    2006-01-01

    Objective: To obtain high quality of mitochondrial DNA (mtDNA) and carry out extra-long PCR (XL-PCR). Methods: Mitochondria were isolated by differential centrifugation, and membranes were disrupted using 10%SDS (pH 7.0). mtDNA was then extracted using phenol and chloroform. Results: The mtDNA obtained by using our improved method can be used as effective template for XL-PCR,and total mtDNA (16 kb) can be amplified easily. Conclusion: Our improved method is effective in preparing high quality of mtDNA, which can be used as template for XL-PCR.

  8. Cloning of cDNA Encoding GRA1 Protein of Tachyzoite Toxoplasma Gondii Local Isolate

    Directory of Open Access Journals (Sweden)

    Erma Sulistyaningsih

    2015-10-01

    Full Text Available Gene encoding GRA1 protein is potent DNA-vaccine candidate against toxoplasmosis. The aim of the researchwas to clone the gene encoding GRA1 protein of tachyzoite Toxoplasma gondii local isolate by DNA recombinanttechnology. Tachyzoite was grown in Balb/c mice in vivo. Messenger RNA was isolated from total RNA and itwas used to synthesis cDNA. Complementary DNA encoding GRA1 protein of tachyzoite Toxoplasma gondii localisolate was amplified and cloned in a prokaryote cloning vector. The recombinant GRA1-encoding gene was thendigesting using EcoRI restriction endonuclease and sequencing. The result showed that the recombinant GRA1-encoding gene consisted of DNA sequences encoding all signal peptide and mature peptide of GRA1 protein.Alignment of recombinant GRA1 sequence to gene encoding GRA1 protein of Toxoplasma gondii RH isolate showed100% homologous.Keywords: GRA1 protein, Toxoplasma gondii, tachyzoite, cloning, cDNA

  9. [Isolation and identification of a bacterial strain JS018 capable of degrading several kinds of organophosphate pesticides].

    Science.gov (United States)

    Jiang, Yu-Ji; Deng, You-Jin; Liu, Xin-Rui; Xie, Bao-Gui; Hu, Fang-Ping

    2006-06-01

    Organophosphate pesticides are used widely all over the world and play an important role in plant pest control. However these pesticides are considered as pollutants and harmful to human health. To search for microorganisms that can degrade organophosphate pesticides with high efficiency, a bacterial strain, coded as JS018, was isolated and screened from the soil in the vicinity of Shanming Pesticides Factory, Shanming, Fujian. Laboratory tests showed that the bacterium could degrade several kinds of organophosphate pesticides, such as Parathion-methyl and phoxin. The strain's degrading rates on phoxin, Parathion-methyl, hostathion and dichlorvos in LB liquid fermentation medium for 36 h were 99%, 96%, 80.4% and 69.0% respectively. The bacterial colonies on LB plate appeared shiny and pale-pink in color. The bacteria were Gram-negative coccoids, 0.5 - 0.7 microm in diameter. They grew well at 30 - 38 degrees C and pH 7.0 - 9.0. The optimal temperature and pH for cell growth was 32 degrees C and pH 7.5 - 8.0, respectively. They did not grow in medium containing 6% or more NaCl. The antibiotic susceptibility tests showed that the strain was resistant to ampicillin, penicillin and lincomycin. It was sensitive to kanamycin, tetracycline and gentamicin. Laboratory tests also showed that the strain could ferment D-glucose, trehalose, melezitose and ethanol. It was negative in the production of indole and hydrogen sulfide. It could not liquefy gelatin, utilize citrate, nor ferment L-arabinose, sucrose, D-mannitol, D-xylose, fructose, D-galactose, maltose or lactose. The catalase, urease and nitrate reduction were positive. Based on its morphological, physiological and biochemical properties as well as the 16S rDNA sequence analysis result, the strain was tentatively identified as Roseomonas sp.

  10. ISOLATION AND CHARACTERIZATION OF BIFENTHRIN CATABOLIZING BACTERIAL STRAIN BACILLUS CIBI FROM SOIL FOR PYRETHROIDS BIODEGRADATION

    Directory of Open Access Journals (Sweden)

    Preeti Pandey

    2014-01-01

    Full Text Available Pyrethroids are commonly used in most parts of the world and are reported to have potential health risks. Bifenthrin, a third generation pyrethroid used as insecticide has caused potential effect on aquatic life and human health. Bioremediation is a practical approach to reduce pesticide in the environment and reports of microbial degradation of bifenthrin are meagre. This study was aimed at isolating and characterizing bacterial isolates for the efficient removal of bifenthrin residues in the environment. A bacterial strain PGS-4 isolated from sewage of pesticide industry was tested for growth at higher concentration of bifenthrin (800 mg L-1 and the optimum pH and temperature were determined. The strain utilized bifenthrin as sole carbon source for growth over a wide range of pH (4.0-9.0 and temperatures (16-37°C. On the basis of growth kinetics studies, the optimal conditions were determined to be pH 7.0-8.0 and 30°C. 16S rRNA gene sequence analysis showed that strain PGS-4 forms a distinct phylogenetic lineage within the evolutionary radiation encompassed by the genus Bacillus and showed 99% similarity to that of Bacillus cibi. This study depicts the ability of B. cibi to utilize bifenthrin at higher concentration under in vitro thereby can be used in eliminating bifenthrin from contaminated soils as a practical approach to reduce pyrethroid toxicity in the environment.

  11. Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench).

    Science.gov (United States)

    Maropola, Mapula Kgomotso Annah; Ramond, Jean-Baptiste; Trindade, Marla

    2015-05-01

    Culture-independent studies rely on the quantity and quality of the extracted environmental metagenomic DNA (mDNA). To fully access the plant tissue microbiome, the extracted plant mDNA should allow optimal PCR applications and the genetic content must be representative of the total microbial diversity. In this study, we evaluated the endophytic bacterial diversity retrieved using different mDNA extraction procedures. Metagenomic DNA from sorghum (Sorghum bicolor L. Moench) stem and root tissues were extracted using two classical DNA extraction protocols (CTAB- and SDS-based) and five commercial kits. The mDNA yields and quality as well as the reproducibility were compared. 16S rRNA gene terminal restriction fragment length polymorphism (t-RFLP) was used to assess the impact on endophytic bacterial community structures observed. Generally, the classical protocols obtained high mDNA yields from sorghum tissues; however, they were less reproducible than the commercial kits. Commercial kits retrieved higher quality mDNA, but with lower endophytic bacterial diversities compared to classical protocols. The SDS-based protocol enabled access to the highest sorghum endophytic diversities. Therefore, "SDS-extracted" sorghum root and stem microbiome diversities were analysed via 454 pyrosequencing, and this revealed that the two tissues harbour significantly different endophytic communities. Nevertheless, both communities are dominated by agriculturally important genera such as Microbacterium, Agrobacterium, Sphingobacterium, Herbaspirillum, Erwinia, Pseudomonas and Stenotrophomonas; which have previously been shown to play a role in plant growth promotion. This study shows that DNA extraction protocols introduce biases in culture-independent studies of environmental microbial communities by influencing the mDNA quality, which impacts the microbial diversity analyses and evaluation. Using the broad-spectrum SDS-based DNA extraction protocol allows the recovery of the most

  12. Large Preferred Region for Packaging of Bacterial DNA by phiC725A, a Novel Pseudomonas aeruginosa F116-Like Bacteriophage

    Science.gov (United States)

    Pourcel, Christine; Midoux, Cédric; Hauck, Yolande; Vergnaud, Gilles; Latino, Libera

    2017-01-01

    Bacteriophage vB_PaeP_PAO1_phiC725A (short name phiC725A) was isolated following mitomycin C induction of C7-25, a clinical Pseudomonas aeruginosa strain carrying phiC725A as a prophage. The phiC725A genome sequence shows similarity to F116, a P. aeruginosa podovirus capable of generalized transduction. Likewise, phiC725A is a podovirus with long tail fibers. PhiC725A was able to lysogenize two additional P. aeruginosa strains in which it was maintained both as a prophage and in an episomal state. Investigation by deep sequencing showed that bacterial DNA carried inside phage particles originated predominantly from a 700-800kb region, immediately flanking the attL prophage insertion site, whether the phages were induced from a lysogen or recovered after infection. This indicates that during productive replication, recombination of phage genomes with the bacterial chromosome at the att site occurs occasionally, allowing packaging of adjacent bacterial DNA. PMID:28060939

  13. Bioremediation potential of a highly mercury resistant bacterial strain Sphingobium SA2 isolated from contaminated soil.

    Science.gov (United States)

    Mahbub, Khandaker Rayhan; Krishnan, Kannan; Megharaj, Mallavarapu; Naidu, Ravi

    2016-02-01

    A mercury resistant bacterial strain, SA2, was isolated from soil contaminated with mercury. The 16S rRNA gene sequence of this isolate showed 99% sequence similarity to the genera Sphingobium and Sphingomonas of α-proteobacteria group. However, the isolate formed a distinct phyletic line with the genus Sphingobium suggesting the strain belongs to Sphingobium sp. Toxicity studies indicated resistance to high levels of mercury with estimated EC50 values 4.5 mg L(-1) and 44.15 mg L(-1) and MIC values 5.1 mg L(-1) and 48.48 mg L(-1) in minimal and rich media, respectively. The strain SA2 was able to volatilize mercury by producing mercuric reductase enzyme which makes it potential candidate for remediating mercury. ICP-QQQ-MS analysis of Hg supplemented culture solutions confirmed that almost 79% mercury in the culture suspension was volatilized in 6 h. A very small amount of mercury was observed to accumulate in cell pellets which was also evident according to ESEM-EDX analysis. The mercuric reductase gene merA was amplified and sequenced. The deduced amino acid sequence demonstrated sequence homology with α-proteobacteria and Ascomycota group.

  14. Characterization of boron tolerant bacteria isolated from a fly ash dumping site for bacterial boron remediation.

    Science.gov (United States)

    Edward Raja, Chellaiah; Omine, Kiyoshi

    2013-08-01

    Boron is an essential micronutrient for plants, but can above certain concentrations be toxic to living organisms. A major environmental concern is the removal of boron from contaminated water and fly ash. For this purpose, the samples were collected from a fly ash dumping site, Nagasaki prefecture, Japan. The chemical characteristics and heavy metal concentration of the samples were performed by X-ray fluorescent analysis and leaching test. For bacterial analysis, samples were collected in sterile plastic sheets and isolation was carried out by serial dilution method. The boron tolerant isolates that showed values of maximum inhibitory concentration toward boron ranging from 100 to 260 mM level were screened. Based on 16S rRNA sequencing and phylogenetic analysis, the isolates were most closely related to the genera Bacillus, Lysinibacillus, Microbacterium and Ralstonia. The boron tolerance of these strains was also associated with resistant to several heavy metals, such as As (III), Cr (VI), Cd, Cu, Pb, Ni, Se (III) and Zn. Indeed, these strains were arsenic oxidizing bacteria confirmed by silver nitrate test. These strains exhibited their salt resistances ranging from 4 to 15 % were determined in Trypticase soy agar medium. The boron tolerant strains were capable of removing 0.1-2.0 and 2.7-3.7 mg l(-1) boron from the medium and fly ash at 168 h. Thus, we have successfully identified the boron tolerant and removal bacteria from a fly ash dumping site for boron remediation.

  15. Disinfectant and antibiotic activities: a comparative analysis in Brazilian hospital bacterial isolates

    Directory of Open Access Journals (Sweden)

    Guimarães Márcia Aparecida

    2000-01-01

    Full Text Available Nosocomial infections are an important cause of morbidity and mortality all over the world. It has been shown that appropriate environmental hygienic and disinfection practices can be very helpful to hospital infection control. The purpose of this study was to evaluate the bactericidal activity of some disinfectants against antibiotic-susceptible and antibiotic-resistant hospital bacterial isolates. The susceptibility of 27 clinical isolates to disinfectants and antibiotics was determined by the Association of Official Analytical Chemist?s (AOAC Use-Dilution method and by the Kirby-Bauer method, respectively. All strains tested were susceptible to sodium hypochlorite, glutaraldehyde and to the association quaternary ammonium - formaldehyde - ethyl alcohol disinfectants. However, the susceptibility of strains to phenol and to one quaternary ammonium compound was variable. Among twenty-one antibiotic-multiresistant strains (methicillin-resistant staphylococci, Enterococcus spp, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens and Escherichia coli eleven (52% and eight (38% strains were resistant to the quaternary ammonium and phenol compounds, respectively. Among six isolates that demonstrated susceptibility to antibiotics (staphylococci, Enterococcus spp, P. mirabilis, E. cloacae and E. coli two strains (33% showed resistance to these disinfectants. The results demonstrated the lack of correlation between antibiotic-susceptibility and susceptibility to disinfectants in hospital strains.

  16. Antimicrobial Activity of Monoramnholipids Produced by Bacterial Strains Isolated from the Ross Sea (Antarctica

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    Pietro Tedesco

    2016-04-01

    Full Text Available Microorganisms living in extreme environments represent a huge reservoir of novel antimicrobial compounds and possibly of novel chemical families. Antarctica is one of the most extraordinary places on Earth and exhibits many distinctive features. Antarctic microorganisms are well known producers of valuable secondary metabolites. Specifically, several Antarctic strains have been reported to inhibit opportunistic human pathogens strains belonging to Burkholderia cepacia complex (Bcc. Herein, we applied a biodiscovery pipeline for the identification of anti-Bcc compounds. Antarctic sub-sea sediments were collected from the Ross Sea, and used to isolate 25 microorganisms, which were phylogenetically affiliated to three bacterial genera (Psychrobacter, Arthrobacter, and Pseudomonas via sequencing and analysis of 16S rRNA genes. They were then subjected to a primary cell-based screening to determine their bioactivity against Bcc strains. Positive isolates were used to produce crude extracts from microbial spent culture media, to perform the secondary screening. Strain Pseudomonas BNT1 was then selected for bioassay-guided purification employing SPE and HPLC. Finally, LC-MS and NMR structurally resolved the purified bioactive compounds. With this strategy, we achieved the isolation of three rhamnolipids, two of which were new, endowed with high (MIC < 1 μg/mL and unreported antimicrobial activity against Bcc strains.

  17. Antimicrobial Activity of Monoramnholipids Produced by Bacterial Strains Isolated from the Ross Sea (Antarctica) †

    Science.gov (United States)

    Tedesco, Pietro; Maida, Isabel; Palma Esposito, Fortunato; Tortorella, Emiliana; Subko, Karolina; Ezeofor, Chidinma Christiana; Zhang, Ying; Tabudravu, Jioji; Jaspars, Marcel; Fani, Renato; de Pascale, Donatella

    2016-01-01

    Microorganisms living in extreme environments represent a huge reservoir of novel antimicrobial compounds and possibly of novel chemical families. Antarctica is one of the most extraordinary places on Earth and exhibits many distinctive features. Antarctic microorganisms are well known producers of valuable secondary metabolites. Specifically, several Antarctic strains have been reported to inhibit opportunistic human pathogens strains belonging to Burkholderia cepacia complex (Bcc). Herein, we applied a biodiscovery pipeline for the identification of anti-Bcc compounds. Antarctic sub-sea sediments were collected from the Ross Sea, and used to isolate 25 microorganisms, which were phylogenetically affiliated to three bacterial genera (Psychrobacter, Arthrobacter, and Pseudomonas) via sequencing and analysis of 16S rRNA genes. They were then subjected to a primary cell-based screening to determine their bioactivity against Bcc strains. Positive isolates were used to produce crude extracts from microbial spent culture media, to perform the secondary screening. Strain Pseudomonas BNT1 was then selected for bioassay-guided purification employing SPE and HPLC. Finally, LC-MS and NMR structurally resolved the purified bioactive compounds. With this strategy, we achieved the isolation of three rhamnolipids, two of which were new, endowed with high (MIC < 1 μg/mL) and unreported antimicrobial activity against Bcc strains. PMID:27128927

  18. Isolation and characteristics analysis of a novel high bacterial cellulose producing strain Gluconacetobacter intermedius CIs26.

    Science.gov (United States)

    Yang, Ying; Jia, Jingjing; Xing, Jianrong; Chen, Jianbing; Lu, Shengmin

    2013-02-15

    A strain producing bacterial cellulose (BC) screened from rotten mandarin fruit was identified as Gluconacetobacter intermedius CIs26 by the examination of general taxonomical characteristics and 16S rDNA sequence analysis. Furthermore, Fourier transform infrared (FT-IR) spectrum showed that pellicle produced by strain CIs26 was composed of glucan, and had the same functional group as a typical BC. X-ray diffractometry (XRD) analysis indicated that the BC was type I in structure with crystallinity index of 75%. BC yields of strain CIs26 in Hestrin-Schramn (HS), citrus waste modified HS (CMHS) and citrus waste solution (CWS) mediums were 2.1 g/L, 5.7 g/L, and 7.2 g/L, respectively. It was shown that citrus waste could stimulate BC production of strain CIs26 efficiently. Based on the ability of utilization of citrus waste, this strain appeared to have potential in BC manufacture on an industrial scale.

  19. The use of nano-sized acicular material, sliding friction, and antisense DNA oligonucleotides to silence bacterial genes

    OpenAIRE

    2014-01-01

    Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons. We have used nano-sized acicular material to establish a novel method for bacterial transformation. Here, we generated penetrons that carried antisense DNA adsorbed on nano-sized acicular material (α-sepiolite) by prov...

  20. Accurate episomal HIV 2-LTR circles quantification using optimized DNA isolation and droplet digital PCR

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    Eva Malatinkova

    2014-11-01

    Full Text Available Introduction: In HIV-infected patients on combination antiretroviral therapy (cART, the detection of episomal HIV 2-LTR circles is a potential marker for ongoing viral replication. Quantification of 2-LTR circles is based on quantitative PCR or more recently on digital PCR assessment, but is hampered due to its low abundance. Sample pre-PCR processing is a critical step for 2-LTR circles quantification, which has not yet been sufficiently evaluated in patient derived samples. Materials and Methods: We compared two sample processing procedures to more accurately quantify 2-LTR circles using droplet digital PCR (ddPCR. Episomal HIV 2-LTR circles were either isolated by genomic DNA isolation or by a modified plasmid DNA isolation, to separate the small episomal circular DNA from chromosomal DNA. This was performed in a dilution series of HIV-infected cells and HIV-1 infected patient derived samples (n=59. Samples for the plasmid DNA isolation method were spiked with an internal control plasmid. Results: Genomic DNA isolation enables robust 2-LTR circles quantification. However, in the lower ranges of detection, PCR inhibition caused by high genomic DNA load substantially limits the amount of sample input and this impacts sensitivity and accuracy. Moreover, total genomic DNA isolation resulted in a lower recovery of 2-LTR templates per isolate, further reducing its sensitivity. The modified plasmid DNA isolation with a spiked reference for normalization was more accurate in these low ranges compared to genomic DNA isolation. A linear correlation of both methods was observed in the dilution series (R2=0.974 and in the patient derived samples with 2-LTR numbers above 10 copies per million peripheral blood mononuclear cells (PBMCs, (R2=0.671. Furthermore, Bland–Altman analysis revealed an average agreement between the methods within the 27 samples in which 2-LTR circles were detectable with both methods (bias: 0.3875±1.2657 log10. Conclusions: 2-LTR

  1. Isolation and sequence analysis of the wheat B genome subtelomeric DNA

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    Huneau Cecile

    2009-09-01

    Full Text Available Abstract Background Telomeric and subtelomeric regions are essential for genome stability and regular chromosome replication. In this work, we have characterized the wheat BAC (bacterial artificial chromosome clones containing Spelt1 and Spelt52 sequences, which belong to the subtelomeric repeats of the B/G genomes of wheats and Aegilops species from the section Sitopsis. Results The BAC library from Triticum aestivum cv. Renan was screened using Spelt1 and Spelt52 as probes. Nine positive clones were isolated; of them, clone 2050O8 was localized mainly to the distal parts of wheat chromosomes by in situ hybridization. The distribution of the other clones indicated the presence of different types of repetitive sequences in BACs. Use of different approaches allowed us to prove that seven of the nine isolated clones belonged to the subtelomeric chromosomal regions. Clone 2050O8 was sequenced and its sequence of 119 737 bp was annotated. It is composed of 33% transposable elements (TEs, 8.2% Spelt52 (namely, the subfamily Spelt52.2 and five non-TE-related genes. DNA transposons are predominant, making up 24.6% of the entire BAC clone, whereas retroelements account for 8.4% of the clone length. The full-length CACTA transposon Caspar covers 11 666 bp, encoding a transposase and CTG-2 proteins, and this transposon accounts for 40% of the DNA transposons. The in situ hybridization data for 2050O8 derived subclones in combination with the BLAST search against wheat mapped ESTs (expressed sequence tags suggest that clone 2050O8 is located in the terminal bin 4BL-10 (0.95-1.0. Additionally, four of the predicted 2050O8 genes showed significant homology to four putative orthologous rice genes in the distal part of rice chromosome 3S and confirm the synteny to wheat 4BL. Conclusion Satellite DNA sequences from the subtelomeric regions of diploid wheat progenitor can be used for selecting the BAC clones from the corresponding regions of hexaploid wheat

  2. Biogenic amine producing capability of bacterial populations isolated during processing of different types of dry fermented sausages

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    M. Vincenzini

    2010-04-01

    Full Text Available In order to assess the distribution of the biogenic amine (BA producing capability within the bacterial populations occurring during production of dry fermented sausages, four different types of sausage processing, three with the use of starter cultures and one without, were investigated. All the main bacterial populations involved in the sausage processing showed a diffuse and strain dependent capability to produce BAs. However, quantitative determination of individual BAs produced by the bacterial isolates suggests an important role of enterococci in the accumulation of tyramine, the most abundant biogenic amine found in all investigated sausages.

  3. Isolation and Characterization of Gut Bacterial Proteases Involved in Inducing Pathogenicity of Bacillus thuringiensis Toxin in Cotton Bollworm, Helicoverpa armigera

    Science.gov (United States)

    Regode, Visweshwar; Kuruba, Sreeramulu; Mohammad, Akbar S.; Sharma, Hari C.

    2016-01-01

    Bacillus thuringiensis toxin proteins are deployed in transgenic plants for pest management. The present studies were aimed at characterization of gut bacterial proteases involved in activation of inactive Cry1Ac protoxin (pro-Cry1Ac) to active toxin in Helicoverpa armigera. Bacterial strains were isolated from H. armigera midgut and screened for their proteolytic activation toward pro-Cry1Ac. Among 12 gut bacterial isolates seven isolates showed proteolytic activity, and proteases from three isolates (IVS1, IVS2, and IVS3) were found to be involved in the proteolytic conversion of pro-Cry1Ac into active toxin. The proteases from IVS1, IVS2, and IVS3 isolates were purified to 11.90-, 15.50-, and 17.20-fold, respectively. The optimum pH and temperature for gut bacterial protease activity was 8.0 and 40°C. Maximum inhibition of total proteolytic activity was exerted by phenylmethane sulfonyl fluoride followed by EDTA. Fluorescence zymography revealed that proteases from IVS1, IVS2, and IVS3 were chymotrypsin-like and showing protease band at ~15, 65, and 15 kDa, respectively. Active Cry1Ac formed from processing pro-Cry1Ac by gut bacterial proteases exhibited toxicity toward H. armigera. The gut bacterial isolates IVS1, IVS2, and IVS3 showed homology with B. thuringiensis (CP003763.1), Vibrio fischeri (CP000020.2), and Escherichia coli (CP011342.1), respectively. Proteases produced by midgut bacteria are involved in proteolytic processing of B. thuringiensis protoxin and play a major role in inducing pathogenicity of B. thuringiensis toxins in H. armigera. PMID:27766093

  4. Isolation and characterization of gut bacterial proteases involved in inducing pathogenicity of Bacillus thuringiensis toxin in cotton bollworm, Helicoverpa armigera

    Directory of Open Access Journals (Sweden)

    Visweshwar Regode

    2016-10-01

    Full Text Available Bacillus thuringiensis (Bt toxin proteins are deployed in transgenic plants for pest management. The present studies were aimed at characterization of gut bacterial proteases involved in activation of inactive Cry1Ac protoxin (pro-Cry1Ac to active toxin in Helicoverpa armigera. Bacterial strains were isolated from H. armigera midgut and screened for their proteolytic activation towards pro-Cry1Ac. Among twelve gut bacterial isolates seven isolates showed proteolytic activity, and proteases from three isolates (IVS1, IVS2 and IVS3 were found to be involved in the proteolytic conversion of pro-Cry1Ac into active toxin. The proteases from IVS1, IVS2 and IVS3 isolates were purified to 11.90-, 15.50- and 17.20-fold, respectively. The optimum pH and temperature for gut bacterial protease activity was 8.0 and 40 oC. Maximum inhibition of total proteolytic activity was exerted by PMSF followed by EDTA. Fluorescence zymography revealed that proteases from IVS1, IVS2, and IVS3 were chymotrypsin-like and showing protease band at ~15, 65 and 15 kDa, respectively. Active Cry1Ac formed from processing pro-Cry1Ac by gut bacterial proteases exhibited toxicity towards H. armigera. The gut bacterial isolates IVS1, IVS2 and IVS3 showed homology with Bacillus thuringiensis (CP003763.1, Vibrio fischeri (CP000020.2 and Escherichia coli (CP011342.1, respectively. Proteases produced by midgut bacteria are involved in proteolytic processing of Bt protoxin and play a major role in inducing pathogenicity of Bt toxins in H. armigera.

  5. Antibacterial Activity of the Isolation Ethyl Acetate-Soluble Extract Noni Fruit (Morindra citrifolia L.) against Meat Bacterial Decay

    Science.gov (United States)

    Nugraheni, E. R.; Nurrakhman, M. B. E.; Munawaroh, H.; Saputri, L.

    2017-02-01

    Noni (Morindra citrifolia L.) is native to Indonesia which have medicinal properties. One of them as an antibacterial. This study aims to determine the antibacterial activity of isolates from the ethanol extract noni fruit to bacterial decay meat is Bacillus licheniformis, Klebsiella pneumonia, Bacillus alvei, Acinetobacter calcoaceticus, and Staphylococcus saprophyticus. The extraction process using the maceration method, and then made a partition by centrifugation ethyl acetate. Soluble part partition showed bacterial growth inhibition activity of the strong to very strong. Furthermore, the ethyl acetate soluble partition on preparative thin layer chromatography produced 5 isolates. Isolates obtained antibacterial activity test performed with a concentration of 20% and 30%. The results of antibacterial test against bacteria test isolates, showing isolates A can not inhibit the growth of bacteria, isolates B and C have medium activity and strong, isolates D and E isolates have activity against bacteria that were tested. MIC and MBC test results showed that the isolates B gives an inhibitory effect (bacteriostatic) against all bacteria. Content analysis of compounds by TLC using the reagents cerium (IV) sulfate indicates a phenol group. Isolates B contains a major compound which can be used as an antibacterial candidate in food preservation replace chemical preservatives.

  6. Evaluation of DNA microarray for detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis isolates

    Institute of Scientific and Technical Information of China (English)

    王峰

    2013-01-01

    Objective To evaluate the performance of DNA microarray for rapid detection resistance to rifampin and isoniazid in Mycobacterium tuberculosis clinical isolates and identify suitable target sites for molecular genetic test. Methods Twenty-four clinical Mycobacterium

  7. Isolation and characterization of a bacterial strain that efficiently degrades sex steroid hormones

    Institute of Scientific and Technical Information of China (English)

    JI Shulan; LIU Zhipei; LIU Zhipeng; REN Haiyan

    2007-01-01

    A bacterial strain,ZY3,growing on sex steroid hormones as the sole source of carbon and energy was isolated from the sewage treatment plant of a prophylactic steroids factory.ZY3 degrades the 3-methoxy-17β-hyclroxy-1,3,5(10),8(9)-δ-4-estren (MHE).This strain was preliminarily identified as Raoultella sp.ZY3 according to its morphology and its 16S rRNA gene sequence.During the experimental period (72 h),the optimum temperature,pH and 3-MHE concentration for the degradation of hydride by the strain ZY3 were 35℃,10 and 10 mg/L,respectively.The degradation rate of the sex steroid hormones increased to 87% and 85% after the addition of maltose and peptone,respectively.

  8. Comparative Antibacterial Efficacy of Vitellaria paradoxa (Shea Butter Tree Extracts Against Some Clinical Bacterial Isolates

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    Kamoldeen Abiodun AJIJOLAKEWU

    2015-09-01

    Full Text Available The antibacterial activities of the ethanolic extracts of seed, leaf and stem bark of Vitellaria paradoxa were investigated. The extracts were tested against three clinical bacterial pathogens, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae using the agar diffusion and the broth dilution techniques. Ethanolic extracts of the plant parts showed activity against all the bacterial pathogens tested. At the highest extract concentration (200 mg/ml, the leaf extract exhibited the highest antimicrobial activity, while no activity was detected at the lowest concentration (3.13 mg/ml against the tested isolates. Escherichia coli and Staphylococcus aureus were more susceptible to all extracts of V. paradoxa, while Klebsiella pneumoniae showed the least sensitivity. The efficacy of ethanolic extracts of Vitellaria paradoxa was compared to a commercial antibiotic streptomycin. There were differences in the minimum inhibitory concentration (MIC of all the Vitellaria paradoxa ethanolic extracts with respect to the type of organism. All extracts exhibited bacteriostatic effects against the tested organisms at the experimented concentrations. Qualitative phytochemical screening of the extracts revealed the presence of saponins, tannins and alkaloids as the active principles of Vitellaria paradoxa's antimicrobial activity. V. paradoxa could be used as a potential source of antibiotic substance for a drug development.

  9. Bacterial Agents Andantibiogram of Most Common Isolated Organisms from Hands of Surgical Team Members after Scrubbing

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    PS Mohseni- Meybodi

    2008-04-01

    Full Text Available Introduction: Many post-surgical wound infections in hospitals cause morbidity and morality of patients and these are usually transmitted via hands of surgical personnel. The aim of the present study was to detect and antibiogram the bacterial agents following scrubbing of hands of surgical personnel before operation. Methods: Hands of 134 personnels of operation room were swabbed following scrubbing with antiseptic Betadine solution. Swab samples were inoculated on selective and differential media such as blood ager, McConky and manitol salt agar(MSA. Following incubation of media at 37c° for 24hr, bacterial species were identified using differential related tests. The isolated species were than antibiogramed and the results together with other data was analysed by SPSS software program. Results: Of the total of 134 cases, 81(60.4% were male and 53(39.6% female. The mean scrub time for each person was (206.1+/-103.2 seconds; 6 to 60 seconds base change. Increasing time of scrub was significantly correlated with decreasing rate of bacteria (P=0.003, (R=-0.254. Contamination was present in 129(96.3% cases following scrubbing. Maximum contamination was observed in nails (92.5%. Average number of bacteria for each individual was between 0 and 159. 62.6% of isolated bacteria were non- staphylococci and 7.7% were S. aureus. Vancomycin and ceftizoxim were the most sensitive, while penicillin was the least sensitive antibiotic. Conclusion: Results revealed that hand contamination was more than the expected standard level. Therefore, regarding the critical task of surgical personnel, training of all operation room staff is highly recommended to minimize the rate of contamination.

  10. NKLP27: a teleost NK-lysin peptide that modulates immune response, induces degradation of bacterial DNA, and inhibits bacterial and viral infection.

    Science.gov (United States)

    Zhang, Min; Li, Mo-fei; Sun, Li

    2014-01-01

    NK-lysin is an antimicrobial protein produced by cytotoxic T lymphocytes and natural killer cells. In this study, we examined the biological property of a peptide, NKLP27, derived from tongue sole (Cynoglossus semilaevis) NK-lysin. NKLP27 is composed of 27 amino acids and shares little sequence identity with known NK-lysin peptides. NKLP27 possesses bactericidal activity against both Gram-negative and Gram-positive bacteria including common aquaculture pathogens. The bactericidal activity of NKLP27 was dependent on the C-terminal five residues, deletion of which dramatically reduced the activity of NKLP27. During its interaction with the target bacterial cells, NKLP27 destroyed cell membrane integrity, penetrated into the cytoplasm, and induced degradation of genomic DNA. In vivo study showed that administration of tongue sole with NKLP27 before bacterial and viral infection significantly reduced pathogen dissemination and replication in tissues. Further study revealed that fish administered with NKLP27 exhibited significantly upregulated expression of the immune genes including those that are known to be involved in antibacterial and antiviral defense. These results indicate that NKLP27 is a novel antimicrobial against bacterial and viral pathogens, and that the observed effect of NKLP27 on bacterial DNA and host gene expression adds new insights to the action mechanism of fish antimicrobial peptides.

  11. NKLP27: a teleost NK-lysin peptide that modulates immune response, induces degradation of bacterial DNA, and inhibits bacterial and viral infection.

    Directory of Open Access Journals (Sweden)

    Min Zhang

    Full Text Available NK-lysin is an antimicrobial protein produced by cytotoxic T lymphocytes and natural killer cells. In this study, we examined the biological property of a peptide, NKLP27, derived from tongue sole (Cynoglossus semilaevis NK-lysin. NKLP27 is composed of 27 amino acids and shares little sequence identity with known NK-lysin peptides. NKLP27 possesses bactericidal activity against both Gram-negative and Gram-positive bacteria including common aquaculture pathogens. The bactericidal activity of NKLP27 was dependent on the C-terminal five residues, deletion of which dramatically reduced the activity of NKLP27. During its interaction with the target bacterial cells, NKLP27 destroyed cell membrane integrity, penetrated into the cytoplasm, and induced degradation of genomic DNA. In vivo study showed that administration of tongue sole with NKLP27 before bacterial and viral infection significantly reduced pathogen dissemination and replication in tissues. Further study revealed that fish administered with NKLP27 exhibited significantly upregulated expression of the immune genes including those that are known to be involved in antibacterial and antiviral defense. These results indicate that NKLP27 is a novel antimicrobial against bacterial and viral pathogens, and that the observed effect of NKLP27 on bacterial DNA and host gene expression adds new insights to the action mechanism of fish antimicrobial peptides.

  12. Influence of DNA isolation method on the investigation of archaeal diversity and abundance in biogas plants.

    Science.gov (United States)

    Theiss, Juliane; Rother, Michael; Röske, Kerstin

    2016-09-01

    Various methods are available for DNA isolation from environmental samples. Because the chemical and biological composition of samples such as soil, sludge, or plant material is different, the effectiveness of DNA isolation can vary depending on the method applied and thus, have a substantial effect on the results of downstream analysis of the microbial community. Although the process of biogas formation is being intensely investigated, a systematic evaluation of kits for DNA isolation from material of biogas plants is still lacking. Since no DNA isolation kit specifically tailored for DNA isolation from sludge of biogas plants is available, this study compares five commercially available kits regarding their influence on downstream analyses such denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qPCR). The results show that not all kits are equally suited for the DNA isolation from samples of different biogas plants, but highly reproducible DGGE fingerprints as well as qPCR results across the tested samples from biogas reactors using different substrate compositions could be produced using selected kits.

  13. DNA barcoding and isolation of vertically transmitted ascomycetes in sorghum from Burkina Faso

    DEFF Research Database (Denmark)

    Stokholm, Michaela Schiller; Wulff, Ednar G.; Zida, Elisabeth P.;

    2016-01-01

    -day-old seedlings was analyzed by 18S ribosomal DNA (rDNA) amplicon sequencing. More than 99% of the fungal rDNA was found to originate from ascomycetes. The distribution of ascomycetes at species level was subsequently analyzed by barcoding of ITS2 rDNA. Eighteen Operational Taxonomic Units (OTUs) were identified......Molecular identification of fungal taxa commonly transmitted through seeds of sorghum in Western Africa is lacking. In the present study, farm-saved seeds, collected from four villages in Northern Burkina Faso, were surface sterilized and the distribution of fungal DNA in seeds and seven...... samples collected in Central Burkina Faso confirming a common occurrence. E. sorghinum was highly predominant in seedlings both measured by DNA analysis and by isolation. The dominance of E. sorghinum was particularly strong in roots from poorly growing seedlings. Pathogenicity of E. sorghinum isolates...

  14. Isolation of high molecular weight DNA suitable for the construction of genomic libraries.

    Science.gov (United States)

    Steven, J; McKechnie, D; Graham, A

    1988-01-01

    Recent advances in molecular biology have made it possible to construct complete gene libraries for any organism that uses DNA as its carrier of genetic information. A gene library should contain a large number of cloned DNA fragments that in total contain the entire donor genome. The construction of a genomic library first requires the isolation of DNA from the donor organism. To be of maximum use in the construction of genomic libraries, DNA isolated from the donor organism should fulfill the following criteria. First, the DNA must represent all sequences in the genome to be cloned. Second, it must be of high molecular weight. Third, no contaminants must taint the DNA so that its use as a substrate for restriction endonucleases and other enzymes used in genetic engineering is uninhibited.

  15. A comparative study of two methods for the isolation of human leucocytes for DNA extraction.

    Science.gov (United States)

    Lim, L H; Ton, S H; Cheong, S K

    1990-06-01

    The 'Dextran' and the 'Buffy-coat' methods for isolation of human leucocytes for DNA extraction were compared on the basis of DNA yield from the same amounts (10 ml) of blood. Human leucocytes from a total of 11 samples were isolated using both methods for each sample after which DNA was extracted. Extracted DNA samples were treated with ribonucleases and proteinase K after which the yields were quantitated by measuring absorbance at 260 nm. The 'Buffy-coat' method yielded a mean concentration of DNA of 476.7 micrograms/ml (range: 212 to 700 micrograms/ml) while the 'Dextran' method yielded 188.4 micrograms/ml (range: 64 to 340 micrograms/ml). The difference was confirmed by subjecting the extracted DNA samples to agarose gel electrophoresis.

  16. Immobilization of microalgae cells in alginate facilitates isolation of DNA and RNA.

    Science.gov (United States)

    Lopez, Blanca R; Hernandez, Juan-Pablo; Bashan, Yoav; de-Bashan, Luz E

    2017-04-01

    Isolation of nucleic acids from Chlorella is difficult, given the chemically complex nature of their cell walls and variable production of metabolites. Immobilization of microalgae in polymers adds additional difficulty. Here, we modified, amended, and standardized methods for isolation of nucleic acids and compared the yield of DNA and RNA from free-living and encapsulated microalgae C. sorokiniana. Isolation of nucleic acids from immobilized cells required two steps in dissolving the alginate matrix, releasing the cells, and mechanical disruption with glass beads. For DNA extraction, we used modified versions of a commercial kit along with the hexadecyltrimethylammonium bromide (CTAB) method. For RNA extraction, we used the commercial TRI reagent procedure and the CTAB-dithiotreitol method. Quantity and quality of nucleic acids in extracts varied with growth conditions, isolation procedures, and time of incubation of the original culture. There were consistently higher amounts of DNA and RNA in extracts from immobilized cells. Quantitatively, the modified procedure with the commercial Promega kit was the most reliable procedure for isolating DNA and a modified commercial TRI reagent procedure was the choice for isolating RNA. All four procedures eliminated proteins efficiently and had low levels of contamination from residual polysaccharides from the matrices and/or metabolites naturally produced by the microalgae. All DNA extracts under both growth conditions, time of incubation, and two isolation methods successfully amplified the 18S ribosomal RNA by PCR and quantitative reverse transcription (RT-qPCR).

  17. Application of real-time PCR to postharvest physiology – DNA isolation

    Science.gov (United States)

    Real-time PCR technology has been widely used in the postharvest plant physiology research. One of the difficulties to isolate DNA from plant martial and pathogen cells is the presence of rigid polysaccharide cell walls and capsules, which physically protect DNA from cell lysis. Many materials requi...

  18. Isolation and study of two mutants of Streptomyces cattleya affected in DNA repair and genetic instability.

    Science.gov (United States)

    Hromic, A; Kirby, R

    1989-01-15

    Two mutants of Streptomyces cattleya affecting DNA repair were isolated. These mutants were analysed using spore survival curves and phage reactivation curves in the presence and absence of caffeine and arsenite. Two DNA repair systems (uvr1 and uvr2) were identified, the latter of which seems to influence genetic instability.

  19. Nε-lysine acetylation of a bacterial transcription factor inhibits Its DNA-binding activity.

    Directory of Open Access Journals (Sweden)

    Sandy Thao

    Full Text Available Evidence suggesting that eukaryotes and archaea use reversible N(ε-lysine (N(ε-Lys acetylation to modulate gene expression has been reported, but evidence for bacterial use of N(ε-Lys acetylation for this purpose is lacking. Here, we report data in support of the notion that bacteria can control gene expression by modulating the acetylation state of transcription factors (TFs. We screened the E. coli proteome for substrates of the bacterial Gcn5-like protein acetyltransferase (Pat. Pat acetylated four TFs, including the RcsB global regulatory protein, which controls cell division, and capsule and flagellum biosynthesis in many bacteria. Pat acetylated residue Lys180 of RcsB, and the NAD(+-dependent Sir2 (sirtuin-like protein deacetylase (CobB deacetylated acetylated RcsB (RcsB(Ac, demonstrating that N(ε-Lys acetylation of RcsB is reversible. Analysis of RcsB(Ac and variant RcsB proteins carrying substitutions at Lys180 provided biochemical and physiological evidence implicating Lys180 as a critical residue for RcsB DNA-binding activity. These findings further the likelihood that reversible N(ε-Lys acetylation of transcription factors is a mode of regulation of gene expression used by all cells.

  20. Serotypes and DNA fingerprint profiles of Pasteurella multocida isolated from raptors

    Science.gov (United States)

    Wilson, M.A.; Duncan, R.M.; Nordholm, G.E.; Berlowski, B.M.

    1995-01-01

    Pasteurella multocida isolates from 21 raptors were examined by DNA fingerprint profile and serotyping methods. Isolates were obtained from noncaptive birds of prey found in 11 states from November 28, 1979, through February 10, 1993. Nine isolates were from bald eagles, and the remaining isolates were from hawks, falcons, and owls. Seven isolates were members of capsule group A, and 14 were nonencapsulated. One isolate was identified as somatic type 3, and another was type 3,4,7; both had unique HhaI DNA fingerprint profiles. Nineteen isolates expressed somatic type 1 antigen; HhaI profiles of all type 1 isolates were identical to each other and to the HhaI profile of the reference somatic type 1, strain X-73. The 19 type 1 isolates were differentiated by sequential digestion of DNA with HpaII; four HpaII fingerprint profiles were obtained. The HpaII profile of one isolate was identical to the HpaII profile of strain X-73. Incidence of P. multocida somatic type 1 in raptors suggests that this type may be prevalent in other wildlife or wildlife environments.

  1. Uranium isotopic fractionation factors during U(VI) reduction by bacterial isolates

    Science.gov (United States)

    Basu, Anirban; Sanford, Robert A.; Johnson, Thomas M.; Lundstrom, Craig C.; Löffler, Frank E.

    2014-07-01

    We experimentally determined the magnitude of uranium isotopic fractionation induced by U(VI) reduction by metal reducing bacterial isolates. Our results indicate that microbial U(VI) reduction induces isotopic fractionation; heavier isotopes (i.e., 238U) partition into the solid U(IV) products. The magnitudes of isotopic fractionation (expressed as ε = 1000‰ * (α-1)) for 238U/235U were 0.68‰ ± 0.05‰ and 0.99‰ ± 0.12‰ for Geobacter sulfurreducens strain PCA and strain IFRC-N, respectively. The ε values for Anaeromyxobacter dehalogenans strain FRC-W, strain FRC-R5, a novel Shewanella isolate, and Desulfitobacterium sp. strain Viet1 were 0.72‰ ± 0.15‰, 0.99‰ ± 0.12‰, 0.96‰ ± 0.16‰ and 0.86‰ ± 0.06‰, respectively. Our results show that the maximum ε values of ∼1.0‰ were obtained with low biomass (∼107 cells/mL) and low electron donor concentrations (∼500 μM). These results provide an initial assessment of 238U/235U shifts induced by microbially-mediated U(VI) reduction, which is needed as 238U/235U data are increasingly applied as redox indicators in various geochemical settings.

  2. Isolation of bacterial strains from bovine fecal microflora capable of degradation of ceftiofur.

    Science.gov (United States)

    Rafii, Fatemeh; Williams, Anna J; Park, Miseon; Sims, Lillie M; Heinze, Thomas M; Cerniglia, Carl E; Sutherland, John B

    2009-10-20

    Ceftiofur, a third-generation cephalosporin used to treat bacterial infections in animals, is degraded in bovine feces but the specific bacteria involved are unknown. To find the bacteria involved in ceftiofur metabolism, the bovine fecal microflora was screened. Twenty-one nonidentical strains of bovine fecal bacteria were isolated on media containing 1-32 microg ml(-1) of ceftiofur. The cultures were incubated with 5 microg ml(-1) ceftiofur for different times, then centrifuged and analyzed by high-performance liquid chromatography. Three strains of Bacillus spp., two strains of Roseomonas spp., and one strain of Azospirillum sp. metabolized 5 microg ml(-1) ceftiofur in broth cultures in less than 24h; ten other strains of Roseomonas and one strain of Bacillus pumilus had metabolized it by 120 h. After the ceftiofur had been metabolized by these bacteria, the filter-sterilized supernatants of centrifuged cultures no longer inhibited the growth of a ceftiofur-sensitive strain of Kocuria rhizophila, which indicated that ceftiofur had been transformed to compounds without bactericidal activity. Each isolate was also found to be able to grow in the presence of other beta-lactams, and a nitrocefin assay showed beta-lactamase activity in the 17 strains that metabolized ceftiofur. The results show that some beta-lactamase-producing bacteria from the bovine fecal microflora are capable of transforming ceftiofur to metabolites lacking bactericidal activity.

  3. Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood.

    Directory of Open Access Journals (Sweden)

    Alexander Tolios

    Full Text Available Telomeres are located at chromosome ends and their length (TL has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated with and without actinomycin D to induce apoptosis as a prototype of sample degradation. DNA was isolated from fresh blood pools and after freezing at -80°C. Commercially available kits using beads (Invitrogen, spin columns (Qiagen, Macherey-Nagel and 5prime or precipitation (Stratec/Invisorb and a published isopropanol precipitation protocol (IPP were used for DNA isolation. TL was assessed by qPCR, and normalized to the single copy reference gene 36B4 using two established single-plex and a new multiplex protocol. We show that the method of DNA isolation significantly affected TL (e.g. 1.86-fold longer TL when comparing IPP vs. Invitrogen. Sample degradation led to an average TL decrease of 22% when using all except for one DNA isolation method (5prime. Preanalytical storage conditions did not affect TL with exception of samples that were isolated with the 5prime kit, where a 27% increase in TL was observed after freezing. Finally, performance of the multiplex qPCR protocol was comparable to the single-plex assays, but showed superior time- and cost-effectiveness and required > 80% less DNA. Findings of the current study highlight the need for standardization of whole blood processing and DNA isolation in clinical study settings to avoid preanalytical bias of TL quantification and show that multiplex assays may improve TL/SCG measurements.

  4. Discrimination of Arcobacter butzleri isolates by polymerase chain reaction-mediated DNA fingerprinting

    DEFF Research Database (Denmark)

    Atabay, H. I.; Bang, Dang Duong; Aydin, F.;

    2002-01-01

    Aims: The objective of this study was to subtype Arcobacter butzleri isolates using RAPD-PCR. Methods and Results: Thirty-five A. butzleri isolates obtained from chicken carcasses were examined. PCR-mediated DNA fingerprinting technique with primers of the variable sequence motifs was used...

  5. Isolation of chromatin DNA tightly bound to the nuclear envelope of HeLa cells.

    Science.gov (United States)

    Kuvichkin, Vasily Vladimirovich

    2012-11-01

    Recent discovery of the role of nuclear pores in transcription, predicted by our early DNA-membrane complex (DMC) model, makes membrane-bound DNA (MBD) isolation from the cell nucleus and analysis of the MBD actual. The method of MBD isolation proposed by us retains DMC integrity during isolation. We used HeLa cells for DMC extraction. Changing the ionic composition of the isolation medium and replacing DNase I, used commonly for chromatin destruction, with a set of restriction enzymes allowed us to isolate the MBD. Treatment of a nuclear membrane with proteinase K and ultrasound has been used to increase the yield of MBD. Electron microscopic analysis of the purified fraction of isolated DMC supports our previous model of nuclear envelope lipid-chromatin interaction in the nuclear pore assembly.

  6. Transgenic Rice Plants Harboring Genomic DNA from Zizania latifolia Confer Bacterial Blight Resistance

    Institute of Scientific and Technical Information of China (English)

    SHEN Wei-wei; SONG Cheng-li; CHEN Jie; Fu Ya-ping; Wu Jian-li; JIANG Shao-mei

    2011-01-01

    Based on the sequence of a resistance gene analog FZ14 derived from Zizania latifolia (Griseb.),a pair of specific PCR primers FZ14P1/FZ14P2 was designed to isolate candidate disease resistance gene.The pooled-PCR approach was adopted using the primer pair to screen a genomic transformation-competent artificial chromosome (TAC) library derived from Z.latifolia.A positive TAC clone (ZR1) was obtained and confirmed by sequence analysis.The results indicated that ZR1 consisted of conserved motifs similar to P-loop (kinase 1a),kinase 2,kinase 3a and GLPL (Gly-Leu-Pro-Leu),suggesting that it could be a portion of NBS-LRR type of resistance gene.Using Agrobacterium-mediated transformation of Nipponbare mature embryo,a total of 48 independent transgenic T0 plants were obtained.Among them,36 plants were highly resistant to the virulent bacterial blight strain P×O71.The results indicate that ZR1 contains at least one functional bacterial blight resistance gene.

  7. Antibacterial and antifouling activities of chitosan/TiO2/Ag NPs nanocomposite films against packaged drinking water bacterial isolates.

    Science.gov (United States)

    Natarajan, Saravanan; Bhuvaneshwari, M; Lakshmi, D Shanthana; Mrudula, P; Chandrasekaran, N; Mukherjee, Amitava

    2016-10-01

    TiO2 and Ag NPs are widely used as antibacterial agents against many bacterial pathogens. Chitosan (polymer) itself acts as a strong antibacterial agent. Hence, chitosan/TiO2/Ag NPs incorporated nanocomposite film was prepared against packed drinking water bacterial strains. A concentration-dependent increase in the reduction of cell viability was observed in all the isolates under UV-C and dark exposure conditions. The bacteria consortium showed greater resistance against antibacterial effects of chitosan/TiO2/Ag nanocomposite as compared to single isolates. Glycocalyx test and mass assessment conclude the effective antibacterial activity by inhibiting bacterial adhesion on the film surface. The release of LDH and generation of ROS act as the predominant antibacterial mechanism induced by TiO2/Ag NPs. Surface characterization of chitosan/TiO2/Ag nanocomposite was studied by FTIR and XRD analyses and SEM analysis after interaction with the bacteria.

  8. ‘Olegusella massiliensis’ strain KHD7, a new bacterial genus isolated from the female genital tract

    Directory of Open Access Journals (Sweden)

    K. Diop

    2016-07-01

    Full Text Available We report the main characteristics of ‘Olegusella massiliensis’ gen. nov., sp. nov., strain KHD7 (= CSUR P2268=DSM 101849, a new member of the Coriobacteriaceae family isolated from the vaginal flora of a patient with bacterial vaginosis.

  9. A simple and rapid method for extracting bacterial DNA from intestinal microflora for ERIC-PCR detection

    Institute of Scientific and Technical Information of China (English)

    Jin-Long Yang; Ming-Shu Wang; An-Chun Cheng; Kang-Cheng Pan; Chuan-Feng Li; Shu-Xuan Deng

    2008-01-01

    AIM: To develop a simple and convenient method for extracting genomic DNA from intestinal microflora for enterobacterial repetitive intergenic consensus (ERIC)-PCR detection.METHODS: Five methods of extracting bacterial DNA,including Tris-EDTA buffer, chelex-100, ultrapure water,2% sodium dodecyl sulfate and 10% Triton-100 with and without sonication, were compared with the commercial fecal DNA extraction kit method, which is considered as the gold standard for DNA extraction. The comparison was based on the yield and purity of DNA and the indexes of the structure and property of micro-organisms that were reflected by ERIC-PCR.RESULTS: The yield and purity of DNA obtained by the chelex method was similar to that obtained with the fecal DNA kit. The ERIC-PCR results obtained for the DNA extracted by the chelex method and those obtained for DNA extracted with the fecal DNA kit were basically the same.CONCLUSION: The chelex method is recommended for ERIC-PCR experiments in view of its simplicity and costeffectiveness; and it is suitable for extracting total DNA from intestinal micro-organisms, particularly for handling a large number of samples.

  10. A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples

    Science.gov (United States)

    The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in th...

  11. Bacterial Mitosis: ParM of Plasmid R1 Moves Plasmid DNA by an Actin-like Insertional Polymerization Mechanism

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette

    2003-01-01

    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system...

  12. Bacterial antigen expression is an important component in inducing an immune response to orally administered Salmonella-delivered DNA vaccines.

    Directory of Open Access Journals (Sweden)

    Michelle E Gahan

    Full Text Available BACKGROUND: The use of Salmonella to deliver heterologous antigens from DNA vaccines is a well-accepted extension of the success of oral Salmonella vaccines in animal models. Attenuated S. typhimurium and S. typhi strains are safe and efficacious, and their use to deliver DNA vaccines combines the advantages of both vaccine approaches, while complementing the limitations of each technology. An important aspect of the basic biology of the Salmonella/DNA vaccine platform is the relative contributions of prokaryotic and eukaryotic expression in production of the vaccine antigen. Gene expression in DNA vaccines is commonly under the control of the eukaryotic cytomegalovirus (CMV promoter. The aim of this study was to identify and disable putative bacterial promoters within the CMV promoter and evaluate the immunogenicity of the resulting DNA vaccine delivered orally by S. typhimurium. METHODOLOGY/PRINCIPAL FINDINGS: The results reported here clearly demonstrate the presence of bacterial promoters within the CMV promoter. These promoters have homology to the bacterial consensus sequence and functional activity. To disable prokaryotic expression from the CMV promoter a series of genetic manipulations were performed to remove the two major bacterial promoters and add a bacteria transcription terminator downstream of the CMV promoter. S. typhimurium was used to immunise BALB/c mice orally with a DNA vaccine encoding the C-fragment of tetanus toxin (TT under control of the original or the modified CMV promoter. Although both promoters functioned equally well in eukaryotic cells, as indicated by equivalent immune responses following intramuscular delivery, only the original CMV promoter was able to induce an anti-TT specific response following oral delivery by S. typhimurium. CONCLUSIONS: These findings suggest that prokaryotic expression of the antigen and co-delivery of this protein by Salmonella are at least partially responsible for the successful

  13. Intracloacal inoculation, an effective screening method for determining the efficacy of probiotic bacterial isolates against Campylobacter colonization in broiler chickens.

    Science.gov (United States)

    Arsi, K; Donoghue, A M; Woo-Ming, A; Blore, P J; Donoghue, D J

    2015-01-01

    Campylobacter is a leading cause of foodborne illness worldwide. It is common in poultry, and human infections are often associated with consumption of contaminated poultry products. One strategy to reduce Campylobacter colonization in poultry is the use of oral probiotics, but this produces variable results, possibly because the probiotics are destroyed in the stomach's acidic environment. Protection (e.g., encapsulation) of isolates may overcome this problem, but there is no assurance that these isolates will have efficacy in the lower gastrointestinal tract. Therefore, screening candidate isolates by directly placing them in the lower intestinal tract via cloacal inoculation may eliminate the time and expense of encapsulating ineffective isolates. Thus, the purpose of this study was to collect bacterial isolates with anti-Campylobacter activity in vitro and evaluate their efficacy in vivo upon either oral or intracloacal administration. Bacterial isolates were collected from healthy birds and were evaluated for efficacy against C. jejuni in vitro. Isolates having generally regarded as safe status and demonstrating in vitro anti-Campylobacter properties were evaluated after oral or intracloacal inoculation into chicks on day 1 (n = 10 birds per isolate per route of administration). On day 7, birds were dosed by oral gavage with a four-strain mixture of wild-type Campylobacter containing at least 1 × 10(7) CFU/ml organisms. On day 14, birds were euthanized and the ceca were collected aseptically for Campylobacter enumeration. When dosed orally, only one isolate had a 1-log reduction in cecal Campylobacter counts, whereas when administered intracloacally, six of these isolates produced a 1- to 3-log reduction in cecal Campylobacter counts in 14-day-old chickens. These results support the strategy of evaluating the efficacy of potential probiotic isolates via cloacal inoculation prior to undergoing the effort of encapsulating isolates for oral administration.

  14. Stability of randomly amplified polymorphic DNA fingerprinting in genotyping clinical isolates of Helicobacter pylori

    Institute of Scientific and Technical Information of China (English)

    Feng-Chan Han; Han-Chong Ng; Bow Ho

    2003-01-01

    AIM: Hpylorigenomes are highly diversified. This project was designed to genotype Hpyloriisolates by the polymerase chain reaction (PCR)-based randomly amplified polymorphic DNA (RAPD) fingerprinting technique and to verify its stability by Southern blotting and DNA sequencing.METHODS: Clinical isolates of Hpyloriwere cultured from gastric antra and cardia of 73 individuals, and genomic DNA was prepared for each isolate. RAPD was carried out under optimized conditions. 23S rDNA was regarded as an internal control, and a 361 bp rDNA fragment (RDF) was used as a probe to screen the RAPD products by Southern blotting.Ten RDFs from different clinical isolates and the flanking regions (both upstream and downstream) of four RDFs were amplified and sequenced.RESULTS: Hpyloriisolates from different individuals had different RAPD profiles, but the profiles for isolates cultured from different gastric sites of a given individual were identical in all but one case. Isolates from 27 individuals were RDF positive by Southern blotting. Sequences of the RDFs and their flanking regions were almost the same between the RDF positive and negative isolates as determined by Southern blotting. There was no binding site for random PCR primer inside the sequences.CONCLUSION: RAPD is very useful in genotyping H pylori grossly on a large scale. However, it seems unstable in amplification of low yield fragments, especially those that do not appear as visible bands on the agarose gel stained with EB, since the palmer is partially matched to the template.

  15. Amplification of pico-scale DNA mediated by bacterial carrier DNA for small-cell-number transcription factor ChIP-seq

    DEFF Research Database (Denmark)

    Jakobsen, Janus S; Bagger, Frederik O; Hasemann, Marie S;

    2015-01-01

    BACKGROUND: Chromatin-Immunoprecipitation coupled with deep sequencing (ChIP-seq) is used to map transcription factor occupancy and generate epigenetic profiles genome-wide. The requirement of nano-scale ChIP DNA for generation of sequencing libraries has impeded ChIP-seq on in vivo tissues of low...... transcription factor (CEBPA) and histone mark (H3K4me3) ChIP. We further demonstrate that genomic profiles are highly resilient to changes in carrier DNA to ChIP DNA ratios. CONCLUSIONS: This represents a significant advance compared to existing technologies, which involve either complex steps of pre...... cell numbers. RESULTS: We describe a robust, simple and scalable methodology for ChIP-seq of low-abundant cell populations, verified down to 10,000 cells. By employing non-mammalian genome mapping bacterial carrier DNA during amplification, we reliably amplify down to 50 pg of ChIP DNA from...

  16. Nucleotide sequence and infectious cDNA clone of the L1 isolate of Pea seed-borne mosaic potyvirus.

    Science.gov (United States)

    Olsen, B S; Johansen, I E

    2001-01-01

    The complete nucleotide sequence of Pea seed-borne mosaic potyvirus isolate L1 has been determined from cloned virus cDNA. The PSbMV L1 genome is 9895 nucleotides in length excluding the poly(A) tail. Computer analysis of the sequence revealed a single long open reading frame (ORF) of 9594 nucleotides. The ORF potentially encodes a polyprotein of 3198 amino acids with a deduced Mr of 363537. Nine putative proteolytic cleavage sites were identified by analogy to consensus sequences and genome arrangement in other potyviruses. Two full-length cDNA clones, p35S-L1-4 and p35S-L1-5, were assembled under control of an enhanced 35S promoter and nopaline synthase terminator. Clone p35S-L1-4 was constructed with four introns and p35S-L1-5 with five introns inserted in the cDNA. Clone p35S-L1-4 was unstable in Escherichia coli often resulting in amplification of plasmids with deletions. Clone p35S-L1-5 was stable and apparently less toxic to Escherichia coli resulting in larger bacterial colonies and higher plasmid yield. Both clones were infectious upon mechanical inoculation of plasmid DNA on susceptible pea cultivars Fjord, Scout, and Brutus. Eight pea genotypes resistant to L1 virus were also resistant to the cDNA derived L1 virus. Both native PSbMV L1 and the cDNA derived virus infected Chenopodium quinoa systemically giving rise to characteristic necrotic lesions on uninoculated leaves.

  17. The use of nano-sized acicular material, sliding friction, and antisense DNA oligonucleotides to silence bacterial genes.

    Science.gov (United States)

    Mitsudome, Yuya; Takahama, Mamiko; Hirose, Jun; Yoshida, Naoto

    2014-01-01

    Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons. We have used nano-sized acicular material to establish a novel method for bacterial transformation. Here, we generated penetrons that carried antisense DNA adsorbed on nano-sized acicular material (α-sepiolite) by providing sliding friction onto the surface of agar gel; we then investigated whether penetron formation was applicable to gene silencing techniques. Antisense DNA was artificially synthesized as 15 or 90mer DNA oligonucleotides based on the sequences around the translation start codon of target mRNAs. Mixtures of bacterial cells with antisense DNA adsorbed on α-sepiolite were stimulated by sliding friction on the surface of agar gel for 60 s. Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively. The numbers of ampicillin resistant colonies and the β-galactosidase activity derived from penetrons bearing antisense DNA (90mer) was repressed to 15% and 25%, respectively, of that of control penetrons which lacked antisense DNA. Biphenyl metabolite, ring cleavage yellow compound produced by Pseudomonas pseudoalcaligenes penetron treated with antisense oligonucleotide DNA targeted to bphD increased higher than that lacking antisense DNA. This result indicated that expression of bphD in P. pseudoalcaligenes penetrons was repressed by antisense DNA that targeted bphD mRNA. Sporulation rates of Bacillus subtilis penetrons treated with antisense DNA (15mer) targeted to spo0A decreased to 24.4% relative to penetrons lacking antisense DNA. This novel method of gene silencing has substantial promise for

  18. Isolation of 24 novel cDNA fragments from microdis—sected human chromosome band

    Institute of Scientific and Technical Information of China (English)

    ZHANGMIN; LONGYU; 等

    1998-01-01

    The strategy of isolating the band0specific expression fragments from a probe pool generated by human chromosome microdissection was reported.A chromosome 14q 24.3 band-specific single copy DNA pool was constructed based on this probe pool.Using total DNA of the pool as probe to hybridize the human marrow cDNA library,68 primary positive clones were selected from 5×105 cDNA clones.Among these primary clones,32 secondary clones were obtained after second-round screening and designed as cFD14-1-32.Finally,24 band-specific expression fragments were identified from these 32 positive clones by DNA hybridization.Those band-specific clones can hybridize to both 14q24.3 DNA and human genomic DNA but cann't hybridize to 17q11-12 DNA,Partial sequences of 13 fragments of them were sequenced and idenfified as novel cDNA sequences,and these sequences were proved to have some homology with known genes in NCBI database.Analysis of expression spectrum of cFD 14-1 suggested that the cDNA fragments thus obtained should be used to isolate the genes can not been cloned in 14q24.3 region.

  19. Analysis of the bacterial diversity existing on animal hide and wool: development of a preliminary PCR-restriction fragment length polymorphism fingerprint database for identifying isolates.

    Science.gov (United States)

    Chen, Yu; Gao, Hongwei; Zhang, Yanming; Deng, Mingjun; Wu, Zhenxing; Zhu, Laihua; Duan, Qing; Xu, Biao; Liang, Chengzhu; Yue, Zhiqin; Xiao, Xizhi

    2012-01-01

    Twenty-one bacterial strains were isolated from imported cattle hide and rabbit wool using two types of media, nutrient broth, and nutrient broth with serum. The bacteria identified were Brevibacillus laterosporus, Leclercia adecarboxylata, Peptococcus niger, Bacillus circulans, Raoultella ornithinolytica, Bacillus subtilis, Bacillus cereus, Bacillus thermobacillus, Bacillus choshinensis, Bacillus sphaericus, Acinetobacter haemolyticus, Sphingomonas paucimobilis, Bacillus thuringiensis, Staphylococcus intermedius, Mycobacteria, Moraxella, Klebsiella pneumoniae, Ralstonia pickettii, Staphylococcus chromogenes, Comamonas testosteroni, and Cupriavidus pauculus. The 16s rDNA gene of each bacterium was amplified using the universal primers 27f and 1492r. The amplicons were digested with AvaI, BamHI, BgII, DraI, EcoRI, EcoRV, HindIII, HinfI, HpaI, PstI, SmaI, TaqII, XbaI, XmaI, AluI, XhoI, and PvuI individually. A specific fingerprint from the PCR-restriction fragment length polymorphism method based on 16s rDNA was obtained for each bacterium. The results showed that the method developed was useful not only for bacterial identification but also for the etiological investigation of pathogens in imported animal hair and wool.

  20. DNA from processed and unprocessed wood: factors influencing the isolation success.

    Science.gov (United States)

    Rachmayanti, Yanti; Leinemann, Ludger; Gailing, Oliver; Finkeldey, Reiner

    2009-06-01

    Molecular genetic markers have numerous potential applications in environmental forensics if DNA can be isolated from 'difficult' non-human biological material such as hairs, feathers, or wood. The identification of the origin of wood is particularly important in order to identify illegally harvested and traded timber and wood products. We describe success rates of DNA isolation from wood based on a simple, previously published extraction protocol. The protocol was used to isolate DNA from a total of 406 wood samples, mainly of the important tropical tree family Dipterocarpaceae. The reliability of the extraction method was confirmed by comparing fragment sizes and sequences after isolation of DNA from leaves and wood of the same trees. We observed the success of amplification of chloroplast DNA (cpDNA) fragments of different lengths by means of PCR, investigated key factors influencing PCR, and conducted inhibitor tests for a subset of the samples. The average rate of successful PCR amplification was 75.7%. Main factors influencing the success of PCR amplification were the size of the amplified fragment and the processing status of the wood. Short fragments and unprocessed wood resulted in higher success rates. The success rate was also dependent on the age (storage duration) of the wood probe and on the investigated species. Amplification success was higher if DNA was isolated from outer sapwood (without cambium) in comparison to DNA isolated from the transition zone between sapwood and heartwood and the inner heartwood. However, inhibitor tests also indicated more PCR inhibitory substances in the outer sapwood in comparison to transition wood and heartwood. The addition of polyvinylpyrolidone (PVP) to the lysis buffer proved to be highly efficient to improve the amplification success if inhibitory substances were present.

  1. Isolation and Characterization of a Thermophilic Oil-Degrading Bacterial Consortium

    Institute of Scientific and Technical Information of China (English)

    Gu Guizhou; Li Zheng; Zhao Dongfeng; Zhao Chaocheng

    2013-01-01

    In this study, a thermophilic oil-degrading bacterial consortium KO8-2 growing within the temperature range of 45-65℃(with 55℃being the optimum temperature) was isolated from oil-contaminated soil of Karamay in Xinjiang, China. Denaturing gradient gel electrophoresis (DGGE) showed that there were nine strains included in KO8-2, which originated from the genera of Bacillus, Geobacillus and Clostridium. They all belonged to thermophilic bacteria, and had been previously proved as degraders of at least one petroleum fraction. The crude oil degraded by KO8-2 was analyzed by infrared spectrophotometry, hydrocarbon group type analysis and gas chromatography. The results indicated that the bacterial consortium KO8-2 was able to utilize 64.33%of saturates, 27.06%of aromatics, 13.24%of resins and the oil removal efifciency reached up to 58.73%at 55℃when the oil concentration was 10 g/L. Detailed analysis showed that KO8-2 was able to utilize the hydrocarbon components before C19, and the n-alkanes ranging from C20-C33 were signiif-cantly degraded. The ratios of nC17/Pr and nC18/Ph were 3.12 and 3.87, respectively, before degradation, whereas after degradation the ratios reduced to 0.21 and 0.38, respectively. Compared with the control sample, the oil removal efifciency in KO8-2 composting reactor reached 50.12%after a degradation duration of 60 days.

  2. A one-step miniprep for the isolation of plasmid DNA and lambda phage particles.

    Directory of Open Access Journals (Sweden)

    George Lezin

    Full Text Available Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments E. coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.

  3. An economical and combined method for rapid and efficient isolation of fungal DNA.

    Science.gov (United States)

    Lech, T; Syguła-Cholewinska, J; Szostak-Kot, J

    2014-12-18

    DNA isolation is a crucial step of conducting genetic studies in any organism. However, this process is quite difficult when studying fungi because of the need to damage the fungal cell walls of specific structures. In this study, we developed a method for the rapid and efficient isolation of fungal DNA based on simultaneous mechanical and enzymatic cell wall degradation. There are several typical modifications of the standard phenol-chloroform DNA extraction method. This method can be modified to degrade the fungal cell wall. The first step of the presented DNA extraction included manual homogenization in modified lysis buffer. Next, enzymatic digestion using 2 enzymes was conducted, including lyticase and proteinase K. To carefully select the most favorable conditions, we developed an economical, rapid, and reliable method for fungal DNA extraction that ensures both high efficiency and proper purity, which are essential for further analyses.

  4. Human cultured cells are capable to incorporate isolated plant mitochondria loaded with exogenous DNA

    Directory of Open Access Journals (Sweden)

    Laktionov P. P.

    2012-07-01

    Full Text Available Aim. To investigate the possibility of human cultured cells to incorporate isolated mitochondria together with exogenous DNA introduced into organelles. Methods. Two approaches were used for this purpose, fluorescent labelling of mitochondria and/or DNA with subsequent analysis of the cells subjected to incubation by microscopy or by quantitative PCR. Results. We have shown that human cultured cells lines, HeLa and HUVEC, are capable to uptake isolated plant mitochondria and that this process depends on the incubation time and concentration of organelles present in medium. The incorporated mitochondria can serve as vehicles to deliver exogenous DNA into human cells, this DNA is then distributed in different cell compartments. Conclusions. These results are preliminary and need further investigations, including testing the possibility of human cells to incorporate the mitochondria of human or animal origin and creating genetic construction which could provide certain selectivity or stability of the transferred exogenous DNA upon cell uptake of the mitochondria as vectors.

  5. Differentiation of environmental aquatic bacterial isolates by MALDI-TOF MS.

    Science.gov (United States)

    Popović, Natalija Topić; Kazazić, Snježana P; Strunjak-Perović, Ivančica; Čož-Rakovac, Rozelindra

    2017-01-01

    Identification of bacteria in aquatic and environmental applications, for monitoring purposes and research, for health assessments and therapy considerations of farmed and free-living aquatic organisms, still relies on conventional phenotypic and biochemical protocols. Although molecular techniques based on DNA amplification and sequencing are finding ways into diagnostic laboratories, they are time-consuming, costly and difficult in the case of multiplex assays. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is a rapid and accurate proteomic method reliable for identification of unknown bacteria to the genus and species level. Upon extension of databases, it will certainly find its position in environmental sciences. The paper presents an overview of the principle of the method, its effectiveness in comparison with conventional and molecular identification procedures, and applicability on environmental and aquatic isolates, discussing its advantages and shortcomings, as well as possible future implementations.

  6. RNA- and single-stranded DNA-binding (SSB) proteins expressed during Drosophila melanogaster oogenesis: a homolog of bacterial and eukaryotic mitochondrial SSBs.

    Science.gov (United States)

    Stroumbakis, N D; Li, Z; Tolias, P P

    1994-06-10

    Little is known about the identity and involvement of single-stranded (ss) DNA-binding (SSB) and RNA-binding proteins in developmental processes that occur during oogenesis in Drosophila melanogaster (Dm). Here, we describe a molecular approach designed to identify such proteins by virtue of their ssDNA-binding activity. We have constructed a directional ovarian cDNA library and conducted expression cloning screens which identified five unique cDNAs that encode proteins capable of binding ssDNA. All five represent previously unreported sequences. The remainder of this paper focuses on one of these cDNAs which encodes a Dm protein displaying significant sequence homology to Escherichia coli ssDNA-binding protein (SSB, involved in DNA replication, repair and recombination), as well as eukaryotic SSBs isolated from the mitochondria (mt) of rats, frogs, humans and yeast. The deduced amino acid (aa) sequence of this 15.6-kDa protein, which we will refer to as Dm mtSSB, displays average identities of 38.3% with eukaryotic mtSSBs and 23.4% with bacterial SSBs. Gel retardation analysis with an affinity-purified GST fusion protein confirms that Dm mtSSB specifically binds ss, but not double stranded DNA. Dm mtSSB is encoded by a nuclear gene whose expression appears to be developmentally regulated. It is expressed as a single 600-nucleotide (nt) transcript during oogenesis and embryogenesis. A larger transcript of 1500 nt is prevalent in some later stages of Dm development.

  7. Frequency of bacterial isolates and pattern of antimicrobial resistance in patients with hematological malignancies: A snapshot from tertiary cancer center

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    M Sengar

    2015-01-01

    Full Text Available BACKGROUND: Infections are the most important cause of mortality in patients with high-risk febrile neutropenia. Emergence of multi-drug resistant organisms (MDROs has become a major challenge for hemato-oncologists. Knowledge of the prevalent organisms and their antimicrobial sensitivity can help deciding the empirical therapy at individual centers and allows timely measures to reduce the risk of antimicrobial resistance. AIMS: To evaluate the frequency of bacterial isolates from all the samples and the pattern of bacterial bloodstream infections and incidence of MDROs. SETTINGS AND DESIGN: This is a retrospective analysis from a tertiary care cancer center. MATERIALS AND METHODS: From January to June 2014 information on all the samples received in Department of Microbiology was collected retrospectively. The data from samples collected from patients with hematological cancers were analyzed for types of bacterial isolates and antimicrobial sensitivity. RESULTS: A total of 739 isolates were identified with 67.9% of isolates being Gram-negative. The predominant Gram-negative organisms were Escherichia coli, Psuedomonas spp. and Klebsiella spp. Among the bacterial bloodstream infections, 66% were Gram-negative isolates. MDROs constituted 22% of all isolates in blood cultures. Incidence of resistant Gram-positive organisms was low in the present dataset (methicillin resistant Staphylococcus aureus and vancomycin-resistant enterococci-1.3%. CONCLUSIONS: The analysis reconfirms the Gram-negative organisms as the predominant pathogens in bacteremia seen in patients with hematological cancers. The high frequency of multi-drug resistance in the dataset calls for the need of emergency measures to curtail further development and propagation of resistant organisms.

  8. Bacterial diversity of soil under eucalyptus assessed by 16S rDNA sequencing analysis Diversidade bacteriana de solo sob eucaliptos obtida por seqüenciamento do 16S rDNA

    Directory of Open Access Journals (Sweden)

    Érico Leandro da Silveira

    2006-10-01

    Full Text Available Studies on the impact of Eucalyptus spp. on Brazilian soils have focused on soil chemical properties and isolating interesting microbial organisms. Few studies have focused on microbial diversity and ecology in Brazil due to limited coverage of traditional cultivation and isolation methods. Molecular microbial ecology methods based on PCR amplified 16S rDNA have enriched the knowledge of soils microbial biodiversity. The objective of this work was to compare and estimate the bacterial diversity of sympatric communities within soils from two areas, a native forest (NFA and an eucalyptus arboretum (EAA. PCR primers, whose target soil metagenomic 16S rDNA were used to amplify soil DNA, were cloned using pGEM-T and sequenced to determine bacterial diversity. From the NFA soil 134 clones were analyzed, while 116 clones were analyzed from the EAA soil samples. The sequences were compared with those online at the GenBank. Phylogenetic analyses revealed differences between the soil types and high diversity in both communities. Soil from the Eucalyptus spp. arboretum was found to have a greater bacterial diversity than the soil investigated from the native forest area.Estudos sobre impacto do Eucalyptus spp. em solos brasileiros têm focalizado propriedades químicas do solo e isolamento de microrganismos de interesse. No Brasil há pouco enfoque em ecologia e diversidade microbiana, devido às limitações dos métodos tradicionais de cultivo e isolamento. A utilização de métodos moleculares no estudo da ecologia microbiana baseados na amplificação por PCR do 16S rDNA têm enriquecido o conhecimento da biodiversidade microbiana dos solos. O objetivo deste trabalho foi comparar e estimar a diversidade bacteriana de comunidades simpátricas em solos de duas áreas: uma floresta nativa (NFA e outra adjacente com arboreto de eucaliptos (EAA. Oligonucleotídeos iniciadores foram utilizados para amplificar o 16S rDNA metagenômico do solo, o qual foi

  9. Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs.

    OpenAIRE

    1988-01-01

    To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, we developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex...

  10. Cloning and Sequencing cDNA Encoding for Rhoptry-2 Toxoplasma Gondii Tachyzoite Local Isolate

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    Murwantoko M

    2015-11-01

    Full Text Available Rhoptry protein belongs to an excretory and secretory antigens (ESAs that play an important role during active penetration of parasite into the cell target. This protein an able Toxoplasma gondii to actively penetrate targeted cell, meanwhile ESAs protein stimulates intracellular vacuole modification. It is, therefore, after the parasite successfully enter the cell target then Granule (GRA proteins are responsible for the formation of parasitophorus vacuole, which is protect the fusion with other intracellular compartments such as lysosomal vacuole. Consequently, this parasite is being able to survive and multiply at the cell target. The current study was aimed to clone and sequens cDNA encoding for ROP-2 of local isolated T. gondii tachizoite through DNA recombinant technique. Total ribonucleic acid (RNA was isolated from tachyzoites of local isolated T. gondii that were grown up in Balb/c mice. Messenger RNA was isolated from total RNA using PolyAtract mRNA Isolation System. Messenger RNA was used as a template for synthesis cDNA using Riboclone cDNA Synthesis System AMV-RT. EcoRI adaptor from Riboclone EcoRI Adaptor Ligation System was added to Complementary DNA and than ligated to pUC19. Recombinant plasmid was transformed into E. coli (XL1-Blue. The transformed E. coli XL-1 Blue were plated on LB agar containing X-Gal, IPTG and ampicillin. Recombinant clones (white colony were picked up and grown up in the LB medium at 37oC overnight. Expression of recombinant protein was analysed by immunoblotting in order to identify cDNA recombinant wich is express ESA of T. gondii local isolate. Recombinant plasmid were isolated using alkalilysis method and were elektroforated in 1% agarose gel. The isolated DNA recombinant plasmid was cut using Eco RI and then sequenced through Big Dye Terminator Mix AB1 377A Sequencer using M13 Forward and M13 Reverse primers. The conclusion of this results showed that the recombinant clone was coding for excretory

  11. Isolation, characterization and phylogenetic analysis of a bacterial strain capable of degrading acetamiprid

    Institute of Scientific and Technical Information of China (English)

    YAO Xiao-hua; MIN Hang

    2006-01-01

    An aerobic bacterium, capable of degrading the new chloronicotine pesticide acetamiprid, was isolated from the sludge of pesticide factory after successive enrichment cultures and named strain FH2 which is a Gram-negative, rod-shaped, obligate aerobic organism with ((0.5-0.7) ×(1.5-3.0))μm of cell size and with monotrichous flagellum. It was identified as a member of Pseudomonas sp. based on morphology, physio-biochemical properties, Biolog GN2, 16S rDNA sequence and phylogenetic characteristic analysis. The isolate could grow optimally at pH 7.0 and 30℃ in acetamiprid-mineral medium with 800 mg/L concentration. About 53.3% acetamiprid was degraded by strain FH2 after incubation for 14 d in acetamiprid-mineral medium and nearly 96.7% degraded when incubated in acetamiprid-yeast mineral medium at 30℃ for 14 d. This paper describes phylogenetic and degradation characterization of a pure bacterium being able to mineralize acetamiprid for the first time.

  12. Genotyping of Single Nucleotide Polymorphisms in DNA Isolated from Serum Using Sequenom MassARRAY Technology.

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    Tess V Clendenen

    Full Text Available Large epidemiologic studies have the potential to make valuable contributions to the assessment of gene-environment interactions because they prospectively collected detailed exposure data. Some of these studies, however, have only serum or plasma samples as a low quantity source of DNA.We examined whether DNA isolated from serum can be used to reliably and accurately genotype single nucleotide polymorphisms (SNPs using Sequenom multiplex SNP genotyping technology. We genotyped 81 SNPs using samples from 158 participants in the NYU Women's Health Study. Each participant had DNA from serum and at least one paired DNA sample isolated from a high quality source of DNA, i.e. clots and/or cell precipitates, for comparison.We observed that 60 of the 81 SNPs (74% had high call frequencies (≥95% using DNA from serum, only slightly lower than the 85% of SNPs with high call frequencies in DNA from clots or cell precipitates. Of the 57 SNPs with high call frequencies for serum, clot, and cell precipitate DNA, 54 (95% had highly concordant (>98% genotype calls across all three sample types. High purity was not a critical factor to successful genotyping.Our results suggest that this multiplex SNP genotyping method can be used reliably on DNA from serum in large-scale epidemiologic studies.

  13. Fermentative hydrogen production from hydrolyzed cellulosic feedstock prepared with a thermophilic anaerobic bacterial isolate

    Energy Technology Data Exchange (ETDEWEB)

    Lo, Yung Chung [Department of Chemical Engineering, National Cheng Kung University, No. 1 University Road, Tainan 701 (China); Huang, Chi-Yu.; Fu, Tzu-Ning [Department of Environmental Engineering and Science, Tunghai University, Taichung 407 (China); Chen, Chun-Yen; Chang, Jo-Shu [Department of Chemical Engineering, National Cheng Kung University, No. 1 University Road, Tainan 701 (China); Sustainable Environment Research Center, National Cheng Kung University, Tainan (China)

    2009-08-15

    Hydrogen gas was produced via dark fermentation from natural cellulosic materials and {alpha}-cellulose via a two-step process, in which the cellulosic substrates were first hydrolyzed by an isolated cellulolytic bacterium Clostridium strain TCW1, and the resulting hydrolysates were then used as substrate for fermentative H{sub 2} production. The TCW1 strain was able to hydrolyze all the cellulosic materials examined to produce reducing sugars (RS), attaining the best reducing sugar production yield of 0.65 g reducing sugar/g substrate from hydrolysis of {alpha}-cellulose. The hydrolysates of those cellulosic materials were successfully converted to H{sub 2} via dark fermentation using seven H{sub 2}-producing bacterial isolates. The bioH{sub 2} production performance was highly dependent on the type of cellulosic feedstock used, the initial reducing sugar concentration (C{sub RS,o}) (ranging from 0.7 to 4.5 mg/l), as well as the composition of sugar and soluble metabolites present in the cellulosic hydrolysates. It was found that Clostridium butyricum CGS5 displayed the highest H{sub 2}-producing efficiency with a cumulative H{sub 2} production of 270 ml/l from {alpha}-cellulose hydrolysate (C{sub RS,o} = 4.52 mg/l) and a H{sub 2} yield of 7.40 mmol/g RS (or 6.66 mmol/g substrate) from napier grass hydrolysate (C{sub RS,o} = 1.22 g/l). (author)

  14. Isolation of plant DNA for PCR and genotyping using organic extraction and CTAB.

    Science.gov (United States)

    Springer, Nathan M

    2010-11-01

    A general difficulty in isolation of DNA from plant cells is the presence of a cell wall. It is necessary to degrade plant cell walls, either physically or enzymatically, in order to effectively isolate plant DNA. Additionally, some tissues (such as endosperm) or some species contain high levels of starches or phenolic compounds that can complicate DNA isolation. A number of plant DNA isolation protocols are designed to overcome species-specific difficulties. This is a relatively simple protocol that uses an extraction buffer containing cetyltrimethylammonium bromide (CTAB); it can be used for many plant species. It provides a substantial amount of high-quality DNA that is suitable for polymerase chain reaction (PCR) procedures and is stable for long periods of time. The cost per sample is very low. In addition, this protocol is relatively robust and can be performed by individuals who have had relatively little training. A typical undergraduate student can perform ~200-300 isolations in a day using this protocol. The disadvantages are that it requires a freeze-dryer and a mill or paint-shaker-like device and that it utilizes an organic extraction step, requiring the use of a fume hood.

  15. A rapid method for isolation of total DNA from pathogenic filamentous plant fungi.

    Science.gov (United States)

    González-Mendoza, D; Argumedo-Delira, R; Morales-Trejo, A; Pulido-Herrera, A; Cervantes-Díaz, L; Grimaldo-Juarez, O; Alarcón, A

    2010-02-02

    DNA isolation from some fungal organisms of agronomic importance is difficult because they have cell walls or capsules that are relatively unsusceptible to lysis. We have developed a fast DNA isolation protocol for Fusarium oxysporum, which causes fusarium wilt disease in more than 100 plant species, and for Pyrenochaeta terrestris, which causes pink root in onions. This protocol was based on the sodium dodecyl sulfate/phenol method, without beta-mercaptoethanol and without maceration in liquid nitrogen; it uses phenol/chloroform extraction to remove proteins and co-precipitated polysaccharides. The A(260/280) absorbance ratios of isolated DNA were around 1.9, suggesting that the DNA fraction was pure and may be used for further analysis. Additionally, the A(260/230) values were higher than 1.8, suggesting negligible contamination by polysaccharides. The DNA isolated by this protocol is of sufficient quality for molecular applications; this technique could be applied to other organisms that have similar substances that hinder DNA extraction.

  16. Investigating bacterial populations in styrene-degrading biofilters by 16S rDNA tag pyrosequencing.

    Science.gov (United States)

    Portune, Kevin J; Pérez, M Carmen; Álvarez-Hornos, F Javier; Gabaldón, Carmen

    2015-01-01

    Microbial biofilms are essential components in the elimination of pollutants within biofilters, yet still little is known regarding the complex relationships between microbial community structure and biodegradation function within these engineered ecosystems. To further explore this relationship, 16S rDNA tag pyrosequencing was applied to samples taken at four time points from a styrene-degrading biofilter undergoing variable operating conditions. Changes in microbial structure were observed between different stages of biofilter operation, and the level of styrene concentration was revealed to be a critical factor affecting these changes. Bacterial genera Azoarcus and Pseudomonas were among the dominant classified genera in the biofilter. Canonical correspondence analysis (CCA) and correlation analysis revealed that the genera Brevundimonas, Hydrogenophaga, and Achromobacter may play important roles in styrene degradation under increasing styrene concentrations. No significant correlations (P > 0.05) could be detected between biofilter operational/functional parameters and biodiversity measurements, although biological heterogeneity within biofilms and/or technical variability within pyrosequencing may have considerably affected these results. Percentages of selected bacterial taxonomic groups detected by fluorescence in situ hybridization (FISH) were compared to results from pyrosequencing in order to assess the effectiveness and limitations of each method for identifying each microbial taxon. Comparison of results revealed discrepancies between the two methods in the detected percentages of numerous taxonomic groups. Biases and technical limitations of both FISH and pyrosequencing, such as the binding of FISH probes to non-target microbial groups and lack of classification of sequences for defined taxonomic groups from pyrosequencing, may partially explain some differences between the two methods.

  17. Bacterial Contamination and Antibiotic Resistance of Staphylococcus Aureus Isolated from Automated Teller Machine

    Directory of Open Access Journals (Sweden)

    Moshtaghi, H. (PhD

    2015-05-01

    Full Text Available Background and Objective: Automated Teller Machine (ATMs is likely to be contaminated with various microorganisms specially pathogen germs. This may be due to their exposure to dust and their vast dermal contact with multiple users. This study investigated the bacterial contamination on the keyboard of ATMs and drug resistance of the bacteria isolated from them. Material and Methods: the keyboards of 50 ATMs in Shahrekord city, Iran, were examined from October 2012 to February 2013. The sterile swab sticks moistened with Triptose soy broth were used for sampling. The bacteriological tests used were culture, biochemical test and agar disk diffusion method for antibiogram. Results: All the samples were found to be contaminated with Coagulase negative staphylococci (57.54%, Bacillus species (21.92%, Staphylococcus aureus (19.18% and coliform bacteria (1.36%. The resistance of Staphylococcus aureus was 92.8% to penicillin, 85.7% to amoxicilin، 71.4% to ampicillin, 57.1% to nytrofuran, 50% to tetracycline, 42.8% to erythromycin, 42.8% to gentamycin, 14.2 % to ciprofloxacin, 7.1% to trimethoprim and sulfamtuksazul. All species were susceptible to, ofloxacine, chloramphenicol, clindamycin, tobramycin, vancomycin and cefotaxime. Conclusion: given the presence of pathogens on ATMs and their role in transferring the contamination, we recommend considering personal hygiene and periodically disinfecting the keyboards to reduce contamination

  18. Textile dye removal from wastewater effluents using bioflocculants produced by indigenous bacterial isolates.

    Science.gov (United States)

    Buthelezi, Simphiwe P; Olaniran, Ademola O; Pillay, Balakrishna

    2012-11-30

    Bioflocculant-producing bacteria were isolated from activated sludge of a wastewater treatment plant located in Durban, South Africa, and identified using standard biochemical tests as well as the analysis of their 16S rRNA gene sequences. The bioflocculants produced by these organisms were ethanol precipitated, purified using 2% (w/v) cetylpyridinium chloride solution and evaluated for removal of wastewater dyes under different pH, temperature and nutritional conditions. Bioflocculants from these indigenous bacteria were very effective for decolourizing the different dyes tested in this study, with a removal rate of up to 97.04%. The decolourization efficiency was largely influenced by the type of dye, pH, temperature, and flocculant concentration. A pH of 7 was found to be optimum for the removal of both whale and mediblue dyes, while the optimum pH for fawn and mixed dye removal was found to be between 9 and 10. Optimum temperature for whale and mediblue dye removal was 35 °C, and that for fawn and mixed dye varied between 40–45 °C and 35–40 °C, respectively. These bacterial bioflocculants may provide an economical and cleaner alternative to replace or supplement present treatment processes for the removal of dyes from wastewater effluents, since they are biodegradable and easily sustainable.

  19. Textile Dye Removal from Wastewater Effluents Using Bioflocculants Produced by Indigenous Bacterial Isolates

    Directory of Open Access Journals (Sweden)

    Balakrishna Pillay

    2012-11-01

    Full Text Available Bioflocculant-producing bacteria were isolated from activated sludge of a wastewater treatment plant located in Durban, South Africa, and identified using standard biochemical tests as well as the analysis of their 16S rRNA gene sequences. The bioflocculants produced by these organisms were ethanol precipitated, purified using 2% (w/v cetylpyridinium chloride solution and evaluated for removal of wastewater dyes under different pH, temperature and nutritional conditions. Bioflocculants from these indigenous bacteria were very effective for decolourizing the different dyes tested in this study, with a removal rate of up to 97.04%. The decolourization efficiency was largely influenced by the type of dye, pH, temperature, and flocculant concentration. A pH of 7 was found to be optimum for the removal of both whale and mediblue dyes, while the optimum pH for fawn and mixed dye removal was found to be between 9 and 10. Optimum temperature for whale and mediblue dye removal was 35 °C, and that for fawn and mixed dye varied between 40–45 °C and 35–40 °C, respectively. These bacterial bioflocculants may provide an economical and cleaner alternative to replace or supplement present treatment processes for the removal of dyes from wastewater effluents, since they are biodegradable and easily sustainable.

  20. Calcite biomineralization by bacterial isolates from the recently discovered pristine karstic herrenberg cave.

    Science.gov (United States)

    Rusznyák, Anna; Akob, Denise M; Nietzsche, Sándor; Eusterhues, Karin; Totsche, Kai Uwe; Neu, Thomas R; Frosch, Torsten; Popp, Jürgen; Keiner, Robert; Geletneky, Jörn; Katzschmann, Lutz; Schulze, Ernst-Detlef; Küsel, Kirsten

    2012-02-01

    Karstic caves represent one of the most important subterranean carbon storages on Earth and provide windows into the subsurface. The recent discovery of the Herrenberg Cave, Germany, gave us the opportunity to investigate the diversity and potential role of bacteria in carbonate mineral formation. Calcite was the only mineral observed by Raman spectroscopy to precipitate as stalactites from seepage water. Bacterial cells were found on the surface and interior of stalactites by confocal laser scanning microscopy. Proteobacteria dominated the microbial communities inhabiting stalactites, representing more than 70% of total 16S rRNA gene clones. Proteobacteria formed 22 to 34% of the detected communities in fluvial sediments, and a large fraction of these bacteria were also metabolically active. A total of 9 isolates, belonging to the genera Arthrobacter, Flavobacterium, Pseudomonas, Rhodococcus, Serratia, and Stenotrophomonas, grew on alkaline carbonate-precipitating medium. Two cultures with the most intense precipitate formation, Arthrobacter sulfonivorans and Rhodococcus globerulus, grew as aggregates, produced extracellular polymeric substances (EPS), and formed mixtures of calcite, vaterite, and monohydrocalcite. R. globerulus formed idiomorphous crystals with rhombohedral morphology, whereas A. sulfonivorans formed xenomorphous globular crystals, evidence for taxon-specific crystal morphologies. The results of this study highlighted the importance of combining various techniques in order to understand the geomicrobiology of karstic caves, but further studies are needed to determine whether the mineralogical biosignatures found in nutrient-rich media can also be found in oligotrophic caves.

  1. Effect of CuO Nanoparticles over Isolated Bacterial Strains from Agricultural Soil

    Directory of Open Access Journals (Sweden)

    Sandra I. Concha-Guerrero

    2014-01-01

    Full Text Available The increased use of the nanoparticles (NPs on several processes is notorious. In contrast the ecotoxicological effects of NPs have been scarcely studied. The main current researches are related to the oxide metallic NPs. In the present work, fifty-six bacterial strains were isolated from soil, comprising 17 different OTUs distributed into 3 classes: Bacilli (36 strains, Flavobacteria (2 strains, and Gammaproteobacteria (18 strains. Copper oxide nanoparticles (CuONPs were synthesized using a process of chemical precipitation. The obtained CuONPs have a spherical shape and primary size less than 17 nm. Twenty-one strains were used to evaluate the cytotoxicity of CuONPs and 11 of these strains showed high sensibility. Among those 11 strains, 4 (Brevibacillus laterosporus strain CSS8, Chryseobacterium indoltheticum strain CSA28, and Pantoea ananatis strains CSA34 and CSA35 were selected to determine the kind of damage produced. The CuONPs toxic effect was observed at expositions over 25 mg·L−1 and the damage to cell membrane above 160 mg·L−1. The electron microscopy showed the formation of cavities, holes, membrane degradation, blebs, cellular collapse, and lysis. These toxic effects may probably be due to the ions interaction, the oxide-reduction reactions, and the generation of reactive species.

  2. DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry.

    Science.gov (United States)

    Dingley, Karen H; Ubick, Esther A; Vogel, John S; Ognibene, Ted J; Malfatti, Michael A; Kulp, Kristen; Haack, Kurt W

    2014-01-01

    Accelerator mass spectrometry (AMS) is a highly sensitive technique used for the quantification of adducts following exposure to carbon-14- or tritium-labeled chemicals, with detection limits in the range of one adduct per 10(11)-10(12) nucleotides. The protocol described in this chapter provides an optimal method for isolating and preparing DNA samples to measure isotope-labeled DNA adducts by AMS. When preparing samples, special precautions must be taken to avoid cross-contamination of isotope among samples and produce a sample that is compatible with AMS. The DNA isolation method described is based upon digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen isolation columns. The extracted DNA is precipitated with isopropanol, washed repeatedly with 70 % ethanol to remove salt, and then dissolved in water. DNA samples are then converted to graphite or titanium hydride and the isotope content measured by AMS to quantify adduct levels. This method has been used to reliably generate good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels.

  3. Variation in Ribosomal DNA among Isolates of the Mycorrhizal Fungus Cenococcum Geophilum FR.

    Science.gov (United States)

    Lobuglio, Katherine Frances

    1990-01-01

    Cenococcum geophilum Fr., a cosmopolitan mycorrhizal fungus, is well-known for its extremely wide host and habitat range. The ecological diversity of C. geophilum sharply contrasts its present taxonomic status as a monotypic form -genus. Restriction fragment length polymorphisms (RFLPs) in nuclear ribosomal DNA (rDNA) was used to assess the degree of genetic variation among 72 isolates of C. geophilum. The probe used in this study was the rDNA repeat cloned from C. geophilum isolate A145 (pCG15). Length of the rDNA repeat was approximately 9 kb. The rDNA clone was mapped for 5 restriction endonucleases. Hybridization with cloned Saccharomyces cerevisiae rDNA (pSR118, and pSR125 containing the 18S, and 5.8-25S rRNA genes respectively), and alignment of restriction endonuclease sites conserved in the rDNA genes of other fungi, were used to position the corresponding rDNAs of C. geophilum. Southern hybridizations with EcoRI, HindIII, XhoI, and PstI digested DNAs indicated extensive variation among the C. geophilum isolates, greater than has been previously reported to occur within a fungal species. Most of the rDNA polymorphisms occurred in the IGS region. Restriction endonuclease site and length polymorphisms were also observed in the 5.8S-26S genic regions. Sixteen size categories of length mutations, 6 restriction endonuclease site additions, and 4 restriction endonuclease site deletions were determined using isolate A145 as a reference. The rDNA repeat length among the isolates varied from approximately 8.5 to 10.2 kb. RFLPs were also observed in the mitochondrial (mt) 24S rRNA gene and flanking regions of HindIII digested DNAs of C. geophilum isolates representing both geographically distinct and similar origins. Among the C. geophilum isolates analyzed there were fewer RFLPs in mt-DNA than in nuclear rDNA. EcoRI rDNA phenotypes between C. geophilum and Elaphomyces anthracinus, its proposed teleomorph or sexual state, did not correspond. In addition, the four

  4. The identification of a bacterial strain BGI-1 isolated from the intestinal flora of Blattella germanica, and its anti-entomopathogenic fungi activity.

    Science.gov (United States)

    Huang, Y H; Wang, X J; Zhang, F; Huo, X B; Fu, R S; Liu, J J; Sun, W B; Kang, D M; Jing, X

    2013-02-01

    A bacterial strain BGI-1 was isolated from the gut of German cockroaches (Blattella germanica L.) and was identified as Bacillus subtilis based on 16S rDNA sequence and morphological, physiological, and biochemical characters. The strain BGI-1 inhibited the growth of Beauveria bassiana; the diameter of the inhibition zone exceeded 30 mm. Vesicles were observed in B. bassiana hyphae on the edge of the inhibition zone. Fermentation of BGI-1 reduced the conidial germination rate by 12%. Further studies demonstrated that B. bassiana infections in German cockroaches orally treated with the extracts of BGI-1 fermentation were significantly weakened. Cumulative mortality rate was 49.5% in the treatment group at the 20 d, while that of the control group was 62.3%. The study intends to understand the relationship between the intestinal flora and the cockroach. Those microbes with anti-entomopathogenic fungi activity might contribute to resisting the infection of pathogenic fungi.

  5. In Vitro Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA.

    Directory of Open Access Journals (Sweden)

    Janet L Smith

    2015-05-01

    Full Text Available DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep sequencing (IDAP-Seq. We used these data to identify 269 binding regions, refine the consensus sequence of the DnaA binding site, and compare the relative affinity of binding regions for ATP-DnaA and ADP-DnaA. Most sites had a slightly higher affinity for ATP-DnaA than ADP-DnaA, but a few had a strong preference for binding ATP-DnaA. Of the 269 sites, only the eight strongest binding ones have been observed to bind DnaA in vivo, suggesting that other cellular factors or the amount of available DnaA in vivo restricts DnaA binding to these additional sites. Conversely, we found several chromosomal regions that were bound by DnaA in vivo but not in vitro, and that the nucleoid-associated protein Rok was required for binding in vivo. Our in vitro characterization of the inherent ability of DnaA to bind the genome at single nucleotide resolution provides a backdrop for interpreting data on in vivo binding and regulation of DnaA, and is an approach that should be adaptable to many other DNA binding proteins.

  6. Bacterial diversity associated with wild caught Anopheles mosquitoes from Dak Nong Province, Vietnam using culture and DNA fingerprint.

    Directory of Open Access Journals (Sweden)

    Chung Thuy Ngo

    Full Text Available Microbiota of Anopheles midgut can modulate vector immunity and block Plasmodium development. Investigation on the bacterial biodiversity in Anopheles, and specifically on the identification of bacteria that might be used in malaria transmission blocking approaches, has been mainly conducted on malaria vectors of Africa. Vietnam is an endemic country for both malaria and Bancroftian filariasis whose parasitic agents can be transmitted by the same Anopheles species. No information on the microbiota of Anopheles mosquitoes in Vietnam was available previous to this study.The culture dependent approach, using different mediums, and culture independent (16S rRNA PCR - TTGE method were used to investigate the bacterial biodiversity in the abdomen of 5 Anopheles species collected from Dak Nong Province, central-south Vietnam. Molecular methods, sequencing and phylogenetic analysis were used to characterize the microbiota.The microbiota in wild-caught Anopheles was diverse with the presence of 47 bacterial OTUs belonging to 30 genera, including bacterial genera impacting Plasmodium development. The bacteria were affiliated with 4 phyla, Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria, the latter being the dominant phylum. Four bacterial genera are newly described in Anopheles mosquitoes including Coxiella, Yersinia, Xanthomonas, and Knoellia. The bacterial diversity per specimen was low ranging from 1 to 4. The results show the importance of pairing culture and fingerprint methods to better screen the bacterial community in Anopheles mosquitoes.Sampled Anopheles species from central-south Vietnam contained a diverse bacterial microbiota that needs to be investigated further in order to develop new malaria control approaches. The combination of both culture and DNA fingerprint methods allowed a thorough and complementary screening of the bacterial community in Anopheles mosquitoes.

  7. Bacterial Diversity Associated with Wild Caught Anopheles Mosquitoes from Dak Nong Province, Vietnam Using Culture and DNA Fingerprint

    Science.gov (United States)

    Ngo, Chung Thuy; Aujoulat, Fabien; Veas, Francisco; Jumas-Bilak, Estelle; Manguin, Sylvie

    2015-01-01

    Background Microbiota of Anopheles midgut can modulate vector immunity and block Plasmodium development. Investigation on the bacterial biodiversity in Anopheles, and specifically on the identification of bacteria that might be used in malaria transmission blocking approaches, has been mainly conducted on malaria vectors of Africa. Vietnam is an endemic country for both malaria and Bancroftian filariasis whose parasitic agents can be transmitted by the same Anopheles species. No information on the microbiota of Anopheles mosquitoes in Vietnam was available previous to this study. Method The culture dependent approach, using different mediums, and culture independent (16S rRNA PCR – TTGE) method were used to investigate the bacterial biodiversity in the abdomen of 5 Anopheles species collected from Dak Nong Province, central-south Vietnam. Molecular methods, sequencing and phylogenetic analysis were used to characterize the microbiota. Results and Discussion The microbiota in wild-caught Anopheles was diverse with the presence of 47 bacterial OTUs belonging to 30 genera, including bacterial genera impacting Plasmodium development. The bacteria were affiliated with 4 phyla, Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria, the latter being the dominant phylum. Four bacterial genera are newly described in Anopheles mosquitoes including Coxiella, Yersinia, Xanthomonas, and Knoellia. The bacterial diversity per specimen was low ranging from 1 to 4. The results show the importance of pairing culture and fingerprint methods to better screen the bacterial community in Anopheles mosquitoes. Conclusion Sampled Anopheles species from central-south Vietnam contained a diverse bacterial microbiota that needs to be investigated further in order to develop new malaria control approaches. The combination of both culture and DNA fingerprint methods allowed a thorough and complementary screening of the bacterial community in Anopheles mosquitoes. PMID:25747513

  8. Adaptation of the neutral bacterial comet assay to assess antimicrobial-mediated DNA double-strand breaks in Escherichia coli

    Science.gov (United States)

    SOLANKY, DIPESH; HAYDEL, SHELLEY E.

    2012-01-01

    This study aimed to determine the mechanism of action of a natural antibacterial clay mineral mixture, designated CB, by investigating the induction of DNA double-strand breaks (DSBs) in Escherichia coli. To quantify DNA damage upon exposure to soluble antimicrobial compounds, we modified a bacterial neutral comet assay, which primarily associates the general length of an electrophoresed chromosome, or comet, with the degree of DSB-associated DNA damage. To appropriately account for antimicrobial-mediated strand fragmentation, suitable control reactions consisting of exposures to water, ethanol, kanamycin, and bleomycin were developed and optimized for the assay. Bacterial exposure to the CB clay resulted in significantly longer comet lengths, compared to water and kanamycin exposures, suggesting that the induction of DNA DSBs contributes to the killing activity of this antibacterial clay mineral mixture. The comet assay protocol described herein provides a general technique for evaluating soluble antimicrobial-derived DNA damage and for comparing DNA fragmentation between experimental and control assays. PMID:22940101

  9. A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples.

    Science.gov (United States)

    Rothrock, Michael J; Hiett, Kelli L; Gamble, John; Caudill, Andrew C; Cicconi-Hogan, Kellie M; Caporaso, J Gregory

    2014-12-10

    The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the "gold standard" enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

  10. Identification of Fic-1 as an enzyme that inhibits bacterial DNA replication by AMPylating GyrB, promoting filament formation.

    Science.gov (United States)

    Lu, Canhua; Nakayasu, Ernesto S; Zhang, Li-Qun; Luo, Zhao-Qing

    2016-01-26

    The morphology of bacterial cells is important for virulence, evasion of the host immune system, and coping with environmental stresses. The widely distributed Fic proteins (filamentation induced by cAMP) are annotated as proteins involved in cell division because of the presence of the HPFx[D/E]GN[G/K]R motif. We showed that the presence of Fic-1 from Pseudomonas fluorescens significantly reduced the yield of plasmid DNA when expressed in Escherichia coli or P. fluorescens. Fic-1 interacted with GyrB, a subunit of DNA gyrase, which is essential for bacterial DNA replication. Fic-1 catalyzed the AMPylation of GyrB at Tyr(109), a residue critical for binding ATP, and exhibited auto-AMPylation activity. Mutation of the Fic-1 auto-AMPylated site greatly reduced AMPylation activity toward itself and toward GyrB. Fic-1-dependent AMPylation of GyrB triggered the SOS response, indicative of DNA replication stress or DNA damage. Fic-1 also promoted the formation of elongated cells when the SOS response was blocked. We identified an α-inhibitor protein that we named anti-Fic-1 (AntF), encoded by a gene immediately upstream of Fic-1. AntF interacted with Fic-1, inhibited the AMPylation activity of Fic-1 for GyrB in vitro, and blocked Fic-1-mediated inhibition of DNA replication in bacteria, suggesting that Fic-1 and AntF comprise a toxin-antitoxin module. Our work establishes Fic-1 as an AMPylating enzyme that targets GyrB to inhibit DNA replication and may target other proteins to regulate bacterial morphology.

  11. Cloning and Sequencing cDNA Encoding for Rhoptry-2 Toxoplasma Gondii Tachyzoite Local Isolate

    Directory of Open Access Journals (Sweden)

    Wayan T. Artama

    2015-10-01

    Full Text Available Rhoptry protein belongs to an excretory and secretory antigens (ESAs that play an important role during activepenetration of parasite into the cell target. This protein an able Toxoplasma gondii to actively penetrate targetedcell, meanwhile ESAs protein stimulates intracellular vacuole modification. It is, therefore, after the parasitesuccessfully enter the cell target then Granule (GRA proteins are responsible for the formation of parasitophorusvacuole, which is protect the fusion with other intracellular compartments such as lysosomal vacuole. Consequently,this parasite is being able to survive and multiply at the cell target. The current study was aimed to clone andsequens cDNA encoding for ROP-2 of local isolated T. gondii tachizoite through DNA recombinant technique.Total ribonucleic acid (RNA was isolated from tachyzoites of local isolated T. gondii that were grown up in Balb/c mice. Messenger RNA was isolated from total RNA using PolyAtract mRNA Isolation System. Messenger RNA wasused as a template for synthesis cDNA using Riboclone cDNA Synthesis System AMV-RT. EcoRI adaptor fromRiboclone EcoRI Adaptor Ligation System was added to Complementary DNA and than ligated to pUC19. Recombinantplasmid was transformed into E. coli (XL1-Blue. The transformed E. coli XL-1 Blue were plated on LB agarcontaining X-Gal, IPTG and ampicillin. Recombinant clones (white colony were picked up and grown up in theLB medium at 37oC overnight. Expression of recombinant protein was analysed by immunoblotting in order toidentify cDNA recombinant wich is express ESA of T. gondii local isolate. Recombinant plasmid were isolatedusing alkalilysis method and were elektroforated in 1% agarose gel. The isolated DNA recombinant plasmid wascut using Eco RI and then sequenced through Big Dye Terminator Mix AB1 377A Sequencer using M13 Forward andM13 Reverse primers. The conclusion of this results showed that the recombinant clone was coding for excretoryand secretory

  12. Antibacterial activity of carbapenems against clinical isolates of respiratory bacterial pathogens in the northeastern region of Japan in 2007.

    Science.gov (United States)

    Gomi, Kazunori; Fujimura, Shigeru; Fuse, Katsuhiro; Takane, Hidenari; Nakano, Yoshihisa; Kariya, Yasuko; Kikuchi, Toshiaki; Kurokawa, Iku; Tokue, Yutaka; Watanabe, Akira

    2011-04-01

    As the increasing prevalence of resistant strains of respiratory bacterial pathogens has recently been reported, continuous monitoring of the susceptibility of clinical isolates to antibacterial agents is important. We performed a surveillance study focusing on the susceptibility of major respiratory bacterial pathogens in the northeastern region of Japan to carbapenems and control drugs. A total of 168 bacterial strains isolated from patients with respiratory tract infections in 2007 were collected and the minimum inhibitory concentration (MIC) determined. MIC data were subjected to pharmacokinetic/pharmacodynamic analysis with Monte Carlo simulation to calculate the probability of achieving the target of time above MIC with each carbapenem. All Moraxella catarrhalis, Streptococcus pneumoniae, and methicillin-sensitive Staphylococcus aureus isolates were susceptible to carbapenems. Despite the increasing prevalence of β-lactamase-nonproducing ampicillin-resistant strains, all Haemophilus influenzae isolates were susceptible to meropenem. For Pseudomonas aeruginosa, the susceptibility rates for meropenem and biapenem were 76.7%, and the highest probability of achieving pharmacodynamic target (40% of the time above MIC) was obtained with meropenem 0.5 g three times daily as a 4-h infusion (89.4%), followed by meropenem 0.5 g four times daily as a 1-h infusion (88.4%). Carbapenems have retained their position as key drugs for severe respiratory tract infections.

  13. Identifying the bacterial community on the surface of Intralox belting in a meat boning room by culture-dependent and culture-independent 16S rDNA sequence analysis.

    Science.gov (United States)

    Brightwell, Gale; Boerema, Jackie; Mills, John; Mowat, Eilidh; Pulford, David

    2006-05-25

    We examined the bacterial community present on an Intralox conveyor belt system in an operating lamb boning room by sequencing the 16S ribosomal DNA (rDNA) of bacteria extracted in the presence or absence of cultivation. RFLP patterns for 16S rDNA clone library and cultures were generated using HaeIII and MspI restriction endonucleases. 16S rDNA amplicons produced 8 distinct RFLP pattern groups. RFLP groups I-IV were represented in the clone library and RFLP groups I and V-VIII were represented amongst the cultured isolates. Partial DNA sequences from each RFLP group revealed that all group I, II and VIII representatives were Pseudomonas spp., group III were Sphingomonas spp., group IV clones were most similar to an uncultured alpha proteobacterium, group V was similar to a Serratia spp., group VI with an Alcaligenes spp., and group VII with Microbacterium spp. Sphingomonads were numerically dominant in the culture-independent clone library and along with the group IV alpha proteobacterium were not represented amongst the cultured isolates. Serratia, Alcaligenes and Microbacterium spp. were only represented with cultured isolates. Pseudomonads were detected by both culture-dependent (84% of isolates) and culture-independent (12.5% of clones) methods and their presence at high frequency does pose the risk of product spoilage if transferred onto meat stored under aerobic conditions. The detection of sphingomonads in large numbers by the culture-independent method demands further analysis because sphingomonads may represent a new source of meat spoilage that has not been previously recognised in the meat processing environment. The 16S rDNA collections generated by both methods were important at representing the diversity of the bacterial population associated with an Intralox conveyor belt system.

  14. Intraspecific variability of Bipolaris sorokiniana isolates determined by random-amplified polymorphic DNA (RAPD).

    Science.gov (United States)

    de Oliveira, Andréia M R; Matsumura, Aida T S; Prestes, Ariano M; Van Der Sand, Sueli T

    2002-01-01

    Isolates of Bipolaris sorokiniana were analyzed by random-amplified polymorphic DNA (RAPD) techniques to determine the amount of intraspecific genetic variability and to study host-pathogen interactions. Ten isolates originated from different regions of Brazil were examined. Plants of the wheat cultivars BR8, BH1146 (original host) and IAC-5 Maringá, classified as resistant, moderately resistant or susceptible to B. sorokiniana, respectively, were inoculated with these 10 isolates. Twenty-seven isolates were recovered from these cultivars and were analyzed by RAPD assay and compared to the RAPD of the original 10 isolates. According to the RAPD profiles there was a high level of genetic variability among the isolates. We detected 69 polymorphic fragments, ranging from 1.6 to 0.54 kb, in the original 10 isolates; 57 fragments with sizes between 1.98 and 0.38 kb from the isolates recovered from BH1146; 47 polymorphic bands, ranging from 1.96-0.54 kb, were detected in the isolates from BR8 and 32 fragments between 1.98 and 0.42 kb in isolates were recovered from IAC-5 Maringá. The number of polymorphic fragments varied, even for the same isolate, when the isolates were recovered from different cultivar hosts.

  15. Development and validation of an rDNA operon based primer walking strategy applicable to de novo bacterial genome finishing.

    Directory of Open Access Journals (Sweden)

    Alexander William Eastman

    2015-01-01

    Full Text Available Advances in sequencing technology have drastically increased the depth and feasibility of bacterial genome sequencing. However, little information is available that details the specific techniques and procedures employed during genome sequencing despite the large numbers of published genomes. Shotgun approaches employed by second-generation sequencing platforms has necessitated the development of robust bioinformatics tools for in silico assembly, and complete assembly is limited by the presence of repetitive DNA sequences and multi-copy operons. Typically, re-sequencing with multiple platforms and laborious, targeted Sanger sequencing are employed to finish a draft bacterial genome. Here we describe a novel strategy based on the identification and targeted sequencing of repetitive rDNA operons to expedite bacterial genome assembly and finishing. Our strategy was validated by finishing the genome of Paenibacillus polymyxa strain CR1, a bacterium with potential in sustainable agriculture and bio-based processes. An analysis of the 38 contigs contained in the P. polymyxa strain CR1 draft genome revealed 12 repetitive rDNA operons with varied intragenic and flanking regions of variable length, unanimously located at contig boundaries and within contig gaps. These highly similar but not identical rDNA operons were experimentally verified and sequenced simultaneously with multiple, specially designed primer sets. This approach also identified and corrected significant sequence rearrangement generated during the initial in silico assembly of sequencing reads. Our approach reduces the required effort associated with blind primer walking for contig assembly, increasing both the speed and feasibility of genome finishing. Our study further reinforces the notion that repetitive DNA elements are major limiting factors for genome finishing. Moreover, we provided a step-by-step workflow for genome finishing, which may guide future bacterial genome finishing

  16. Persistent Staphylococcus aureus isolates from two independent cases of bacteremia display increased bacterial fitness and novel immune evasion phenotypes.

    Science.gov (United States)

    Richards, R L; Haigh, R D; Pascoe, B; Sheppard, S K; Price, F; Jenkins, D; Rajakumar, K; Morrissey, J A

    2015-08-01

    Staphylococcus aureus bacteremia cases are complicated by bacterial persistence and treatment failure despite the confirmed in vitro susceptibility of the infecting strain to administered antibiotics. A high incidence of methicillin-resistant S. aureus (MRSA) bacteremia cases are classified as persistent and are associated with poorer patient outcomes. It is still unclear how S. aureus evades the host immune system and resists antibiotic treatment for the prolonged duration of a persistent infection. In this study, the genetic changes and associated phenotypic traits specific to S. aureus persistent bacteremia were identified by comparing temporally dispersed isolates from persistent infections (persistent isolates) originating from two independent persistent S. aureus bacteremia cases with the initial infection isolates and with three resolved S. aureus bacteremia isolates from the same genetic background. Several novel traits were associated specifically with both independent sets of persistent S. aureus isolates compared to both the initial isolates and the isolates from resolved infections (resolved isolates). These traits included (i) increased growth under nutrient-poor conditions; (ii) increased tolerance of iron toxicity; (iii) higher expression of cell surface proteins involved in immune evasion and stress responses; and (iv) attenuated virulence in a Galleria mellonella larva infection model that was not associated with small-colony variation or metabolic dormancy such as had been seen previously. Whole-genome sequence analysis identified different single nucleotide mutations within the mprF genes of all the isolates with the adaptive persistence traits from both independent cases. Overall, our data indicate a novel role for MprF function during development of S. aureus persistence by increasing bacterial fitness and immune evasion.

  17. Persistent Staphylococcus aureus Isolates from Two Independent Cases of Bacteremia Display Increased Bacterial Fitness and Novel Immune Evasion Phenotypes

    Science.gov (United States)

    Richards, R. L.; Haigh, R. D.; Pascoe, B.; Sheppard, S. K.; Price, F.; Jenkins, D.; Rajakumar, K.

    2015-01-01

    Staphylococcus aureus bacteremia cases are complicated by bacterial persistence and treatment failure despite the confirmed in vitro susceptibility of the infecting strain to administered antibiotics. A high incidence of methicillin-resistant S. aureus (MRSA) bacteremia cases are classified as persistent and are associated with poorer patient outcomes. It is still unclear how S. aureus evades the host immune system and resists antibiotic treatment for the prolonged duration of a persistent infection. In this study, the genetic changes and associated phenotypic traits specific to S. aureus persistent bacteremia were identified by comparing temporally dispersed isolates from persistent infections (persistent isolates) originating from two independent persistent S. aureus bacteremia cases with the initial infection isolates and with three resolved S. aureus bacteremia isolates from the same genetic background. Several novel traits were associated specifically with both independent sets of persistent S. aureus isolates compared to both the initial isolates and the isolates from resolved infections (resolved isolates). These traits included (i) increased growth under nutrient-poor conditions; (ii) increased tolerance of iron toxicity; (iii) higher expression of cell surface proteins involved in immune evasion and stress responses; and (iv) attenuated virulence in a Galleria mellonella larva infection model that was not associated with small-colony variation or metabolic dormancy such as had been seen previously. Whole-genome sequence analysis identified different single nucleotide mutations within the mprF genes of all the isolates with the adaptive persistence traits from both independent cases. Overall, our data indicate a novel role for MprF function during development of S. aureus persistence by increasing bacterial fitness and immune evasion. PMID:26056388

  18. Identification of DNA motifs implicated in maintenance of bacterial core genomes by predictive modeling.

    Science.gov (United States)

    Halpern, David; Chiapello, Hélène; Schbath, Sophie; Robin, Stéphane; Hennequet-Antier, Christelle; Gruss, Alexandra; El Karoui, Meriem

    2007-09-01

    Bacterial biodiversity at the species level, in terms of gene acquisition or loss, is so immense that it raises the question of how essential chromosomal regions are spared from uncontrolled rearrangements. Protection of the genome likely depends on specific DNA motifs that impose limits on the regions that undergo recombination. Although most such motifs remain unidentified, they are theoretically predictable based on their genomic distribution properties. We examined the distribution of the "crossover hotspot instigator," or Chi, in Escherichia coli, and found that its exceptional distribution is restricted to the core genome common to three strains. We then formulated a set of criteria that were incorporated in a statistical model to search core genomes for motifs potentially involved in genome stability in other species. Our strategy led us to identify and biologically validate two distinct heptamers that possess Chi properties, one in Staphylococcus aureus, and the other in several streptococci. This strategy paves the way for wide-scale discovery of other important functional noncoding motifs that distinguish core genomes from the strain-variable regions.

  19. Single-stranded DNA bound to bacterial cold-shock proteins: preliminary crystallographic and Raman analysis.

    Science.gov (United States)

    Bienert, Ralf; Zeeb, Markus; Dostál, Lubomir; Feske, Anette; Magg, Christine; Max, Klaas; Welfle, Heinz; Balbach, Jochen; Heinemann, Udo

    2004-04-01

    The cold-shock response has been described for several bacterial species. It is characterized by distinct changes in intracellular protein patterns whereby a set of cold-shock-inducible proteins become abundant. The major cold-shock proteins of Bacillus subtilis (Bs-CspB) and Bacillus caldolyticus (Bc-Csp) are small oligonucleotide/oligosaccharide-binding (OB) fold proteins that have been described as binding single-stranded nucleic acids. Bs-CspB (Mr = 7365) and Bc-Csp (Mr = 7333) were crystallized in the presence of the deoxyhexanucleotide (dT)6. Crystals of (dT)6 with Bs-CspB grew in the orthorhombic space group C222(1), with unit-cell parameters a = 49.0, b = 53.2, c = 77.0 A. Crystals with Bc-Csp grew in the primitive orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 74.3, b = 64.9, c = 31.2 A. These crystals diffract to maximal resolutions of 1.78 and 1.29 A, respectively. The presence of protein and DNA in the crystals was demonstrated by Raman spectroscopy.

  20. Identification of DNA motifs implicated in maintenance of bacterial core genomes by predictive modeling.

    Directory of Open Access Journals (Sweden)

    David Halpern

    2007-09-01

    Full Text Available Bacterial biodiversity at the species level, in terms of gene acquisition or loss, is so immense that it raises the question of how essential chromosomal regions are spared from uncontrolled rearrangements. Protection of the genome likely depends on specific DNA motifs that impose limits on the regions that undergo recombination. Although most such motifs remain unidentified, they are theoretically predictable based on their genomic distribution properties. We examined the distribution of the "crossover hotspot instigator," or Chi, in Escherichia coli, and found that its exceptional distribution is restricted to the core genome common to three strains. We then formulated a set of criteria that were incorporated in a statistical model to search core genomes for motifs potentially involved in genome stability in other species. Our strategy led us to identify and biologically validate two distinct heptamers that possess Chi properties, one in Staphylococcus aureus, and the other in several streptococci. This strategy paves the way for wide-scale discovery of other important functional noncoding motifs that distinguish core genomes from the strain-variable regions.

  1. Cloning of Thermostable DNA Polymerase Gene from a Thermophilic Brevibacillus sp. Isolated from Sikidang Crater, Dieng Plateu, Central Java

    Directory of Open Access Journals (Sweden)

    Lucia Dhiantika Witasari

    2015-11-01

    Full Text Available Thermostable DNA polymerase has an important role for amplifying small amount of DNA through polymerase chain reaction (PCR. Thermophillic bacteria Brevibacillus sp. was isolated from Sikidang Crater, Dieng Plateu, Central Java. Previous study showed that crude protein of the isolate could be used in PCR. Unfortunately, like most native thermostable enzymes, the thermostable DNA polymerase of the isolate is synthesized in a very low level and therefore is cumbersome to purify. The purpose of this research is to clone thermostable DNA polymerase gene of the isolate. The DNA polymerase gene was amplified by means of PCR using spesific primers. The amplified fragment was then isolated, purified, and ligated into the pGEM-T cloning vector. The recombinant plasmid was then transformed to competent E. coli JM109 cells using heat shock method. The cloned thermostable DNA polymerase gene from the thermophilic isolate was then characterized for its nucleotide base sequence. The result showed that the DNA Pol I gene was successfully be amplified from the isolate DNA genom, resulting in ± 2,7 kb DNA fragment in length. Sequence analysis of segment of targeted gene showed high similarity to that of thermostable DNA polymerase genes from other Bacillus.Key words : Thermostable DNA Pol I, Brevibacillus sp., PCR, cloning

  2. A robust and cost-effective method for DNA isolation from Satureja species (Lamiaceae)

    OpenAIRE

    2014-01-01

    Aromatic species of the genus Satureja are rich in secondary metabolites that interfere with DNA isolation procedures. Four protocols based on the standard CTDNA extraction protocol of Doyle and Doyle (1987) were tested in six savory taxa. The polyphenol adsorbents activated charcoal and/or polyvinylpyrrolidone 10 were employed in three procedures (B, C and D); for the elimination of polysaccharides, 4M NaCl was applied in the latter two. The highest DNA yi...

  3. A biogeochemical and genetic survey of acetylene fermentation by environmental samples and bacterial isolates

    Science.gov (United States)

    Miller, Laurence G.; Baesman, Shaun M.; Kirshtein, Julie; Voytek, Mary A.; Oremland, Ronald S.

    2013-01-01

    Anoxic samples (sediment and groundwater) from 13 chemically diverse field sites were assayed for their ability to consume acetylene (C2H2). Over incubation periods ranging from ˜ 10 to 80 days, selected samples from 7 of the 13 tested sites displayed significant C2H2 removal. No significant formation of ethylene was noted in these incubations; therefore, C2H2 consumption could be attributed to acetylene hydratase (AH) rather than nitrogenase activity. This putative AH (PAH) activity was observed in only 21% of the total of assayed samples, while amplification of AH genes from extracted DNA using degenerate primers derived from Pelobacter acetylenicus occurred in even fewer (9.8%) samples. Acetylene-fermenting bacteria were isolated as a pure culture from the sediments of a tidal mudflat in San Francisco Bay (SFB93) and as an enrichment culture from freshwater Searsville Lake (SV7). Comparison of 16S rDNA clone libraries revealed that SFB93 was closely related to P. carbolinicus, while SV7 consisted of several unrelated bacteria. AH gene was amplified from SFB93 but not SV7. The inability of the primers to generate amplicons in the SV7 enrichment, as well as from several of the environmental samples that displayed PAH activity, implied that either the primers were too highly constrained in their specificity or that there was a different type of AH gene in these environmental samples than occurs in P. acetylenicus. The significance of this work with regard to the search for life in the outer Solar System, where C2HL2 is abundant, is discussed.

  4. Immunity induced shortly after DNA vaccination of rainbow trout against rhabdoviruses protects against heterologous virus but not against bacterial pathogens

    DEFF Research Database (Denmark)

    Lorenzen, Niels; Lorenzen, Ellen; Einer-Jensen, Katja;

    2002-01-01

    It was recently reported that DNA vaccination of rainbow trout fingerlings against viral hemorrhagic septicaemia virus (VHSV) induced protection within 8 days after intramuscular injection of plasmid DNA. In order to analyse the specificity of this early immunity, fish were vaccinated with plasmid...... DNA encoding the VHSV or the infectious haematopoietic necrosis virus (IHNV) glycoprotein genes and later challenged with homologous or heterologous pathogens. Challenge experiments revealed that immunity established shortly after vaccination was cross-protective between the two viral pathogens...... whereas no increased survival was found upon challenge with bacterial pathogens. Within two months after vaccination, the cross-protection disappeared while the specific immunity to homologous virus remained high. The early immunity induced by the DNA vaccines thus appeared to involve short-lived non...

  5. Characterization of bacterial isolates from rubber dump site and their use in biodegradation of isoprene in batch and continuous bioreactors.

    Science.gov (United States)

    Srivastva, Navnita; Shukla, Awadhesh Kumar; Singh, Ram Sharan; Upadhyay, Siddh Nath; Dubey, Suresh Kumar

    2015-01-01

    Bacterial isolates from contaminated soil of a waste rubber dumping site were isolated and characterized using biochemical and molecular approaches. Isoprene degradation kinetics in batch mode (isoprene concentration: 100-1000 ppm) revealed the degradation efficiency of isolates as: Pseudomonas sp. (83%)>Alcaligenes sp. (70%)>Klebsiella sp. (68.5%). The most efficient isolate Pseudomonas sp. was finally inoculated in a specifically designed bioreactor system comprising a bioscrubber and a biofilter packed with polyurethane foam connected in series. The bioscrubber and biofilter units when operated in a series showed more than 90% removal efficiency up to the inlet loading rate (IL) of 371.1g/m(3)/h. Maximum elimination capacity (EC) of biofilter was found to be an order of magnitude greater than that for bioscrubber. Oxidative cleavage of the double bond of isoprene has been revealed through IR spectra of the leachate.

  6. Characterization of novel extracellular protease produced by marine bacterial isolate from the Indian Ocean

    Directory of Open Access Journals (Sweden)

    Rachana Fulzele

    2011-12-01

    Full Text Available Out of the vast pool of enzymes, proteolytic enzymes from microorganisms are the most widely used in different industries such as detergent, food, peptide production etc. Several marine microorganisms are known to produce proteases with commercially desirable characteristics. We have isolated nine different cultures from marine samples of the Indian Ocean. All of them were i motile ii rod shaped iii non spore forming iv catalase and amylase positive v able to grow in presence of 10 % NaCl. They produced acid from glucose, fructose and maltose and grew optimally at 30 0C temperature and pH 7.0-8.0. None of them could grow above 45 0C and below 15 0C. Only one of them (MBRI 7 exhibited extracellular protease activity on skim milk agar plates. Based on 16S rDNA sequencing, it belonged to the genus Marinobacter (98% sequence similarity, 1201 bp. The cell free extract was used to study effects of temperature and pH on protease activity. The optimum temperature and pH for activity were found to be 40 0C and 7.0 respectively. The crude enzyme was stable at temperature range of 30-80 0C and pH 5.0-9.0. It retained 60 % activity at 80 0C after 4 h and more than 70 % activity at 70 0C after 1 h. D value was found to be 342 minutes and 78 minutes for 40 0C and 80 0C respectively. Interestingly the enzyme remained 50 % active at pH 9.0 after 1 h. Comparison with other proteases from different microbial sources indicated that the neutral protease from the halotolerant marine isolate MBRI 7 is a novel enzyme with high thermostability.

  7. Randomly Amplified Polymorphic DNA of Trichoderma isolates and antagonism against Rhizoctonia solani

    OpenAIRE

    Larissa Brandão Góes; Ana Bolena Lima da Costa; Laurineide Lopes de Carvalho Freire; Neiva Tinti de Oliveira

    2002-01-01

    Random Amplified Polymorphic DNA (RAPD) procedure was used to examine the genetic variability among fourteen isolates of Trichoderma and their ability to antagonize Rhizoctonia solani using a dual-culture assay for correlation among RAPD products and their hardness to R. solani. Seven oligodeoxynucleotide primers were selected for the RAPD assays which resulted in 197 bands for 14 isolates of Trichoderma. The data were entered into a binary matrix and a similarity matrix was constructed using...

  8. Molecular mechanism of immune cells activated by bacterial DNA%细菌DNA激活免疫细胞的分子机制

    Institute of Scientific and Technical Information of China (English)

    王良喜; 周红

    2003-01-01

    Bacterial DNA taken up by immune cells in a CpG motif- independent manner is translo-cated into endosome. Endosomal maturation is essential for subsequent bacterial DNA - mediated signal trans-duction. TLR9 is recruited into endosome to recognize bacterial DNA and initiate the TLB/IL- 1R signal transduction pathway. As a result , transcription factors NF - κB and AP- 1 are activated, which, in tum,leads to proinflammatory cytokine expression and induces a strong acute Th1 - like inflammatory response.

  9. Pattern of Bacterial Pathogens and Their Susceptibility Isolated from Surgical Site Infections at Selected Referral Hospitals, Addis Ababa, Ethiopia

    Directory of Open Access Journals (Sweden)

    Walelign Dessie

    2016-01-01

    Full Text Available Background. The emergence of multidrug resistant bacterial pathogens in hospitals is becoming a challenge for surgeons to treat hospital acquired infections. Objective. To determine bacterial pathogens and drug susceptibility isolated from surgical site infections at St. Paul Specialized Hospital Millennium Medical College and Yekatit 12 Referral Hospital Medical College, Addis Ababa, Ethiopia. Methods. A cross-sectional study was conducted between October 2013 and March 2014 on 107 surgical site infected patients. Wound specimens were collected using sterile cotton swab and processed as per standard operative procedures in appropriate culture media; and susceptibility testing was done using Kirby-Bauer disc diffusion technique. The data were analyzed by using SPSS version 20. Result. From a total of 107 swabs collected, 90 (84.1% were culture positive and 104 organisms were isolated. E. coli (24 (23.1% was the most common organism isolated followed by multidrug resistant Acinetobacter species (23 (22.1%. More than 58 (75% of the Gram negative isolates showed multiple antibiotic resistance (resistance ≥ 5 drugs. Pan-antibiotic resistance was noted among 8 (34.8% Acinetobacter species and 3 (12.5% E. coli. This calls for abstinence from antibiotic abuse. Conclusion. Gram negative bacteria were the most important isolates accounting for 76 (73.1%. Ampicillin, amoxicillin, penicillin, cephazoline, and tetracycline showed resistance while gentamicin and ciprofloxacin were relatively effective antimicrobials.

  10. Bacterial microflora isolated from the bark surface of poplars growing in areas where air pollution is very high

    Directory of Open Access Journals (Sweden)

    Krystyna Przybył

    2015-05-01

    Full Text Available In the autumn of 1976 bacteria of the genera Bacillus, Pseudomonas, Flavobacterium, Erwinia and Cellulomonas were isolated from the bark surface of poplars growing in protective belts around several industrial plants. It was found that the qualitative and quantitative composition of the surface bacterial microflora changes in dependence on the degree of resistance of the poplars to the action of the dust emitted by the industrial establishment and containing high amounts of heavy metals.

  11. Borrelia burgdorferi EbfC defines a newly-identified, widespread family of bacterial DNA-binding proteins.

    Science.gov (United States)

    Riley, Sean P; Bykowski, Tomasz; Cooley, Anne E; Burns, Logan H; Babb, Kelly; Brissette, Catherine A; Bowman, Amy; Rotondi, Matthew; Miller, M Clarke; DeMoll, Edward; Lim, Kap; Fried, Michael G; Stevenson, Brian

    2009-04-01

    The Lyme disease spirochete, Borrelia burgdorferi, encodes a novel type of DNA-binding protein named EbfC. Orthologs of EbfC are encoded by a wide range of bacterial species, so characterization of the borrelial protein has implications that span the eubacterial kingdom. The present work defines the DNA sequence required for high-affinity binding by EbfC to be the 4 bp broken palindrome GTnAC, where 'n' can be any nucleotide. Two high-affinity EbfC-binding sites are located immediately 5' of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA. Consensus EbfC-binding sites are abundantly distributed throughout the B. burgdorferi genome, occurring approximately once every 1 kb. These and other features of EbfC suggest that this small protein and its orthologs may represent a distinctive type of bacterial nucleoid-associated protein. EbfC was shown to bind DNA as a homodimer, and site-directed mutagenesis studies indicated that EbfC and its orthologs appear to bind DNA via a novel alpha-helical 'tweezer'-like structure.

  12. Comparison of two DNA microarrays for detection of plasmid-mediated antimicrobial resistance and virulence factor genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae.

    LENUS (Irish Health Repository)

    Walsh, Fiona

    2010-06-01

    A DNA microarray was developed to detect plasmid-mediated antimicrobial resistance (AR) and virulence factor (VF) genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae. The array was validated with the following bacterial species: Escherichiacoli (n=17); Klebsiellapneumoniae (n=3); Enterobacter spp. (n=6); Acinetobacter genospecies 3 (n=1); Acinetobacterbaumannii (n=1); Pseudomonasaeruginosa (n=2); and Stenotrophomonasmaltophilia (n=2). The AR gene profiles of these isolates were identified by polymerase chain reaction (PCR). The DNA microarray consisted of 155 and 133 AR and VF gene probes, respectively. Results were compared with the commercially available Identibac AMR-ve Array Tube. Hybridisation results indicated that there was excellent correlation between PCR and array results for AR and VF genes. Genes conferring resistance to each antibiotic class were identified by the DNA array. Unusual resistance genes were also identified, such as bla(SHV-5) in a bla(OXA-23)-positive carbapenem-resistant A. baumannii. The phylogenetic group of each E. coli isolate was verified by the array. These data demonstrate that it is possible to screen simultaneously for all important classes of mobile AR and VF genes in Enterobacteriaceae and non-Enterobacteriaceae whilst also assigning a correct phylogenetic group to E. coli isolates. Therefore, it is feasible to test clinical Gram-negative bacteria for all known AR genes and to provide important information regarding pathogenicity simultaneously.

  13. Monoclonal antibodies against DNA-binding tips of DNABII proteins disrupt biofilms in vitro and induce bacterial clearance in vivo

    Directory of Open Access Journals (Sweden)

    Laura A. Novotny

    2016-08-01

    Full Text Available The vast majority of chronic and recurrent bacterial diseases are attributed to the presence of a recalcitrant biofilm that contributes significantly to pathogenesis. As such, these diseases will require an innovative therapeutic approach. We targeted DNABII proteins, an integral component of extracellular DNA (eDNA which is universally found as part of the pathogenic biofilm matrix to develop a biofilm disrupting therapeutic. We show that a cocktail of monoclonal antibodies directed against specific epitopes of a DNABII protein is highly effective to disrupt diverse biofilms in vitro as well as resolve experimental infection in vivo, in both a chinchilla and murine model. Combining this monoclonal antibody cocktail with a traditional antibiotic to kill bacteria newly released from the biofilm due to the action of the antibody cocktail was highly effective. Our results strongly support these monoclonal antibodies as attractive candidates for lead optimization as a therapeutic for resolution of bacterial biofilm diseases.

  14. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, Edward M.; Cullen, Bryan R., E-mail: bryan.cullen@duke.edu

    2015-05-15

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  15. Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein.

    OpenAIRE

    Jiang, H P; Serrero, G

    1992-01-01

    We have previously isolated from a 1246 adipocyte cDNA library a cDNA clone called 154, corresponding to a mRNA that increases abundantly at a very early time during the differentiation of 1246 adipocytes and in adipocyte precursors in primary culture. We show here that the mRNA encoded by this cDNA is expressed abundantly and preferentially in mouse fat pads. A full-length cDNA for clone 154 was isolated by the RACE (rapid amplification of cDNA ends) protocol. Sequence analysis of this cDNA ...

  16. Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein

    OpenAIRE

    Jiang, Hui-Ping; Serrero, Ginette

    1992-01-01

    We have previously isolated from a 1246 adipocyte cDNA library a cDNA clone called 154, corresponding to a mRNA that increases abundantly at a very early time during the differentiation of 1246 adipocytes and in adipocyte precursors in primary culture. We show here that the mRNA encoded by this cDNA is expressed abundantly and preferentially in mouse fat pads. A full-length cDNA for clone 154 was isolated by the RACE (rapid amplification of cDNA ends) protocol. Sequence analysis of this cDNA ...

  17. Efficiency of silver nanoparticles against bacterial contaminants isolated from surface and ground water in Egypt

    Directory of Open Access Journals (Sweden)

    Reem Dosoky

    2015-06-01

    Full Text Available The bactericidal efficiency of silver nanoparticles (AgNP was evaluated against bacteria isolated from surface and ground water samples in Egypt. The AgNP were synthesized by typical one-step synthesis protocol, and were characterized using transmission electron microscopy and atomic absorption spectrophotometer. The bactericidal efficiency of AgNP was evaluated by its application in three concentrations i.e., 0.1, 0.05 and 0.01 ppm to water sample, and allowed to interact with bacteria for different duration e.g., 5 min 15 min, 30 min, 1 h and 2 h. Then, the bactericidal efficiency of AgNPs was determined by comparing the counted bacteria before and after the treatments. Higher mean values of total bacterial count (TBC, total coliform count (TCC, and total streptococcal count (TFS were detected in surface water than in ground water. Also, the results showed that TBC, TCC and TFS exceeded permissible limits. Application of AgNP at different concentration, the number of bacteria in TBC was significantly reduced in all AgNP-exposed samples as compared to the control group (p<0.05. The highest concentration of AgNP exhibited highest bactericidal efficiency in TBC, where, after two hours, 0.1, 0.05 and 0.01 mg/L AgNP was found to be sufficient to inhibit 91.85, 89.14 and 74.92%, and 92.33, 85.23 and 53.17% in TBC of surface and ground water, respectively. Moreover, the inhibition efficiency of the highest concentration (0.1 ppm against TCC reached to 98.10 and 99.88% in surface water and 95.54 and 99.20% in ground water after 1 h and 2 h, respectively. Similar results were found against TFS count. The AgNPs were found to be effective against bacteria of water origin.

  18. Qualitative toxicity assessment of silver nanoparticles on the fresh water bacterial isolates and consortium at low level of exposure concentration.

    Science.gov (United States)

    Kumar, Deepak; Kumari, Jyoti; Pakrashi, Sunandan; Dalai, Swayamprava; Raichur, Ashok M; Sastry, T P; Mandal, A B; Chandrasekaran, N; Mukherjee, Amitava

    2014-10-01

    Silver nanoparticles (AgNPs) pose a high risk of exposure to the natural environment owing to their extensive usage in various consumer products. In the present study we attempted to understand the harmful effect of AgNPs at environmentally relevant low concentration levels (≤1ppm) towards two different freshwater bacterial isolates and their consortium. The standard plate count assay suggested that the AgNPs were toxic towards the fresh water bacterial isolates as well as the consortium, though toxicity was significantly reduced for the cells in the consortium. The oxidative stress assessment and membrane permeability studies corroborated with the toxicity data. The detailed electron microscopic studies suggested the cell degrading potential of the AgNPs, and the FT-IR studies confirmed the involvement of the surface groups in the toxic effects. No significant ion leaching from the AgNPs was observed at the applied concentration levels signifying the dominant role of the particle size, and size distribution in bacterial toxicity. The reduced toxicity for the cells in the consortium than the individual isolates has major significance in further studies on the ecotoxicity of the AgNPs.

  19. Resistance trends among clinical isolates in China reported from CHINET surveillance of bacterial resistance, 2005-2014.

    Science.gov (United States)

    Hu, F-P; Guo, Y; Zhu, D-M; Wang, F; Jiang, X-F; Xu, Y-C; Zhang, X-J; Zhang, C-X; Ji, P; Xie, Y; Kang, M; Wang, C-Q; Wang, A-M; Xu, Y-H; Shen, J-L; Sun, Z-Y; Chen, Z-J; Ni, Y-X; Sun, J-Y; Chu, Y-Z; Tian, S-F; Hu, Z-D; Li, J; Yu, Y-S; Lin, J; Shan, B; Du, Y; Han, Y; Guo, S; Wei, L-H; Wu, L; Zhang, H; Kong, J; Hu, Y-J; Ai, X-M; Zhuo, C; Su, D-H; Yang, Q; Jia, B; Huang, W

    2016-03-01

    With the aim of gathering temporal trends on bacterial epidemiology and resistance from multiple laboratories in China, the CHINET surveillance system was organized in 2005. Antimicrobial susceptibility testing was carried out according to a unified protocol using the Kirby-Bauer method or automated systems. Results were analyzed according to Clinical and Laboratory Standards Institute (CLSI) 2014 definitions. Between 2005 and 2014, the number of bacterial isolates ranged between 22,774 and 84,572 annually. Rates of extended-spectrum β-lactamase production among Escherichia coli isolates were stable, between 51.7 and 55.8%. Resistance of E. coli and Klebsiella pneumoniae to amikacin, ciprofloxacin, piperacillin/tazobactam and cefoperazone/sulbactam decreased with time. Carbapenem resistance among K. pneumoniae isolates increased from 2.4 to 13.4%. Resistance of Pseudomonas aeruginosa strains against all of antimicrobial agents tested including imipenem and meropenem decreased with time. On the contrary, resistance of Acinetobacter baumannii strains to carbapenems increased from 31 to 66.7%. A marked decrease of methicillin resistance from 69% in 2005 to 44.6% in 2014 was observed for Staphylococcus aureus. Carbapenem resistance rates in K. pneumoniae and A. baumannii in China are high. Our results indicate the importance of bacterial surveillance studies.

  20. Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories

    DEFF Research Database (Denmark)

    Godschalk, Roger W L; Ersson, Clara; Stępnik, Maciej;

    2014-01-01

    This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred...... to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were......%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference...

  1. Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies.

    Science.gov (United States)

    Scollo, Francesco; Egea, Leticia A; Gentile, Alessandra; La Malfa, Stefano; Dorado, Gabriel; Hernandez, Pilar

    2016-12-15

    Olive oil is considered a premium product for its nutritional value and health benefits, and the ability to define its origin and varietal composition is a key step towards ensuring the traceability of the product. However, isolating the DNA from such a matrix is a difficult task. In this study, the quality and quantity of olive oil DNA, isolated using four different DNA isolation protocols, was evaluated using the qRT-PCR and ddPCR techniques. The results indicate that CTAB-based extraction methods were the best for unfiltered oil, while Nucleo Spin-based extraction protocols showed greater overall reproducibility. The use of both qRT-PCR and ddPCR led to the absolute quantification of the DNA copy number. The results clearly demonstrate the importance of the choice of DNA-isolation protocol, which should take into consideration the qualitative aspects of DNA and the evaluation of the amplified DNA copy number.

  2. DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques

    Directory of Open Access Journals (Sweden)

    Vicente-Carbajosa Jesús

    2008-10-01

    Full Text Available Abstract Background High throughput applications of the reverse transcriptase quantitative PCR (RT-qPCR for quantification of gene expression demand straightforward procedures to isolate and analyze a considerable number of DNA-free RNA samples. Published protocols are labour intensive, use toxic organic chemicals and need a DNase digestion once pure RNAs have been isolated. In addition, for some tissues, the amount of starting material may be limiting. The convenience of commercial kits is often prohibitive when handling large number of samples. Findings We have established protocols to isolate DNA-free RNA from Arabidopsis thaliana tissues ready for RT-qPCR applications. Simple non-toxic buffers were used for RNA isolation from Arabidopsis tissues with the exception of seeds and siliques, which required the use of organic extractions. The protocols were designed to minimize the number of steps, labour time and the amount of starting tissue to as little as 10–20 mg without affecting RNA quality. In both protocols genomic DNA (gDNA can be efficiently removed from RNA samples before the final alcohol precipitation step, saving extra purification steps before cDNA synthesis. The expression kinetics of previously characterized genes confirmed the robustness of the procedures. Conclusion Here, we present two protocols to isolate DNA-free RNA from Arabidopsis tissues ready for RT-qPCR applications that significantly improve existing ones by reducing labour time and the use of organic extractions. Accessibility to these protocols is ensured by its simplicity and the low cost of the materials used.

  3. Bacteria and bacterial DNA in atherosclerotic plaque and aneurysmal wall biopsies from patients with and without periodontitis

    Directory of Open Access Journals (Sweden)

    Zahra Armingohar

    2014-05-01

    Full Text Available Background: Several studies have reported an association between chronic periodontitis (CP and cardiovascular diseases. Detection of periodontopathogens, including red complex bacteria (RCB, in vascular lesions has suggested these bacteria to be involved in the pathogenesis of atherosclerosis and abdominal aortic aneurysms. Objective: In this study, we investigate bacteria and their DNA in vascular biopsies from patients with vascular diseases (VD; i.e. abdominal aortic aneurysms, atherosclerotic carotid, and common femoral arteries, with and without CP. Methods: DNA was extracted from vascular biopsies selected from 40 VD patients: 30 with CP and 10 without CP. The V3-V5 region of the 16S rDNA (V3-V5 was polymerase chain reaction (PCR-amplified, and the amplicons were cloned into Escherichia coli, sequenced, and classified (GenBank and the Human Oral Microbiome database. Species-specific primers were used for the detection of Porphyromonas gingivalis. In addition, 10 randomly selected vascular biopsies from the CP group were subjected to scanning electron microscopy (SEM for visualization of bacteria. Checkerboard DNA–DNA hybridization was performed to assess the presence of RCB in 10 randomly selected subgingival plaque samples from CP patients. Results: A higher load and mean diversity of bacteria were detected in vascular biopsies from VD patients with CP compared to those without CP. Enterobacteriaceae were frequently detected in vascular biopsies together with cultivable, commensal oral, and not-yet-cultured bacterial species. While 70% of the subgingival plaque samples from CP patients showed presence of RCB, only P. gingivalis was detected in one vascular biopsy. Bacterial cells were seen in all 10 vascular biopsies examined by SEM. Conclusions: A higher bacterial load and more diverse colonization were detected in VD lesions of CP patients as compared to patients without CP. This indicated that a multitude of bacterial species both

  4. In vitro anti-proliferative, anti-bacterial potential and induction of DNA strand break of partially purified Cuscuta reflexa Roxb.

    Directory of Open Access Journals (Sweden)

    Madhulika Bhagat

    2011-01-01

    Full Text Available Cuscuta reflexa is an important medicinal plant, mentioned in Ayurveda, an ancient Indian system of medicine. The plant is selected to evaluate the possibility for novel pharmaceuticals for anticancer and antibiotics drugs. Since most of these drugs had developed resisitance against currently used chemotherapeutics. This study describes the in vitro anti-proliferative, anti-bacterial and single stand DNA break of the holoprasitic plant Cuscuta reflexa. Bioassay-guided fractionation and partial purification of the plant were done and evaluated for antiproliferative activity against human cancer cell lines by SRB assay and single strand DNA break by comet assay. Further antibacterial activity was also performed by agar well diffusion assay. The alcoholic extract, chloroform fraction and partially purified ethylacetate-methanol (1:1 sub-fraction of C. reflexa showed anti-proliferative potential against IMR-32 and 502713 human cancer cell lines. Alcoholic extract exhibited anti-proliferative activity of 74% and 72%, chloroform fraction demonstrated 91% and 95% against neuroblastoma (IMR-32 and colon (502713 cancer cell lines at 100 μg/ml. Single strand DNA break of the chloroform fraction was also demonstrated using comet assay, indicating that possible mode of cell death may be apoptosis. Anti-microbial properties were evaluated against eight species of pathogenic and non-pathogenic microorganisms and maximum zone of inhibition for anti-bacterial activity was found against Staphylococcus aureus (22 mm by alcoholic extract, 21 mm by chloroform fraction and 12 mm by ethylacetate-methanol (1:1 sub-fraction. Minimum inhibitory concentration (MIC of the chloroform fraction was 1500 μg/ml for S. aureus. The plant was found to be equally effective against gram-positive and negative bacteria. Studies are well underway to isolate and identify active compounds from chloroform fraction and ethyl acetate:methanol (1:1 sub-fraction, which can be used as

  5. An improved DNA isolation technique for PCR detection of Strongyloides stercoralis in stool samples.

    Science.gov (United States)

    Repetto, S A; Alba Soto, C D; Cazorla, S I; Tayeldin, M L; Cuello, S; Lasala, M B; Tekiel, V S; González Cappa, S M

    2013-05-01

    Strongyloides stercoralis is a nematode that causes severe infections in immunocompromised patients. The low parasitic burden of chronically infected patients makes diagnosis difficult to achieve by conventional methods. Here, an in-house (IH) method for the isolation of parasite DNA from stools and a PCR assay for the molecular diagnosis of S. stercoralis were optimized. DNA yield and purity improved with the IH method which included a step of incubation of stool samples with a glycine-SDS buffer and mechanical disruption prior to DNA extraction. For the PCR assay, the addition of bovine serum albumin was required to neutralize inhibitors present in stool. The analytical sensitivity of the PCR using DNA as template, isolated with the IH method, was superior to the commercial one. This study demonstrates that a combined method that adds the step of glycine-SDS buffer incubation plus mechanical disruption prior to DNA isolation with the commercial kit increased PCR sensitivity to levels of the IH method. Finally, our assay was tested on 17 clinical samples. With the IH method for DNA isolation, a S. stercoralis specific band was detected by PCR in the first stool sample in all patients (17/17), while with the commercial kit, our S. stercoralis-specific band was only observed in 7 samples. The superior efficiency of the IH and combined methods over the commercial kit was demonstrated when applied to clinical samples with low parasitic burden. These results show that the DNA extraction procedure is a key to increase sensitivity of the S. stercoralis PCR assay in stool samples. The method developed here could help to improve the molecular diagnosis of S. stercoralis.

  6. Simultaneous isolation of DNA, RNA, and proteins for genetic, epigenetic, transcriptomic, and proteomic analysis.

    Science.gov (United States)

    Radpour, Ramin; Sikora, Michal; Grussenmeyer, Thomas; Kohler, Corina; Barekati, Zeinab; Holzgreve, Wolfgang; Lefkovits, Ivan; Zhong, Xiao Yan

    2009-11-01

    Analysis of DNA, RNA, and proteins for downstream genetic, epigenetic, transcriptomic, and proteomic analysis holds an important place in the field of medical care and life science. This is often hampered by the limited availability of sample material. For this reason, there exists an increasing interest for simultaneous isolation of DNA, RNA and proteins from a single sample aliquot. Several kit-systems allowing such a procedure have been introduced to the market. We present an approach using the AllPrep method for simultaneous isolation of DNA, RNA and proteins from several human specimens, such as whole blood, buffy coat, serum, plasma and tissue samples. The quantification and qualification of the isolated molecular species were assessed by different downstream methods: NanoDrop for measuring concentration and purity of all molecular species; DNA and RNA LabChip for fractionation analysis of nucleic acids; quantitative PCR for quantification analysis of DNA and RNA; thymidine-specific cleavage mass array on MALDI-TOF silico-chip for epigenetic analysis; Protein LabChip and two-dimensional (2D) gel electrophoresis for proteomic analysis. With our modified method, we can simultaneously isolate DNA, RNA and/or proteins from one single sample aliquot. We could overcome to some method limitations like low quality or DNA fragmentation using reamplification strategy for performing high-throughput downstream assays. Fast and easy performance of the procedure makes this method interesting for all fields of downstream analysis, especially when using limited sample resources. The cost-effectiveness of the procedure when material is abundantly available has not been addressed. This methodological improvement enables to execute such experiments that were not performable with standard procedure, and ensures reproducible outcome.

  7. DNA-based diagnosis of isolated sulfite oxidase deficiency by denaturing high-performance liquid chromatography.

    Science.gov (United States)

    Lam, Ching-Wan; Li, Chi-Keung; Lai, Chi-Kong; Tong, Sui-Fan; Chan, Kwok-Yin; Ng, Grace Sui-Fun; Yuen, Yuet-Ping; Cheng, Anna Wai-Fun; Chan, Yan-Wo

    2002-01-01

    Isolated sulfite oxidase deficiency is a rare autosomal recessive disease, characterized by severe neurological abnormalities, seizures, mental retardation, and dislocation of the ocular lenses, that often leads to death in infancy. There is a special demand for prenatal diagnosis, since no effective treatment is available for isolated sulfite oxidase deficiency. Until now, the cDNA sequence of the sulfite oxidase (SUOX) gene has been available, but the genomic sequence of the SUOX gene has not been published. In this study, we have performed a DNA-based diagnosis of isolated sulfite oxidase deficiency in a Chinese patient. To do so, we designed oligonucleotide primers for amplification of the predicted exons and intron-exon boundaries of the SUOX gene obtained from the completed draft version of the human genome. Using overlapping PCR products, we confirmed the flanking intronic sequences of the coding exons and that the entire 466-residue mature peptide is encoded by the last exon of the gene. We then performed mutation detection using denaturing high-performance liquid chromatography (DHPLC). The DHPLC chromatogram of exon 2b showed the presence of heteroduplex peaks only after mixing of the mutant DNA with the wild-type DNA, indicating the presence of a homozygous mutation. Direct DNA sequencing showed a homozygous base substitution at codon 160, changing the codon from CGG to CAG, which changes the amino acid from arginine to glutamine, i.e., R160Q. The DNA-based diagnosis of isolated sulfite oxidase deficiency will enable us to make an accurate determination of carrier status and to perform prenatal diagnosis of this disease. The availability of the genomic sequences of human genes from the completed draft human genome sequence will simplify the development of molecular genetic diagnoses of human diseases from peripheral blood DNA.

  8. 16S ribosomal DNA clone libraries to reveal bacterial diversity in anaerobic reactor-degraded tetrabromobisphenol A.

    Science.gov (United States)

    Peng, Xingxing; Zhang, Zaili; Zhao, Ziling; Jia, Xiaoshan

    2012-05-01

    Microorganisms able to rapidly degrade tetrabromobisphenol A (TBBPA) were domesticated in an anaerobic reactor and added to gradually increased concentrations of TBBPA. After 240 days of domestication, the degradation rate reached 96.0% in cultivated batch experiments lasting 20 days. The optimum cultivating temperature and pH were 30°C and 7.0. The bacterial community's composition and diversity in the reactor was studied by comparative analysis with 16S ribosomal DNA clone libraries. Amplified rDNA restriction analysis of 200 clones from the library indicate that the rDNA richness was high (Coverage C 99.5%) and that evenness was not high (Shannon-Weaver index 2.42). Phylogenetic analysis of 63 bacterial sequences from the reactor libraries demonstrated the presence of Betaproteobacteria (33.1%), Gammaproteobacteria (18.7%), Bacteroidetes (13.9%), Firmicutes (11.4%), Chloroflexi (3.6%), Actinobacteria (0.6%), the candidate division TM7 (4.2%) and other unknown, uncultured bacterial groups (14.5%). Comamonas, Achromobacter, Pseudomonas and Flavobacterium were the dominant types.

  9. Construction and characterization of two bacterial artificial chromosome libraries of pea (Pisum sativum L.) for the isolation of economically important genes.

    Science.gov (United States)

    Coyne, C J; McClendon, M T; Walling, J G; Timmerman-Vaughan, G M; Murray, S; Meksem, K; Lightfoot, D A; Shultz, J L; Keller, K E; Martin, R R; Inglis, D A; Rajesh, P N; McPhee, K E; Weeden, N F; Grusak, M A; Li, C-M; Storlie, E W

    2007-09-01

    Pea (Pisum sativum L.) has a genome of about 4 Gb that appears to share conserved synteny with model legumes having genomes of 0.2-0.4 Gb despite extensive intergenic expansion. Pea plant inventory (PI) accession 269818 has been used to introgress genetic diversity into the cultivated germplasm pool. The aim here was to develop pea bacterial artificial chromosome (BAC) libraries that would enable the isolation of genes involved in plant disease resistance or control of economically important traits. The BAC libraries encompassed about 3.2 haploid genome equivalents consisting of partially HindIII-digested DNA fragments with a mean size of 105 kb that were inserted in 1 of 2 vectors. The low-copy oriT-based T-DNA vector (pCLD04541) library contained 55 680 clones. The single-copy oriS-based vector (pIndigoBAC-5) library contained 65 280 clones. Colony hybridization of a universal chloroplast probe indicated that about 1% of clones in the libraries were of chloroplast origin. The presence of about 0.1% empty vectors was inferred by white/blue colony plate counts. The usefulness of the libraries was tested by 2 replicated methods. First, high-density filters were probed with low copy number sequences. Second, BAC plate-pool DNA was used successfully to PCR amplify 7 of 9 published pea resistance gene analogs (RGAs) and several other low copy number pea sequences. Individual BAC clones encoding specific sequences were identified. Therefore, the HindIII BAC libraries of pea, based on germplasm accession PI 269818, will be useful for the isolation of genes underlying disease resistance and other economically important traits.

  10. Antimicrobial susceptibility pattern of bacterial isolates from surgical wound infections in Tertiary Care Hospital in Allahabad, India

    Directory of Open Access Journals (Sweden)

    A K Kapoor

    2012-01-01

    Full Text Available The aim of present study to analyze the occurrence and in-vitro antimicrobial susceptibility of bacterial pathogens isolated from surgical wound infections. Specimens from a total of 129 patients undergoing either emergency or elective surgery were collected from infected sites or stitch lines and inoculated onto appropriate media. The bacterial cultures were identified utilizing standard microbiological and biochemical methods. Isolates were tested for susceptibility to antimicrobials using the Kirby Bauer disk diffusion method. Statistical analysis was performed using the chi-square test. Of 129 patients investigated (62 emergency and 67 elective surgery cases, bacterial isolates were isolated with almost equal frequency both from emergency and elective surgery cases. Of 108 (83.72% culture positive samples, 62 (57.41% were Gram negative, 39 (36.11% Gram positive, and 7 (6.48% showed multiple organisms. Of total 115 bacteria isolated (101 single and 7 double organisms culture positive, 33 (28.69% were Escherichia coli and were also the commonest; followed by Staphylococcus aureus, 30 (26.09% cases. S. aureus and Streptococcus spp. showed maximum susceptibility (100% to linezolid and vancomycin. Maximum susceptibility of E. coli was observed to ciprofloxacin (75.7%, followed by gentamicin (54.5%; of Klebsiella spp. to ceftriaxone and gentamicin (66.6% each, of Proteus spp. to gentamicin (70% followed by ciprofloxacin (60%, and of Pseudomonas aeruginosa to piperacillin (100% and tobramycin (71.4%. E. coli and S. aureus were the most common and Salmonella spp. and Acinetobacter spp. were the least common organism causing surgical site infections. The definitive therapy included ciprofloxacin and gentamicin for E. coli; linezolid and vancomycin for S. aureus and Streptococcus spp; ceftriaxone and ciprofloxacin for Klebsiella spp., Citrobacter spp., acinetobacter spp and Salmonella spp.

  11. Isolation and characterization of different bacterial strains for bioremediation of n-alkanes and polycyclic aromatic hydrocarbons.

    Science.gov (United States)

    Guermouche M'rassi, A; Bensalah, F; Gury, J; Duran, R

    2015-10-01

    Crude oil is a common environmental pollutant composed of a large number of both aromatic and aliphatic hydrocarbons. Biodegradation is carried out by microbial communities that are important in determining the fate of pollutants in the environment. The intrinsic biodegradability of the hydrocarbons and the distribution in the environment of competent degrading microorganisms are crucial information for the implementation of bioremediation processes. In the present study, the biodegradation capacities of various bacteria toward aliphatic and aromatic hydrocarbons were determined. The purpose of the study was to isolate and characterize hydrocarbon-degrading bacteria from contaminated soil of a refinery in Arzew, Algeria. A collection of 150 bacterial strains was obtained; the bacterial isolates were identified by 16S rRNA gene sequencing and their ability to degrade hydrocarbon compounds characterized. The isolated strains were mainly affiliated to the Gamma-Proteobacteria class. Among them, Pseudomonas spp. had the ability to metabolize high molecular weight hydrocarbon compounds such as pristane (C19) at 35.11 % by strain LGM22 and benzo[a] pyrene (C20) at 33.93 % by strain LGM11. Some strains were able to grow on all the hydrocarbons tested including octadecane, squalene, phenanthrene, and pyrene. Some strains were specialized degrading only few substrates. In contrast, the strain LGM2 designated as Pseudomonas sp. was found able to degrade both linear and branched alkanes as well as low and high poly-aromatic hydrocarbons (PAHs). The alkB gene involved in alkane degradation was detected in LGM2 and other Pseudomonas-related isolates. The capabilities of the isolated bacterial strains to degrade alkanes and PAHs should be of great practical significance in bioremediation of oil-contaminated environments.

  12. Naphthalimides Selectively Inhibit the Activity of Bacterial, Replicative DNA Ligases and Display Bactericidal Effects against Tubercle Bacilli

    Directory of Open Access Journals (Sweden)

    Malgorzata Korycka-Machala

    2017-01-01

    Full Text Available The DNA ligases, enzymes that seal breaks in the backbones of DNA, are essential for all organisms, however bacterial ligases essential for DNA replication use β-nicotinamide adenine dinucleotide as their co-factor, whereas those that are essential in eukaryotes and viruses use adenosine-5′-triphosphate. This fact leads to the conclusion that NAD+-dependent DNA ligases in bacteria could be targeted by their co-factor specific inhibitors. The development of novel alternative medical strategies, including new drugs, are a top priority focus areas for tuberculosis research due to an increase in the number of multi-drug resistant as well as totally drug resistant tubercle bacilli strains. Here, through the use of a virtual high-throughput screen and manual inspection of the top 200 records, 23 compounds were selected for in vitro studies. The selected compounds were evaluated in respect to their Mycobacterium tuberculosis NAD+ DNA ligase inhibitory effect by a newly developed assay based on Genetic Analyzer 3500 Sequencer. The most effective agents (e.g., pinafide, mitonafide inhibited the activity of M. tuberculosis NAD+-dependent DNA ligase A at concentrations of 50 µM. At the same time, the ATP-dependent (phage DNA LigT4 was unaffected by the agents at concentrations up to 2 mM. The selected compounds appeared to also be active against actively growing tubercle bacilli in concentrations as low as 15 µM.

  13. Chromosomal DNA probes for the identification of asaccharolytic anaerobic pigmented bacterial rods from the oral cavity of cats.

    Science.gov (United States)

    Love, D N; Bailey, G D; Bastin, D

    1992-06-01

    A dot-blot hybridisation assay using isolated high molecular weight DNA as whole chromosomal probes of the cat pigmented asaccharolytic Bacteroides/Porphyromonas species was used against both purified high molecular weight DNA and DNA released on membranes from whole cells for the identification of B. salivosus and for its differentiation from the other anaerobic species isolated from normal and diseased mouths of cats and horses. 32P-labelled probes were compared with digoxigenin (DIG)-labelled probes (Boehringer-Mannheim). The whole chromosomal probes were specific--differentiating B. salivosus from a variety of species (including members of the genera Bacteroides, Fusobacterium, Eubacterium, and Prevotella) found in normal and abnormal mouths of cats and horses. Likewise, asaccharolytic black pigmented Group 2 strains were distinguishable from all strains tested. However, cat strains of P. gingivalis which show 68-76% DNA-DNA homology with human strain P. gingivalis ATCC 33277T, were not distinguishable from each other using either 32P-labelled or DIG-labelled probes. The minimum amount of pure Bacteroides DNA which could be detected by the 32P-labelled probe was 100-300 pg, while the amount of pure DNA detected by the DIG system was 1-3 mg after room temperature colour development for 1 h and 100-300 pg after 6 h colour development.

  14. Isolation of carrot Argonaute1 from subtractive somatic embryogenesis cDNA library.

    Science.gov (United States)

    Takahata, Kiminori

    2008-03-01

    Carrot Argonaute1 (C-Ago1) was isolated from a subtractive cDNA library to obtain somatic embryogenesis related genes. C-Ago1 has three conserved domains, which are found in all other Argonautes. C-Ago1 has specific expression during somatic embryogenesis, which indicates that microRNA gene expression controlling system is required for somatic embryogenesis.

  15. Inflammatory Bowel Diseases: When Natural Friends Turn into Enemies—The Importance of CpG Motifs of Bacterial DNA in Intestinal Homeostasis and Chronic Intestinal Inflammation

    Directory of Open Access Journals (Sweden)

    Florian Obermeier

    2010-01-01

    Full Text Available From numerous studies during the last years it became evident that bacteria and bacterial constituents play a decisive role both in the maintenance of intestinal immune homeostasis as well as in the development and perpetuation of chronic intestinal inflammation. In this review we focus on the role of bacterial DNA which is a potent immunomodulatory component of the bacterial flora. Bacterial DNA has been shown to be protective against experimental colitis. In contrast bacterial DNA essentially contributes to the perpetuation of an already established chronic intestinal inflammation in a Toll-like receptor (TLR9-dependent manner. This dichotomic action may be explained by a different activation status of essential regulators of TLR signaling like Glycogen synthase kinase 3- (GSK3- depending on the pre-activation status of the intestinal immune system. In this review we suggest that regulators of TLR signaling may be interesting therapeutic targets in IBD aiming at the restoration of intestinal immune homeostasis.

  16. Isolation of genomic DNA using magnetic nanoparticles as a solid-phase support

    Science.gov (United States)

    Saiyed, Z. M.; Ramchand, C. N.; Telang, S. D.

    2008-05-01

    In recent years, techniques employing magnetizable solid-phase supports (MSPS) have found application in numerous biological fields. This magnetic separation procedure offers several advantages in terms of subjecting the analyte to very little mechanical stress compared to other methods. Secondly, these methods are non-laborious, cheap, and often highly scalable. The current paper details a genomic DNA isolation method optimized in our laboratory using magnetic nanoparticles as a solid-phase support. The quality and yields of the isolated DNA from all the samples using magnetic nanoparticles were higher or equivalent to the traditional DNA extraction procedures. Additionally, the magnetic method takes less than 15 min to extract polymerase chain reaction (PCR) ready genomic DNA as against several hours taken by traditional phenol-chloroform extraction protocols. Moreover, the isolated DNA was found to be compatible in PCR amplification and restriction endonuclease digestion. The developed procedure is quick, inexpensive, robust, and it does not require the use of organic solvents or sophisticated instruments, which makes it more amenable to automation and miniaturization.

  17. Characterization of bacterial isolates from the microbiota of mothers' breast milk and their infants.

    Science.gov (United States)

    Kozak, Kimberly; Charbonneau, Duane; Sanozky-Dawes, Rosemary; Klaenhammer, Todd

    2015-01-01

    This investigation assessed the potential of isolating novel probiotics from mothers and their infants. A subset of 21 isolates among 126 unique bacteria from breast milk and infant stools from 15 mother-infant pairs were examined for simulated GI transit survival, adherence to Caco-2 cells, bacteriocin production, and lack of antibiotic resistance. Of the 21 selected isolates a Lactobacillus crispatus isolate and 3 Lactobacillus gasseri isolates demonstrated good profiles of in vitro GI transit tolerance and Caco-2 cell adherence. Bacteriocin production was observed only by L. gasseri and Enterococcus faecalis isolates. Antibiotic resistance was widespread, although not universal, among isolates from infants. Highly similar isolates (≥ 97% similarity by barcode match) of Bifidobacterium longum subsp. infantis (1 match), Lactobacillus fermentum (2 matches), Lactobacillus gasseri (6 matches), and Enterococcus faecalis (1 match) were isolated from 5 infant-mother pairs. Antibiotic resistance profiles between these isolate matches were similar, except in one case where the L. gasseri isolate from the infant exhibited resistance to erythromycin and tetracycline, not observed in matching mother isolate. In a second case, L. gasseri isolates differed in resistance to ampicillin, chloramphenicol and vancomycin between the mother and infant. In this study, gram positive bacteria isolated from mothers' breast milk as well as their infants exhibited diversity in GI transit survival and acid inhibition of pathogens, but demonstrated limited ability to produce bacteriocins. Mothers and their infants offer the potential for identification of probiotics; however, even in the early stages of development, healthy infants contain isolates with antibiotic resistance.

  18. Near-atomic structural model for bacterial DNA replication initiation complex and its functional insights.

    Science.gov (United States)

    Shimizu, Masahiro; Noguchi, Yasunori; Sakiyama, Yukari; Kawakami, Hironori; Katayama, Tsutomu; Takada, Shoji

    2016-12-13

    Upon DNA replication initiation in Escherichia coli, the initiator protein DnaA forms higher-order complexes with the chromosomal origin oriC and a DNA-bending protein IHF. Although tertiary structures of DnaA and IHF have previously been elucidated, dynamic structures of oriC-DnaA-IHF complexes remain unknown. Here, combining computer simulations with biochemical assays, we obtained models at almost-atomic resolution for the central part of the oriC-DnaA-IHF complex. This complex can be divided into three subcomplexes; the left and right subcomplexes include pentameric DnaA bound in a head-to-tail manner and the middle subcomplex contains only a single DnaA. In the left and right subcomplexes, DnaA ATPases associated with various cellular activities (AAA+) domain III formed helices with specific structural differences in interdomain orientations, provoking a bend in the bound DNA. In the left subcomplex a continuous DnaA chain exists, including insertion of IHF into the DNA looping, consistent with the DNA unwinding function of the complex. The intervening spaces in those subcomplexes are crucial for DNA unwinding and loading of DnaB helicases. Taken together, this model provides a reasonable near-atomic level structural solution of the initiation complex, including the dynamic conformations and spatial arrangements of DnaA subcomplexes.

  19. Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition

    DEFF Research Database (Denmark)

    Knudsen, Berith Elkær; Bergmark, Lasse; Munk, Patrick

    2016-01-01

    Explorations of complex microbiomes using genomics greatly enhance our understanding about their diversity, biogeography, and function. The isolation of DNA from microbiome specimens is a key prerequisite for such examinations, but challenges remain in obtaining sufficient DNA quantities required...... for certain sequencing approaches, achieving accurate genomic inference of microbiome composition, and facilitating comparability of findings across specimen types and sequencing projects. These aspects are particularly relevant for the genomics-based global surveillance of infectious agents and antimicrobial...... that in standard protocols. Based on this insight, we designed an improved DNA isolation procedure optimized for microbiome genomics that can be used for the three examined specimen types and potentially also for other biological specimens. A standard operating procedure is available from https://dx.doi.org/10...

  20. Isolation of subtelomeric DNA sequences labelling sheep and goat chromosome ends

    Directory of Open Access Journals (Sweden)

    Cribiu Edmond P

    2000-11-01

    Full Text Available Abstract Two techniques that make it possible to isolate telomere DNA are presented, using sheep as an example. The first technique is based upon the screening of a sheep BAC library with PCR amplified DNA segments preserved from high-power laser beam irradiation. Twenty-three BACs hybridising to 13 subtelomeric regions in sheep and goats were obtained (out of 27 in the sheep complement, of which 13 recognised more than one region, telomeric or not. Twenty-three microsatellites were isolated from these BACs and 22 were genetically mapped on the sheep international genetic map, always consistently with the cytogenetical localisation in 17 cases out of 22. These results are discussed. The second technique is based upon the selective cloning of subtelomeric enriched DNA. Preliminary results were obtained by this approach.

  1. Mitochondrial DNA assessment of Phytophthora infestans isolates from potato and tomato in Ethiopia reveals unexpected diversity.

    Science.gov (United States)

    Shimelash, Daniel; Hussien, Temam; Fininsa, Chemeda; Forbes, Greg; Yuen, Jonathan

    2016-08-01

    Mitochondrial DNA (mtDNA) haplotypes were determined using restriction fragment length polymorphism (RFLP) for P. infestans sampled from 513 foliar lesions of late blight found on potato and tomato in different regions of Ethiopia. Among the four reported mitochondrial haplotypes of Phytophthora infestans, Ia, Ib and IIb were detected in 93 % of the samples analyzed but the vast majority of these were Ia. The remaining 7 % represented a previously unreported haplotype. DNA sequencing of this new haplotype also confirmed a single base nucleotide substitution that resulted in loss of EcoRI restriction site and gain of two additional MspI sites in cox1 and atp1 genes, respectively. There were 28 polymorphic sites among all nucleotide sequences including five reference isolates. Sites with alignment gaps were observed in P4 with one nucleotide deletion in 11 Ethiopian isolates. None of the reference sequence produced frame-shifts, with the exception of the 3-nucleotide deletion in the P4 region by Phytophthora andina, a feature that can be used to distinguish the new Ethiopian isolates from P. andina. While a distinguishing molecular data presented here clearly separated them from P. infestans, 7 % of the isolates that share this feature formed an important component of the late blight pathogen causing disease on Solanum tuberosum in Ethiopia. Thus, these Ethiopian isolates could represent a novel Phytophthora species reported for the first time here.

  2. Feline cholecystitis and acute neutrophilic cholangitis: clinical findings, bacterial isolates and response to treatment in six cases.

    Science.gov (United States)

    Brain, Philip H; Barrs, Vanessa R; Martin, Patricia; Baral, Randolph; White, Joanna D; Beatty, Julia A

    2006-04-01

    Clinicopathological findings from six cats with confirmed cholecystitis or acute neutrophilic cholangitis are presented. Historical findings included lethargy and anorexia or inappetence of up to five days duration. On physical examination all cats were pyrexic and four out of six were jaundiced and had cranial abdominal pain. Bile samples were obtained by cholecystocentesis at exploratory coeliotomy (two cases) or by percutaneous, ultrasound-guided cholecystocentesis (four cases). Gall bladder rupture and bile peritonitis occurred subsequent to ultrasound-guided cholecystocentesis in one case. The most common bacterial isolate was Escherichia coli (four cases); E coli was isolated alone in two cases, in combination with a Streptococcus species (one case) and in combination with a Clostridium species (one case). Streptococcus species alone was isolated from one case, as was Salmonella enterica serovar Typhimurium. The latter is the first reported case of Salmonella-associated cholecystitis in a cat. Concurrent pancreatic or intestinal disease was detected histologically in three cases. All cases were treated with antimicrobials based on in vitro susceptibility results. Treatment was successful in five cases. One cat with concurrent diffuse epitheliotropic intestinal lymphoma was euthanased. Percutaneous ultrasound-guided cholecystocentesis is an effective, minimally-invasive technique enabling identification of bacterial isolates in cats with inflammatory hepatobiliary disease.

  3. A role for the weak DnaA binding sites in bacterial replication origins

    DEFF Research Database (Denmark)

    Charbon, Godefroid; Løbner-Olesen, Anders

    2011-01-01

    DnaA initiates the chromosomal DNA replication in nearly all bacteria, and replication origins are characterized by binding sites for the DnaA protein (DnaA-boxes) along with an ‘AT-rich’ region. However, great variation in number, spatial organization and specificity of DnaA-boxes is observed...... between species. In the study by Taylor et al. (2011), new and unexpectedly weak DnaA-boxes were identified within the Caulobacter crescentus origin of replication (Cori). The position of weak and stronger DnaA-boxes follows a pattern seen in Escherichia coli oriC. This raises the possibility...

  4. Isolation of "Caenorhabditis elegans" Genomic DNA and Detection of Deletions in the "unc-93" Gene Using PCR

    Science.gov (United States)

    Lissemore, James L.; Lackner, Laura L.; Fedoriw, George D.; De Stasio, Elizabeth A.

    2005-01-01

    PCR, genomic DNA isolation, and agarose gel electrophoresis are common molecular biology techniques with a wide range of applications. Therefore, we have developed a series of exercises employing these techniques for an intermediate level undergraduate molecular biology laboratory course. In these exercises, students isolate genomic DNA from the…

  5. A robust and cost-effective method for DNA isolation from Satureja species (Lamiaceae

    Directory of Open Access Journals (Sweden)

    Dodoš Tanja

    2014-01-01

    Full Text Available Aromatic species of the genus Satureja are rich in secondary metabolites that interfere with DNA isolation procedures. Four protocols based on the standard CTDNA extraction protocol of Doyle and Doyle (1987 were tested in six savory taxa. The polyphenol adsorbents activated charcoal and/or polyvinylpyrrolidone 10 were employed in three procedures (B, C and D; for the elimination of polysaccharides, 4M NaCl was applied in the latter two. The highest DNA yield was obtained with Protocol D and averaged 1420.7±398.3 μg DNA/g of dry leaf tissue. Optimal values of the absorbance ratio 260/280 of all DNA solutions revealed the absence or only negligible contamination by proteins. Contamination by polysaccharides inferred from the absorbance ratio 260/230 showed that Protocol C provided the least contaminated material (average of 1.7±0.4. Enzymatic reactions of DNA solutions obtained by Protocol D showed amplification of both loci in all individuals. In conclusion, Protocol D is suitable for the isolation of high quantities of pure DNA from Satureja spp. [Projekat Ministarstva nauke Republike Srbije, br. 173029 i br. 173005

  6. DNA repair in Haemophilus influenzae: isolation and characterization of an ultraviolet sensitive mutator mutant

    Energy Technology Data Exchange (ETDEWEB)

    Walter, R.B.

    1985-01-01

    DNA repair in Haemophilus influenzae appears to be quite different from that seen in Escherichia coli in that H. influenzae shows neither SOS nor adaptation phenomena. Repair of DNA lesions in H. influenzae has been seen to occur via recombinational, excision, and mismatch repair pathways acting independently of one another. The author has isolated an ultraviolet (UV)-sensitive mutator mutant (mutB1) of H. influenzae Rd which shows deficiencies in both recombinational and mismatch repair pathways. This mutant is sensitive to a variety of DNA damaging agents as well as being hypermutable by alkylating agents and base analogues. MutB1 cells do not show post-UV DNA breakdown but do begin excision after UV irradiation. Genetic transformation with UV-irradiated DNA on mut B1 recipients shows that high (HE) and low (LE) efficiency markers are transformed at a ratio of 1.0 as in the mismatch repair deficient hex 1 mutant; however, kinetics of UV-inactivation experiments indicate that HE markers are sensitized and act as LE markers do on wild type recipients. Thus, the mutB gene product appears to play a role in both DNA repair and genetic transformation. A model is outlined which presents a role for a DNA helicase in both DNA repair and genetic transformation of H. influenzae.

  7. Antibiotic Resistance Pattern Of Bacterial Pathogens Isolated From Poultry Manure Used To Fertilize Fish Ponds In New Bussa, Nigeria

    Directory of Open Access Journals (Sweden)

    Funso Omojowo

    2013-02-01

    Full Text Available This study was carried out to isolate and identify antibiotic resistant bacteria from poultry manure usually used for pond fertilization. Poultry manure from 120 Chickens in National Institute for Freshwater Fisheries Research (NIFFR integrated fish farms, New-Bussa, Nigeria was collected. Five bacterial pathogens; Salmonella typhi, Escherichia coli, Shigella dysenteriae, Staphylococcus aureus and Aeromonas hydrophila were isolated. Antibiotic susceptibility testing carried out using the disk diffusion technique. Antibiotics used were; ofloxacin, amoxicillin, tetracycline, ampicillin, erythromycin, gentamicin, nalidixic acid and chloramphenicol. All the isolated organisms were 100% sensitive to ofloxacin. The multiple resistance pattern revealed that 100% were resistant to tetracycline, 84.34% resistant to ampicillin, 76.68% resistant to amoxicillin, 66% resistant to chloramphenicol, 66% resistant to gentamicin, 29% resistant to erythromycin, 28.34% resistant to nalidixic acid. The risk posed by untreated poultry manure used in fish pond fertilization and the public health implications of these results were discussed.

  8. Antimicrobial susceptibility pattern of bacterial isolates from wound infection and their sensitivity to antibiotic agents at super specialty hospital, Amravati city, India

    Directory of Open Access Journals (Sweden)

    Hrishikesh Sawdekar

    2015-02-01

    Full Text Available Background: Wound infection is one of the health problems that is caused and aggravated by the invasion of pathogenic organisms. Information on local pathogens and sensitivity to antimicrobial agent is crucial for successful treatment of wounds. So the present study was conducted to determine antimicrobial susceptibility pattern of bacterial isolates from wound infection and their sensitivity to antimicrobial agents. Methods: A retrospective study was conducted among patients with wound infection in Suyash super speciality hospital, from January 2012 to December 2013. Wound swab was collected using sterile cotton swabs and processed for bacterial isolation and susceptibility testing to Systemic antimicrobial agents. Results: In this study 78 bacterial isolates were recovered from 258 specimens showing an isolation rate of 31.2%. The predominant bacteria isolated from wounds were gram positive staphylococci 36 (46.2%, followed by gram negative streptococci 18 (23.1% gram negative pseudomonas 12 (15.4 % and gram negative proteus 8 (10.4%. The gram positive and gram negative bacteria constituted 68 (87.2% and 10 (12.8% of bacterial isolates; respectively. Conclusion: In the present study most of the pathogens isolated from wound isolates showed high rate of resistance to most commonly used newer antibiotics used to treat bacterial infections. Therefore, rational use of antibiotics should be practiced. [Int J Res Med Sci 2015; 3(2.000: 433-439

  9. A full-length group 1 bacterial sigma factor adopts a compact structure incompatible with DNA binding.

    Science.gov (United States)

    Schwartz, Edmund C; Shekhtman, Alexander; Dutta, Kaushik; Pratt, Matthew R; Cowburn, David; Darst, Seth; Muir, Tom W

    2008-10-20

    The sigma factors are the key regulators of bacterial transcription initiation. Through direct read-out of promoter DNA sequence, they recruit the core RNA polymerase to sites of initiation, thereby dictating the RNA polymerase promoter-specificity. The group 1 sigma factors, which direct the vast majority of transcription initiation during log phase growth and are essential for viability, are autoregulated by an N-terminal sequence known as sigma1.1. We report the solution structure of Thermotoga maritima sigmaA sigma1.1. We additionally demonstrate by using chemical crosslinking strategies that sigma1.1 is in close proximity to the promoter recognition domains of sigmaA. We therefore propose that sigma1.1 autoinhibits promoter DNA binding of free sigmaA by stabilizing a compact organization of the sigma factor domains that is unable to bind DNA.

  10. Two poplar-associated bacterial isolates induce additive favorable responses in a constructed plant-microbiome system

    Directory of Open Access Journals (Sweden)

    Collin M Timm

    2016-04-01

    Full Text Available The biological function of the plant-microbiome system is the result of contributions from the host plant and microbiome members. The Populus root microbiome is a diverse community that has high abundance of β- and γ-Proteobacteria, both classes which include multiple plant-growth promoting representatives. To understand the contribution of individual microbiome members in a community, we studied the function of a simplified community consisting of Pseudomonas and Burkholderia bacterial strains isolated from Populus hosts and inoculated on axenic Populus cutting in controlled laboratory conditions. Both strains increased lateral root formation and root hair production in Arabidopsis plate assays and are predicted to encode for different functions related to growth and plant growth promotion in Populus hosts. Inoculation individually, with either bacterial isolate, increased root growth relative to uninoculated controls, and while root area was increased in mixed inoculation, the interaction term was insignificant indicating additive effects of root phenotype. Complementary data including photosynthetic efficiency, whole-transcriptome gene expression and GC-MS metabolite expression data in individual and mixed inoculated treatments indicate that the effects of these bacterial strains are unique and additive. These results suggest that the function of a microbiome community may be predicted from the additive functions of the individual members.

  11. Direct DNA isolation from solid biological sources without pretreatments with proteinase-K and/or homogenization through automated DNA extraction.

    Science.gov (United States)

    Ki, Jang-Seu; Chang, Ki Byum; Roh, Hee June; Lee, Bong Youb; Yoon, Joon Yong; Jang, Gi Young

    2007-03-01

    Genomic DNA from solid biomaterials was directly isolated with an automated DNA extractor, which was based on magnetic bead technology with a bore-mediated grinding (BMG) system. The movement of the bore broke down the solid biomaterials, mixed crude lysates thoroughly with reagents to isolate the DNA, and carried the beads to the next step. The BMG system was suitable for the mechanical homogenization of the solid biomaterials and valid as an automated system for purifying the DNA from the solid biomaterials without the need for pretreatment or disruption procedures prior to the application of the solid biomaterials.

  12. A potent fish pathogenic bacterial killer Streptomyces sp. isolated from the soils of east coast region, South India

    Directory of Open Access Journals (Sweden)

    Durairaj Thirumurugan

    2013-10-01

    Full Text Available Objective: To investigate the potentiality of the marine actinobacteria isolated from marine soil against fish pathogenic bacteria. Methods: In the present study, a total of 33 soil samples were collected from the Bay of Bengal, east coast region (ECR of Tamilnadu, South India. Then they were used for the isolation of actinobacteria by using conventional serial dilution technique on starch casein agar medium. The antibacterial activities of the actinobacteria were screened primarily by using cross streak plate method against fish pathogenic bacteria namely Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio cholera, Aeromonas sp. and Pseudomonas sp. The antimicrobial efficacy of the selected isolates was carried out with various organic solvents, and finally the active compound was subjected to chromatographic techniques including TLC and GC-MS. Results: Of the 82 actinobacteria isolated, 21 (26% isolates were possessed antibacterial activity against fish pathogenic bacteria. Out of 21 antibacterial isolates, the isolate ECR77 was selected for further study based on its potential activity against fish pathogenic bacteria. Of the various solvents tested, the ethyl acetate extract had good antibacterial activity against the tested bacterial pathogens. The isolate ECR77 grew well on oat meal agar medium with 2% salt level at 35 °C. GC-MS study found that the presence of bioactive compounds namely tetradecanoic acid, n-hexadecanoic acid and octadecanoic acid. The morphological, physiological, biochemical and cultural characteristics of the potential isolate were supported the identity up to generic level as Streptomyces sp. ECR77. Conclusions: The results obtained from this study concludes that the ECR soils of South India is a hot spot of novel bioactive compound producing marine actinobacteria with great pharmaceutical values.

  13. A potent fish pathogenic bacterial killer Streptomyces sp. isolated from the soils of east coast region, South India

    Institute of Scientific and Technical Information of China (English)

    Durairaj Thirumurugan; Ramasamy Vijayakumar

    2013-01-01

    Objective: To investigate the potentiality of the marine actinobacteria isolated from marine soil against fish pathogenic bacteria.Methods:east coast region (ECR) of Tamilnadu, South India. Then they were used for the isolation of actinobacteria by using conventional serial dilution technique on starch casein agar medium. The antibacterial activities of the actinobacteria were screened primarily by using cross streak plate method against fish pathogenic bacteria namely Vibrio alginolyticus, Vibrio parahaemolyticus,Vibrio cholera, Aeromonas sp. and Pseudomonas sp. The antimicrobial efficacy of the selected isolates was carried out with various organic solvents, and finally the active compound was subjected to chromatographic techniques including TLC and GC-MS.Results:In the present study, a total of 33 soil samples were collected from the Bay of Bengal, against fish pathogenic bacteria. Out of 21 antibacterial isolates, the isolate ECR77 was selected for further study based on its potential activity against fish pathogenic bacteria. Of the various solvents tested, the ethyl acetate extract had good antibacterial activity against the tested bacterial pathogens. The isolate ECR77 grew well on oat meal agar medium with 2% salt level at 35 °C. GC-MS study found that the presence of bioactive compounds namely tetradecanoic acid,n-hexadecanoic acid and octadecanoic acid. The morphological, physiological, biochemical and cultural characteristics of the potential isolate were supported the identity up to generic level asStreptomyces sp. ECR77. Conclusions: The results obtained from this study concludes that the ECR soils of South India is a hot spot of novel bioactive compound producing marine actinobacteria with great pharmaceutical values. Of the 82 actinobacteria isolated, 21 (26%) isolates were possessed antibacterial activity.

  14. Designing universal primers for the isolation of DNA sequences encoding Proanthocyanidins biosynthetic enzymes in Crataegus aronia

    Directory of Open Access Journals (Sweden)

    Zuiter Afnan

    2012-08-01

    Full Text Available Abstract Background Hawthorn is the common name of all plant species in the genus Crataegus, which belongs to the Rosaceae family. Crataegus are considered useful medicinal plants because of their high content of proanthocyanidins (PAs and other related compounds. To improve PAs production in Crataegus tissues, the sequences of genes encoding PAs biosynthetic enzymes are required. Findings Different bioinformatics tools, including BLAST, multiple sequence alignment and alignment PCR analysis were used to design primers suitable for the amplification of DNA fragments from 10 candidate genes encoding enzymes involved in PAs biosynthesis in C. aronia. DNA sequencing results proved the utility of the designed primers. The primers were used successfully to amplify DNA fragments of different PAs biosynthesis genes in different Rosaceae plants. Conclusion To the best of our knowledge, this is the first use of the alignment PCR approach to isolate DNA sequences encoding PAs biosynthetic enzymes in Rosaceae plants.

  15. Serine/threonine/tyrosine phosphorylation regulates DNA binding of bacterial transcriptional regulators

    DEFF Research Database (Denmark)

    Kalantari, Aida; Derouiche, Abderahmane; Shi, Lei;

    2015-01-01

    Reversible phosphorylation of bacterial transcriptional regulators (TRs) belonging to the family of two-component systems (TCSs) is a well-established mechanism for regulating gene expression. Recent evidence points to the fact that reversible phosphorylation of bacterial TRs on other types...

  16. Final Scientific Report: Bacterial Nanowires and Extracellular Electron Transfer to Heavy Metals and Radionuclides by Bacterial Isolates from DOE Field Research Centers

    Energy Technology Data Exchange (ETDEWEB)

    Nealson, Kenneth [Univ. of Southern California, Los Angeles, CA (United States)

    2016-12-20

    This proposal involved the study of bacteria capable of transferring electrons from the bacterial cells to electron acceptors located outside the cell. These could be either insoluble minerals that were transformed into soluble products upon the addition of electrons, or they could be soluble salts like uranium or chromium, that become insoluble upon the addition of electrons. This process is called extracellular electron transport or EET, and can be done directly by cellular contact, or via conductive appendages called bacterial nanowires. In this work we examined a number of different bacteria for their ability to perform EET, and also looked at their ability to produce conductive nanowires that can be used for EET at a distance away from the EET-capable cells. In the work, new bacteria were isolated, new abilities of EET were examined, and many new methods were developed, and carefully described in the literature. These studies set the stage for future work dealing with the bioremediation of toxic metals like uranium and chromium. They also point out that EET (and conductive nanowires) are far more common that had been appreciated, and may be involved with energy transfer not only in sediments, but in symbioses between different bacteria, and in symbiosis/pathogenesis between bacteria and higher organisms.

  17. Characteristics of Bacterial Strains from Pseudomonas Genera Isolated from Diseased Plum Trees

    Directory of Open Access Journals (Sweden)

    Veljko Gavrilović

    2008-01-01

    Full Text Available Characteristics of Pseudomonas syringae strains isolated from diseased plum trees are presented is this paper. Based on pathogenic, biochemical and physiological characteristics, isolated starins were divided into two groups: First group of strains, isolated from diseased plum branches with symptoms of suden decay, was simillar to Pseudomonas syringae pv. syringae; second group of strains, isolated from necrotic flower buds on plum trees, exhibited characteristics simillar to Pseudomonas syringae pv. morsprunorum. In addition, phytopathogenic fungi belonging to genera Phomopsis, Botryosphaeria and Leucostoma, were also isolated from diseased plum trees. Further study of these pathogens and their role in the epidemiology of suden plum trees decay is in progress.

  18. Host Characteristics and Bacterial Traits Predict Experimental Virulence for Escherichia coli Bloodstream Isolates From Patients With Urosepsis.

    Science.gov (United States)

    Johnson, James R; Porter, Stephen; Johnston, Brian; Kuskowski, Michael A; Spurbeck, Rachel R; Mobley, Harry L T; Williamson, Deborah A

    2015-09-01

    Background.  Extraintestinal Escherichia coli infections are common, costly, and potentially serious. A better understanding of their pathogenesis is needed. Methods.  Sixty-seven E coli bloodstream isolates from adults with urosepsis (Seattle, WA; 1980s) underwent extensive molecular characterization and virulence assessment in 2 infection models (murine subcutaneous sepsis and moth larval lethality). Statistical comparisons were made among host characteristics, bacterial traits, and experimental virulence. Results.  The 67 source patients were diverse for age, sex, and underlying medical and urological conditions. The corresponding E coli isolates exhibited diverse phylogenetic backgrounds and virulence profiles. Despite the E coli isolates' common bloodstream origin, they exhibited a broad range of experimental virulence in mice and moth larvae, in patterns that (for the murine model only) corresponded significantly with host characteristics and bacterial traits. The most highly mouse-lethal strains were enriched with classic "urovirulence" traits and typically were from younger women with anatomically and functionally normal urinary tracts. The 2 animal models corresponded poorly with one another. Conclusions.  Host compromise, including older age and urinary tract abnormalities, allows comparatively low-virulence E coli strains to cause urosepsis. Multiple E coli traits predict both experimental and epidemiological virulence. The larval lethality model cannot be a substitute for the murine sepsis model.

  19. Isolation and characterization of high quality DNA from marine benthic macroalgae.

    Science.gov (United States)

    Chakraborty, S; Vijayan, K; Nair, C V; Santra, S C; Bhattacharya, T

    2008-11-01

    The isolation of high quality DNA is essential for many molecular biology applications including polymerase chain reaction (PCR) and endonuclease restriction digestion based techniques. An easy and inexpensive protocol has been developed for extracting genomic DNA from seven species of algae viz. Lola capillaries, Enteromorpha intestinalis, Ulva lactuca and Rhizoclonium sp belonging to Chlorophyceae, Catenella nipae, Polysiphonia mollis belonging to Rhodophyceae and Dictyota ceylanica belonging to Phaeophyceae group were collected from the coastal regions of Sunderban delta in West Bengal, India dominantly growing on mud flats, bark of different mangrove trees, pneumatophores, stilt roots, concrete surfaces, wooden and bamboo poles, sides of the boats and other water vehicles inundated during high tides. The DNA was found suitable for restriction endonuclease digestion and PCR amplification with randomely amplified polymorphic DNA (RAPD) primers. The A260/A280 ratio of 1.15 0.14 to 1.94 indicated little contamination from proteins and polysaccharides. The PCR amplification with RAPD primers showed its suitability in PCR based techniques and the restriction digestion with Eco RV confirmed its suitability for hybridization based techniques. The protocol is equally good for isolating DNA from both fresh as well as preserved materials.

  20. A rapid and inexpensive method for isolation of total DNA from Trichoderma spp (Hypocreaceae).

    Science.gov (United States)

    Vazquez-Angulo, J C; Mendez-Trujillo, V; González-Mendoza, D; Morales-Trejo, A; Grimaldo-Juarez, O; Cervantes-Díaz, L

    2012-05-15

    Extraction of high-quality genomic DNA for PCR amplification from filamentous fungi is difficult because of the complex cell wall and the high concentrations of polysaccharides and other secondary metabolites that bind to or co-precipitate with nucleic acids. We developed a modified sodium dodecyl sulfate/phenol protocol, without maceration in liquid nitrogen and without a final ethanol precipitation step. The A(260/280) absorbance ratios of isolated DNA were approximately 1.7-1.9, demonstrating that the DNA fraction is pure and can be used for analysis. Additionally, the A(260/230) values were higher than 1.6, demonstrating negligible contamination by polysaccharides. The DNA isolated by this protocol is of sufficient quality for molecular applications; this technique could be applied to other organisms that have similar substances that hinder DNA extraction. The main advantages of the method are that the mycelium is directly recovered from culture medium and it does not require the use of expensive and specialized equipment.

  1. Germinal transmission of site-specific excised genomic DNA by the bacterial ParA resolvase

    Science.gov (United States)

    Genome engineering is an essential tool in research and product development. Behind some of the recent advances in plant gene transfer is the development of site-specific recombination systems that enable the precise manipulation of DNA, e.g. the deletion, integration or translocation of DNA. DNA ...

  2. Kinetics of kill of bacterial conjunctivitis isolates with moxifloxacin, a fluoroquinolone, compared with the aminoglycosides tobramycin and gentamicin

    Directory of Open Access Journals (Sweden)

    Rudolph S Wagner

    2010-01-01

    Full Text Available Rudolph S Wagner1, David B Granet2, Steven J Lichtenstein3, Tiffany Jamison4, Joseph J Dajcs4, Robert D Gross5, Paul Cockrum41New Jersey Medical School, Newark, NJ, USA; 2Ratner Children’s Eye Center, University of California – San Diego, La Jolla, CA, USA; 3University of Illinois College of Medicine at Peoria, Peoria, Illinois, USA; 4Alcon Research, Ltd, Fort Worth, TX, USA; 5Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX, USAPurpose: To compare the kinetics and speed of kill of Streptococcus pneumoniae and Haemophilus influenzae on exposure to three topical ophthalmic antibiotic solutions.Materials and methods: Bacterial conjunctivitis isolates of S. pneumoniae and H. influenzae were exposed to 1:1000 dilutions of moxifloxacin 0.5%, tobramycin 0.3%, gentamicin 0.3%, and water (control. At 15, 30, 60, 120, and 180 minutes after exposure, aliquots were collected, cells were cultured, and viable cell counts were determined using standard microbiological methods.Results: Moxifloxacin achieved 99.9% kill (3-log reduction at approximately 2 hours for S. pneumoniae and at 15 minutes for H. influenzae. Tobramycin and gentamicin did not achieve 3-log reduction of S. pneumoniae during the 180-minute study period. An increase in bacterial growth was noted for these isolates. Gentamicin took more than 120 minutes to achieve the 3-log reduction of H. influenzae and tobramycin did not reach the 3-log reduction of this pathogen during the 180-minute study period.Conclusion: Moxifloxacin killed S. pneumoniae and H. influenzae in vitro faster than tobramycin and gentamicin, suggesting its potential clinical benefit as a first-line treatment for bacterial conjunctivitis to minimize patient symptoms and to limit the contagiousness of the disease.Keywords: kinetics of kill, bacterial conjunctivitis, in vitro, Streptococcus pneumoniae, Haemophilus influenzae, fluoroquinolones, aminoglycosides

  3. Chromatin Isolation and DNA Sequence Analysis in Large Undergraduate Laboratory Sections

    Science.gov (United States)

    Hagerman, Ann E.

    1999-10-01

    A pair of exercises that introduce undergraduate students to basic techniques and concepts of molecular biology and that are appropriate for classes with large enrollments are described. One exercise is a simple laboratory experiment in which chromatin is isolated from chicken liver and is resolved into histone proteins and DNA by ion-exchange chromatography. The other is a series of computer simulations that introduce DNA sequencing, mapping, and sequence analysis to the students. The final step of the simulation is submission of a sequence to a database on the World Wide Web for identification of the protein product of the gene.

  4. Comparison of 16S rDNA-PCR Amplification and Culture of Cerebrospinal Fluid for Diagnosis of Bacterial Meningitis

    Directory of Open Access Journals (Sweden)

    Farshad Foroughi

    2010-12-01

    Full Text Available Objective:Early and accurate diagnosis of bacterial meningitis is of critical concern. Optimum and rapid laboratory facilities are not routinely available for detecting the etiologic agents of meningitis. The objective of this study was to compare polymerase chain reaction (PCR assay with culture for detection of bacteria in central nervous system (CNS samples from patients suspected to have meningitis. Methods: One-hundred CSF samples were obtained and divided into two parts. One part of samples was used for standard bacterial culture and gram staining. The remaining was used for DNA extraction. PCR assay was performed with universal primers for 16S rDNA gene of bacteria. Performance characteristics of the test were determined. Findings:The PCR method was able to detect bacteria in all 36 culture-positive and in 38 of 64 culture-negative cases showing sensitivity and specificity of 100% and 40.6% respectively. Positive predictive value was 48.6% and negative predictive value 100%, however, Kappa coefficient showed the correlation of the 2 methods to be at 0.33. Conclusion:There are advantages and disadvantages in performance characteristics of the conventional CSF culture and universal CSF 16S rDNA PCR. Therefore, it is recommended to use both methods in clinical practice, particularly in suspicious contaminated samples, with presumable presence of fastidious or slow growing bacteria because of antibiotic consumption.

  5. Mutational Analysis of Bacterial NAD+-dependent DNA Ligase:Role of Motif Ⅳ in Ligation Catalysis

    Institute of Scientific and Technical Information of China (English)

    Hong FENG

    2007-01-01

    The bacterial DNA ligase as a multiple domain protein is involved in DNA replication, repair and recombination. Its catalysis of ligation can be divided into three steps. To delineate the roles of amino acid residues in motif Ⅳ in ligation catalysis, site-directed mutants were constructed in a bacterial NAD+-dependent DNA ligase from Thermus sp. TAK16D. It was shown that four conserved residues (D286, G287, V289 and K291) in motif Ⅳ had significant roles on the overall ligation. Under single turnover conditions, the observed apparent rates of D286E, G287A, V289I and K291R mutants were clearly reduced compared with that of WT ligase on both match and mismatch nicked substrates. The effects of D286E mutation on overall ligation may not only be ascribed to the third step. The G287A mutation has a major effect on the second step. The effects of V289I and K291R mutation on overall ligation are not on the third step, perhaps other aspects, such as conformation change of ligase protein in ligation catalysis, are involved. Moreover, the amino acid substitutions of above four residues were more sensitive on mismatch nicked substrate, indicating an enhanced ligation fidelity.

  6. Prevalence of antimicrobial resistance among bacterial pathogens isolated from cattle in different European countries: 2002–2004

    Directory of Open Access Journals (Sweden)

    Stärk Katharina

    2008-07-01

    Full Text Available Abstract Background The project "Antibiotic resistance in bacteria of animal origin – II" (ARBAO-II was funded by the European Union (FAIR5-QLK2-2002-01146 for the period 2003–2005, with the aim to establish a continuous monitoring of antimicrobial susceptibility among veterinary laboratories in European countries based on validated and harmonised methodologies. Available summary data of the susceptibility testing of the bacterial pathogens from the different laboratories were collected. Method Antimicrobial susceptibility data for several bovine pathogens were obtained over a three year period (2002–2004. Each year the participating laboratories were requested to fill in excel-file templates with national summary data on the occurrence of antimicrobial resistance from different bacterial species. A proficiency test (EQAS – external quality assurance system for antimicrobial susceptibility testing was conducted each year to test the accuracy of antimicrobial susceptibility testing in the participating laboratories. The data from this testing demonstrated that for the species included in the EQAS the results are comparable between countries. Results Data from 25,241 isolates were collected from 13 European countries. For Staphylococcus aureus from bovine mastitis major differences were apparent in the occurrence of resistance between countries and between the different antimicrobial agents tested. The highest frequency of resistance was observed for penicillin. For Mannheimia haemolytica resistance to ampicillin, tetracycline and trimethoprim/sulphonamide were observed in France, the Netherlands and Portugal. All isolates of Pasteurella multocida isolated in Finland and most of those from Denmark, England (and Wales, Italy and Sweden were susceptible to the majority of the antimicrobials. Streptococcus dysgalactiae and Streptococcus uberis isolates from Sweden were fully susceptible. For the other countries some resistance was observed to

  7. Removal of 2,4,5-trichlorophenol by bacterial isolates from the secondary sludge of pulp and paper mill.

    Science.gov (United States)

    Karn, Santosh Kr; Reddy, M Sudhakara

    2013-09-01

    2,4,5-trichlorophenol (2,4,5-TCP) mineralizing bacteria were isolated from the secondary sludge of pulp and paper industry. These isolates used 2,4,5-TCP as a source of carbon and energy and were capable of degrading this compound, as indicated by stoichimetric release of chloride and biomass formation. Based on 16S rRNA sequence analysis, these bacteria were identified as Kocuria sp. (CL2), Bacillus pumillus (CL5), Pseudomonas stutzeri (CL7). HPLC analysis revealed that these isolates were able to degrade 2,4,5-TCP at higher concentrations (600 mg/l or 3.0 mM). A consortia of these isolates completely removed 2,4,5-TCP from the sludge obtained from pulp and paper mill within 2 weeks when supplemented at a rate of 100 mg l(-1) . Bacterial consortium also significantly reduced absorbable organic halogen (AOX) and extractable organic halogen (EOX) by 61% and 63%, respectively from the sludge. These isolates have high potential to remove 2,4,5-TCP and may be used for remediation of pulp paper mill waste containing 2,4,5-TCP.

  8. DistAMo: A web-based tool to characterize DNA-motif distribution on bacterial chromosomes

    Directory of Open Access Journals (Sweden)

    Patrick eSobetzko

    2016-03-01

    Full Text Available Short DNA motifs are involved in a multitude of functions such as for example chromosome segregation, DNA replication or mismatch repair. Distribution of such motifs is often not random and the specific chromosomal pattern relates to the respective motif function. Computational approaches which quantitatively assess such chromosomal motif patterns are necessary. Here we present a new computer tool DistAMo (Distribution Analysis of DNA Motifs. The algorithm uses codon redundancy to calculate the relative abundance of short DNA motifs from single genes to entire chromosomes. Comparative genomics analyses of the GATC-motif distribution in γ-proteobacterial genomes using DistAMo revealed that (i genes beside the replication origin are enriched in GATCs, (ii genome-wide GATC distribution follows a distinct pattern and (iii genes involved in DNA replication and repair are enriched in GATCs. These features are specific for bacterial chromosomes encoding a Dam methyltransferase. The new software is available as a stand-alone or as an easy-to-use web-based server version at http://www.computational.bio.uni-giessen.de/distamo.

  9. Self-Assembled Functional Nanostructure of Plasmid DNA with Ionic Liquid [Bmim][PF₆]: Enhanced Efficiency in Bacterial Gene Transformation.

    Science.gov (United States)

    Soni, Sarvesh K; Sarkar, Sampa; Mirzadeh, Nedaossadat; Selvakannan, P R; Bhargava, Suresh K

    2015-04-28

    The electrostatic interaction between the negatively charged phosphate groups of plasmid DNA and the cationic part of hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF6]), initiates spontaneous self-assembly to form the functional nanostructures made up of DNA and ionic liquid (IL). These functional nanostructures were demonstrated as promising synthetic nonviral vectors for the efficient bacterial pGFP gene transformation in cells. In particular, the functional nanostructures that were made up of 1 μL of IL ([Bmim][PF6]) and 1 μg of plasmid DNA can increase the transformation efficiency by 300-400% in microbial systems, without showing any toxicity for E. coli DH5α cells. (31)P nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron (XPS) spectroscopic analysis revealed that the electrostatic interaction between negatively charged phosphate oxygen and cationic Bmim(+) tends to initiate the self-assembly process. Thermogravimetric analysis of the DNA-IL functional nanostructures showed that these nanostructures consist of ∼16 wt % ionic liquid, which is considered to provide the stability to the plasmid DNA that eventually enhanced the transformation efficiency.

  10. Human Mitochondrial DNA and Endogenous Bacterial Surrogates for Risk Assessment of Graywater Reuse

    Science.gov (United States)

    Previous graywater risk assessment studies have focused on fecal contamination, yet the low density of fecal indicators may not provide the most useful approach to assess pathogen removal during graywater treatment. In this study, we employed high throughput bacterial sequencing ...

  11. Characterization of Methicillin-Resistant Staphylococcus aureus Isolated from Healthy Turkeys and Broilers Using DNA Microarrays

    Science.gov (United States)

    El-Adawy, Hosny; Ahmed, Marwa; Hotzel, Helmut; Monecke, Stefan; Schulz, Jochen; Hartung, Joerg; Ehricht, Ralf; Neubauer, Heinrich; Hafez, Hafez M.

    2016-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a major human health problem and recently, domestic animals are described as carriers and possible reservoirs. Twenty seven S. aureus isolates from five turkey farms (n = 18) and two broiler farms (n = 9) were obtained by culturing of choana and skin swabs from apparently healthy birds, identified by Taqman-based real-time duplex nuc-mecA-PCR and characterized by spa typing as well as by a DNA microarray based assay which covered, amongst others, a considerable number of antibiotic resistance genes, species controls, and virulence markers. The antimicrobial susceptibility profiles were tested by agar diffusion assays and genotypically confirmed by the microarray. Five different spa types (3 in turkeys and 2 in broilers) were detected. The majority of MRSA isolates (24/27) belonged to clonal complex 398-MRSA-V. The most frequently occurring spa types were accordingly t011, t034, and t899. A single CC5-MRSA-III isolated from turkey and CC398-MRSA with an unidentified/truncated SCCmec element in turkey and broiler were additionally detected. The phenotypic antimicrobial resistance profiles of S. aureus isolated from both turkeys and broilers against 14 different antimicrobials showed that all isolates were resistant to ampicillin, cefoxitin, oxacillin, doxycycline, and tetracycline. Moreover, all S. aureus isolated from broilers were resistant to erythromycin and azithromycin. All isolates were susceptible to gentamicin, chloramphenicol, sulphonamides, and fusidic acid. The resistance rate against ciprofloxacin was 55.6% in broiler isolates and 42.1% in turkey isolates. All tetracycline resistant isolates possessed genes tetK/M. All erythromycin-resistant broiler isolates carried ermA. Only one broiler isolate (11.1%) carried genes ermA, ermB, and ermC, while 55.6% of turkey isolates possessed ermA and ermB genes. Neither PVL genes (lukF/S-PV), animal-associated leukocidin (lukM and luk-P83) nor the gene encoding

  12. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment.

    Science.gov (United States)

    Kennedy, Edward M; Cullen, Bryan R

    2015-05-01

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  13. Carboxyl-functionalized magnetic microparticle carrier for isolation and identification of DNA in dairy products

    Energy Technology Data Exchange (ETDEWEB)

    Horak, Daniel [Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovskeho Sq. 2, 162 06 Prague 6 (Czech Republic)]. E-mail: horak@imc.cas.cz; Rittich, Bohuslav [Masaryk University Brno, Tvrdeho 14, 602 00 Brno (Czech Republic)]. E-mail: rittich@sci.muni.cz; Spanova, Alena [Masaryk University Brno, Tvrdeho 14, 602 00 Brno (Czech Republic)]. E-mail: spanova@sci.muni.cz

    2007-04-15

    Magnetite nanoparticles about 14nm in diameter were obtained by chemical coprecipitation of Fe(II) and Fe(III) salts with aqueous ammonia in the presence of poly(ethylene glycol) (PEG). Magnetic poly(glycidyl methacrylate) (PGMA) microspheres about 1{mu}m in diameter were prepared by dispersion polymerization of GMA in aqueous ethanol in the presence of PEG-coated magnetite nanoparticles. The microspheres were hydrolyzed and carboxyl groups introduced by oxidation with KMnO{sub 4}. The particles reversibly bound bacterial DNA of Bifidobacterium and Lactobacillus genera in the presence of high concentrations of PEG 6000 and sodium chloride from crude cell lysates of various dairy products (butter milk, cheese, yoghurt, probiotic tablets) or from cell lyophilisates. The presence of Bifidobacterium and Lactobacillus DNA in samples was confirmed by PCR amplification.

  14. Priming the immune system of Penaeid shrimp by bacterial HSP70 (DnaK).

    Science.gov (United States)

    Phuoc, L H; Hu, B; Wille, M; Hien, N T; Phuong, V H; Tinh, N T N; Loc, N H; Sorgeloos, P; Bossier, P

    2016-05-01

    This study was conducted to test the effect of DnaK on priming immune responses in Penaeid shrimp. Juvenile-specific pathogen-free (SPF) P. vannamei shrimp were injected with 0.05 μg recombinant DnaK. One hour post-DnaK priming, a non-lethal dose of Vibrio campbellii (10(5) CFU shrimp(-1)) was injected. Other treatments include only DnaK or V. campbellii injection or control with blank inocula. The haemolymph of three shrimp from each treatment was collected at 1.5, 6, 9 and 12 h post-DnaK priming (hpp). It was verified that injection with DnaK and V. campbellii challenge affected the transcription of 3 immune genes, transglutaminase-1 (TGase-1), prophenoloxidase-2 (proPO-2) and endogenous HSP70 (lvHSP70). In P. monodon, shrimp were first injected with DnaK at a dose of 10 μg shrimp(-1) and one hour later with 10(6) CFU of V. harveyi (BB120) shrimp(-1). Shrimp injected with DnaK showed a significant increase in proPO expression compared to the control (P < 0.05). Yet a double injection (DnaK and Vibrio) seemed to cause an antagonistic response at the level of expression, which was not equalled at the level of PO activity. Those results suggest that DnaK is able to modulate immune responses in P. vannamei and P. monodon.

  15. A Simple and Rapid System for DNA and RNA Isolation from Diverse Plants Using Handmade Kit

    OpenAIRE

    sprotocols

    2016-01-01

    This protocol describes a rapid DNA and RNA extraction from plant tissues as a handmade kit. Hexadecyltrimethylammonium bromide (CTAB), Sodium chloride (NaCl), Tris base, and Ethylenediaminetetraacetic acid (EDTA) are the main components of this extraction buffer. In contrast to previous works and all reported protocols, the required components of this protocol have established without providing any stock solutions and it will be prepared just by adding them directly. This isolation buffer ca...

  16. Biodegradation of complex hydrocarbons in spent engine oil by novel bacterial consortium isolated from deep sea sediment.

    Science.gov (United States)

    Ganesh Kumar, A; Vijayakumar, Lakshmi; Joshi, Gajendra; Magesh Peter, D; Dharani, G; Kirubagaran, R

    2014-10-01

    Complex hydrocarbon and aromatic compounds degrading marine bacterial strains were isolated from deep sea sediment after enrichment on spent engine (SE) oil. Phenotypic characterization and phylogenetic analysis of 16S rRNA gene sequences showed the isolates were related to members of the Pseudoalteromonas sp., Ruegeria sp., Exiguobacterium sp. and Acinetobacter sp. Biodegradation using 1% (v/v) SE oil with individual and mixed strains showed the efficacy of SE oil utilization within a short retention time. The addition of non-ionic surfactant 0.05% (v/v) Tween 80 as emulsifying agent enhanced the solubility of hydrocarbons and renders them more accessible for biodegradation. The degradation of several compounds and the metabolites formed during the microbial oxidation process were confirmed by Fourier transform infrared spectroscopy and Gas chromatography-mass spectrometry analyses. The potential of this consortium to biodegrade SE oil with and without emulsifying agent provides possible application in bioremediation of oil contaminated marine environment.

  17. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

    DEFF Research Database (Denmark)

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.;

    2015-01-01

    organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below -10ºC. Multivariate statistical analysis of the bacterial diversity data (DNA......N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable...... was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June...

  18. Bacterial spores as particulate carriers for gene gun delivery of plasmid DNA.

    Science.gov (United States)

    Aps, Luana R M M; Tavares, Milene B; Rozenfeld, Julio H K; Lamy, M Teresa; Ferreira, Luís C S; Diniz, Mariana O

    2016-06-20

    Bacillus subtilis spores represent a suitable platform for the adsorption of proteins, enzymes and viral particles at physiological conditions. In the present work, we demonstrate that purified spores can also adsorb DNA on their surface after treatment with cationic molecules. In addition, we demonstrate that DNA-coated B. subtilis spores can be used as particulate carriers and act as an alternative to gold microparticles for the biolistic (gene gun) administration of plasmid DNA in mice. Gene gun delivery of spores pre-treated with DODAB (dioctadecyldimethylammonium bromide) allowed efficient plasmid DNA absorption and induced protein expression levels similar to those obtained with gold microparticles. More importantly, we demonstrated that a DNA vaccine adsorbed on spores can be loaded into biolistic cartridges and efficiently delivered into mice, which induced specific cellular and antibody responses. Altogether, these data indicate that B. subtilis spores represent a simple and low cost alternative for the in vivo delivery of DNA vaccines by the gene gun technology.

  19. Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatography.

    Science.gov (United States)

    Hilbrig, Frank; Freitag, Ruth

    2012-01-01

    Hydroxyapatite and related stationary phases increasingly play a role in the downstream processing of high-value biological materials, such as recombinant proteins, therapeutic antibodies and pharmaceutical-grade plasmid DNA. Chromatographic hydroxyapatite is an inorganic, ceramic material identical in composition, if not in structure, to calcium phosphate found in human bones and teeth. The interaction of hydroxyapatite with biomacromolecules is complex and highly dynamic, which can make predicting performance difficult, but also allows the design of very selective isolation processes. This review discusses the currently commercially available chromatographic materials, different retention mechanisms supported by these materials and differential exploitation for the design of highly specific isolation procedures. The state of the art of antibody purification by hydroxy- and fluoroapatite is reviewed together with tested routines for method development and implementation. Finally, the isolation of plasmid DNA is discussed, since the purification of DNA therapeutics at a sufficiently large scale is an emerging need in bioprocess development and perhaps the area in bioseparation where apatite chromatography can make its most important contribution to date.

  20. Potential of Polycyclic Aromatic Hydrocarbon-Degrading Bacterial Isolates to Contribute to Soil Fertility

    Science.gov (United States)

    Chirima, George Johannes

    2016-01-01

    Restoration of polycyclic aromatic hydrocarbon- (PAH-) polluted sites is presently a major challenge in agroforestry. Consequently, microorganisms with PAH-degradation ability and soil fertility improvement attributes are sought after in order to achieve sustainable remediation of polluted sites. This study isolated PAH-degrading bacteria from enriched cultures of spent automobile engine-oil polluted soil. Isolates' partial 16S rRNA genes were sequenced and taxonomically classified. Isolates were further screened for their soil fertility attributes such as phosphate solubilization, atmospheric nitrogen fixation, and indoleacetic acid (IAA) production. A total of 44 isolates were obtained and belong to the genera Acinetobacter, Arthrobacter, Bacillus, Flavobacterium, Microbacterium, Ochrobactrum, Pseudomonas, Pseudoxanthomonas, Rhodococcus, and Stenotrophomonas. Data analysed by principal component analysis showed the Bacillus and Ochrobactrum isolates displayed outstanding IAA production. Generalized linear modelling statistical approaches were applied to evaluate the contribution of the four most represented genera (Pseudomonas, Acinetobacter, Arthrobacter, and Rhodococcus) to soil fertility. The Pseudomonas isolates were the most promising in all three soil fertility enhancement traits evaluated and all isolates showed potential for one or more of the attributes evaluated. These findings demonstrate a clear potential of the isolates to participate in restorative bioremediation of polluted soil, which will enhance sustainable agricultural production and environmental protection. PMID:27774456

  1. Christensenella timonensis, a new bacterial species isolated from the human gut

    Directory of Open Access Journals (Sweden)

    S. Ndongo

    2016-09-01

    Full Text Available We propose a new species, Christensenella timonensis, strain Marseille-P2437T (CSUR P2437T, which was isolated from gut microbiota of a 66-year-old patient as a part of culturomics study. C. timonensis represents the second species isolated within the Christensenella genus.

  2. Isolation and Characterization of Phytoene Desaturase cDNA from Stigma of Crocus sativus

    Institute of Scientific and Technical Information of China (English)

    Bai Jie(白洁); Xu Ying; Tang Lin; Zeng Yu; Feng Yun; Wang Shenghua; Chen Fang

    2004-01-01

    Phytoene desaturase (PDS) has recently been identified as an important enzyme in carotenoid biosynthesis pathway. A cDNA clone encoding phytoene desaturase gene is isolated from stigma of saffron (Crocus sativus L.) using RT-PCR technique. Sequence analysis shows 83% similarity to Narcissus pseudonarcissus, 79% to Zea mays, 78% to Arabidopsis thaliana, 77% to Lycopersicon esculentum. A new full-length cDNA is obtained by 5'-RACE and 3' -RACE techniques. The cDNA is 2149bp long with an open reading frame of 1697bp, which encodes a polypeptide of 565 amino acids. Southern analysis shows that the PDS gene is a single copy in saffron. Northern blot analysis shows higher expression level of PDS gene in stigma and anther than in leaves and stem.

  3. A simple model for DNA bridging proteins and bacterial or human genomes: bridging-induced attraction and genome compaction

    Science.gov (United States)

    Johnson, J.; Brackley, C. A.; Cook, P. R.; Marenduzzo, D.

    2015-02-01

    We present computer simulations of the phase behaviour of an ensemble of proteins interacting with a polymer, mimicking non-specific binding to a piece of bacterial DNA or eukaryotic chromatin. The proteins can simultaneously bind to the polymer in two or more places to create protein bridges. Despite the lack of any explicit interaction between the proteins or between DNA segments, our simulations confirm previous results showing that when the protein-polymer interaction is sufficiently strong, the proteins come together to form clusters. Furthermore, a sufficiently large concentration of bridging proteins leads to the compaction of the swollen polymer into a globular phase. Here we characterise both the formation of protein clusters and the polymer collapse as a function of protein concentration, protein-polymer affinity and fibre flexibility.

  4. Isolation and cDNA cloning of somatolactin in rabbitfish (Siganus guttatus).

    Science.gov (United States)

    Ayson, F G; de Jesus, E G; Amemiya, Y; Moriyama, S; Hirano, T; Kawauchi, H

    1999-08-01

    We report the isolation and cDNA cloning of somatolactin (SL) from rabbitfish, Siganus guttatus. Rabbitfish SL was isolated from an alkaline extract of the pituitary glands by gel filtration chromatography on Sephadex G-100 and reversed-phase high-performance liquid chromatography. SL was monitored by immunoblotting with flounder SL antiserum. The preparation (yield: 0.86 mg/g wet tissues) contained two immunoreactive bands of 24 and 28 kDa on SDS-PAGE. Overlapping partial cDNA clones corresponding to teleost SLs were amplified by PCR from single-strand cDNA from pituitary glands. Excluding the poly(A) tail, rabbitfish SL cDNA is 1605 bp long. It contains a 693-bp open reading frame encoding a signal peptide of 24 amino acids (aa) and a mature protein of 207 aa. Rabbitfish SL has two possible N-glycosylation sites at positions 11 and 121 and seven half Cys residues. The deduced amino acid sequence shows over 80% identity with those of advanced teleosts like sea bream, red drum, and flounder, 76% with the salmonids, 57% with the eel, and 46% with the goldfish SL.

  5. Characterization of mitochondrial DNA in various Candida species: isolation, restriction endonuclease analysis, size, and base composition.

    Science.gov (United States)

    Su, C S; Meyer, S A

    1991-01-01

    A practical and effective method for the extraction of mitochondrial DNA from Candida species was developed. Zymolyase was used to induce yeast protoplasts, and mitochondrial DNA was extracted from DNase I-treated mitochondrial preparations. Restriction endonuclease analyses of mitochondrial DNAs from 19 isolates representing seven species of Candida (C. albicans, C. kefyr, C. lusitaniae, C. maltosa, C. parapsilosis, C. shehatae, and C. tropicalis) and Lodderomyces elongisporus revealed different cleavage patterns that appeared to be specific for the species. Few common restriction fragments were evident. The genome sizes of the mitochondrial DNAs ranged from 26.4 to 51.4 kilobase pairs, and the guanine-plus-cytosine contents ranged from 20.7 to 36.8 mol%. There was no correlation between the base compositions of nuclear and mitochondrial DNAs. Eight isolates of C. parapsilosis, including the type culture, and an ascosporogenous strain of L. elongisporus, which was once proposed as the teleomorph of C. parapsilosis, had similar mitochondrial DNA molecular sizes (30.2 and 28.8 kilobase pairs); however, restriction endonuclease patterns of these organisms were distinct. These data provide additional support for discrimination of these two species. The results of our experiments demonstrate that mitochondrial DNA analyses may provide useful criteria for the differentiation of yeast species.

  6. An Improved Multiplex Real-Time SYBR Green PCR Assay for Analysis of 24 Target Genes from 16 Bacterial Species in Fecal DNA Samples from Patients with Foodborne Illnesses.

    Science.gov (United States)

    Kawase, Jun; Etoh, Yoshiki; Ikeda, Tetsuya; Yamaguchi, Keiji; Watahiki, Masanori; Shima, Tomoko; Kameyama, Mitsuhiro; Horikawa, Kazumi; Fukushima, Hiroshi; Goto, Ryoichi; Shirabe, Komei

    2016-05-20

    Here, we developed a new version of our original screening system (Rapid Foodborne Bacterial Screening 24; RFBS24), which can simultaneously detect 24 genes of foodborne pathogens in fecal DNA samples. This new version (RFBS24 ver. 5) detected all known stx2 subtypes, enterotoxigenic Escherichia coli (STh genotype), and Vibrio parahaemolyticus (trh2), which were not detected by the original RFBS24 assay. The detection limits of RFBS24 ver. 5 were approximately 5.6 × 10(-2)-5.6 × 10(-5) (ng DNA)/reaction, significantly lower (10- to 100-fold) than those of the original RFBS24 for the 22 target genes analyzed here. We also tested the new assay on fecal DNA samples from patients infected with Salmonella, Campylobacter, or enterohemorrhagic E. coli. The number of bacterial target genes detected by RFBS24 ver. 5 was greater than that detected by RFBS24. RFBS24 ver. 5 combined with an Ultra Clean Fecal DNA Isolation Kit showed adequate performance (sensitivity and specificity 89% and 100%, respectively, for Salmonella spp. and 100% and 83%, respectively, for Campylobacter jejuni) in terms of rapid detection of a causative pathogen during foodborne-illness outbreaks. Thus, RFBS24 ver. 5 is more useful than the previous assay system for detection of foodborne pathogens and offers quick simultaneous analysis of many targets and thus facilitates rapid dissemination of information to public health officials.

  7. Activity of Norspermidine on Bacterial Biofilms of Multidrug-Resistant Clinical Isolates Associated with Persistent Extremity Wound Infections.

    Science.gov (United States)

    Cardile, Anthony P; Woodbury, Ronald L; Sanchez, Carlos J; Becerra, Sandra C; Garcia, Rebecca A; Mende, Katrin; Wenke, Joseph C; Akers, Kevin S

    2016-11-19

    Biofilm formation is a major virulence factor for numerous pathogenic bacteria and is cited as a central event in the pathogenesis of chronic human infections, which is in large part due to excessive extracellular matrix secretion and metabolic changes that occur within the biofilm rendering them highly tolerant to antimicrobial treatments. Polyamines, including norspermidine, play central roles in bacterial biofilm development, but have also recently been shown to inhibit biofilm formation in select strains of various pathogenic bacteria. The aim of this study was to evaluate in vitro the biofilm dispersive and inhibitory activities of norspermidine against multidrug-resistant clinical isolates of Acinetobacter baumannii(n = 4), Klebsiella pneumoniae (n = 3), Pseudomonas aeruginosa (n = 5) and Staphylococcus aureus (n = 4) associated with chronic extremity wound infections using the semi-quantitative 96-well plate method and confocal laser microscopy. In addition to the antibiofilm activity, biocompatibility of norspermidine was also evaluated by measuring toxicity in vitro to human cell lines and whole porcine tissue explants using MTT viability assay and histological analysis. Norspermidine (5-20 mM) had variable dispersive and inhibitory activity on biofilms which was dependent on both the strain and species. Of the clinical bacterial species evaluated herein, A. baumannii isolates were the most sensitive to the effect of norspermidine, which was in part due to the inhibitory effects of norspermidine on bacterial motility and expression of genes involved in the production of homoserine lactones and quorum sensing molecules both essential for biofilm formation. Importantly, exposure of cell lines and whole tissues to norspermidine for prolonged periods of time (≥24 h) was observed to reduce viability and alter tissue histology in a time and concentration dependent manner, with 20 mM exposure having the greatest negative effects on both

  8. The uptake of Ni2+ and Ag+ by bacterial strains isolated from a boreal nutrient-poor bog

    Directory of Open Access Journals (Sweden)

    Merja Lusa

    2016-05-01

    Full Text Available We studied the uptake of Ni2+ and Ag+ by bacterial strains of Paenibacillus, Pseudomonas, Burkholderia and Rhodococcus isolated from an acidic nutrient-poor boreal bog. The tests were run in two different growth media at two temperatures; +4 °C and +20 °C. All bacterial strains removed Ni2+ and Ag+ from the solution with highest efficiencies shown by one of the Pseudomonas sp. and one of the Paenibacillus sp. strains. Highest Ni2+ uptake was found in 1% Tryptone solution, whereas the highest removal of Ag+ was obtained using 1% Yeast extract. Temperature affected the uptake of Ni2+ and Ag+, but statistically significant difference was found only for Ni2+. Based on tests carried out for the bacteria in nutrient broths and for fresh samples taken from varying depth up to seven meters from the ombrotrophic bog, from which the bacteria were isolated, we estimated that in in situ conditions of the bog the uptake of Ni2+ by bacteria accounts for approximately 0.02% of the total sorption in the uppermost moss layer, 0.01% in the peat layer, 0.02% in the gyttja layer and 0.1% in the bottom clay layer of the bog. For Ag+ the corresponding values were 2.3% in the moss layer, 0.04% in the peat layer, 0.2% in the gyttja and 0.03% in the clay layer.

  9. A model to explain plant growth promotion traits: a multivariate analysis of 2,211 bacterial isolates.

    Directory of Open Access Journals (Sweden)

    Pedro Beschoren da Costa

    Full Text Available Plant growth-promoting bacteria can greatly assist sustainable farming by improving plant health and biomass while reducing fertilizer use. The plant-microorganism-environment interaction is an open and complex system, and despite the active research in the area, patterns in root ecology are elusive. Here, we simultaneously analyzed the plant growth-promoting bacteria datasets from seven independent studies that shared a methodology for bioprospection and phenotype screening. The soil richness of the isolate's origin was classified by a Principal Component Analysis. A Categorical Principal Component Analysis was used to classify the soil richness according to isolate's indolic compound production, siderophores production and phosphate solubilization abilities, and bacterial genera composition. Multiple patterns and relationships were found and verified with nonparametric hypothesis testing. Including niche colonization in the analysis, we proposed a model to explain the expression of bacterial plant growth-promoting traits according to the soil nutritional status. Our model shows that plants favor interaction with growth hormone producers under rich nutrient conditions but favor nutrient solubilizers under poor conditions. We also performed several comparisons among the different genera, highlighting interesting ecological interactions and limitations. Our model could be used to direct plant growth-promoting bacteria bioprospection and metagenomic sampling.

  10. A model to explain plant growth promotion traits: a multivariate analysis of 2,211 bacterial isolates.

    Science.gov (United States)

    da Costa, Pedro Beschoren; Granada, Camille E; Ambrosini, Adriana; Moreira, Fernanda; de Souza, Rocheli; dos Passos, João Frederico M; Arruda, Letícia; Passaglia, Luciane M P

    2014-01-01

    Plant growth-promoting bacteria can greatly assist sustainable farming by improving plant health and biomass while reducing fertilizer use. The plant-microorganism-environment interaction is an open and complex system, and despite the active research in the area, patterns in root ecology are elusive. Here, we simultaneously analyzed the plant growth-promoting bacteria datasets from seven independent studies that shared a methodology for bioprospection and phenotype screening. The soil richness of the isolate's origin was classified by a Principal Component Analysis. A Categorical Principal Component Analysis was used to classify the soil richness according to isolate's indolic compound production, siderophores production and phosphate solubilization abilities, and bacterial genera composition. Multiple patterns and relationships were found and verified with nonparametric hypothesis testing. Including niche colonization in the analysis, we proposed a model to explain the expression of bacterial plant growth-promoting traits according to the soil nutritional status. Our model shows that plants favor interaction with growth hormone producers under rich nutrient conditions but favor nutrient solubilizers under poor conditions. We also performed several comparisons among the different genera, highlighting interesting ecological interactions and limitations. Our model could be used to direct plant growth-promoting bacteria bioprospection and metagenomic sampling.

  11. Diversity and antimicrobial potential of bacterial isolates associated with the soft coral Alcyonium digitatum from the Baltic Sea.

    Science.gov (United States)

    Pham, Thi Mien; Wiese, Jutta; Wenzel-Storjohann, Arlette; Imhoff, Johannes F

    2016-01-01

    It is well recognized that microorganisms associated with marine invertebrates, in particular sponges and hard corals, are an excellent source of new natural products. Therefore, the diversity of bacteria associated with marine invertebrates and their potential to produce bioactive compounds have received much attention in recent years. We report here for the first time on the biodiversity of bacteria associated with the soft coral Alcyonium digitatum, which is abundant in the Baltic Sea. In order to increase the cultured diversity, bacteria were isolated using four different media, identified with support of 16S rRNA gene sequences and screened for antimicrobial activity using two different media. Activity of crude extracts was tested against Bacillus subtilis, Staphylococcus epidermidis, Escherichia coli, and the yeast Candida albicans. A total of 251 coral-associated bacterial isolates were classified and found to belong to 41 species in 14 genera of the Firmicutes, Actinobacteria, Gammaproteobacteria, and Alphaproteobacteria. The genus Bacillus was most abundant and diverse with 17 recognized species. Forty-eight percent of all 251 isolates exhibited antimicrobial activity. All isolates of Bacillus methylotrophicus and Bacillus amyloliquefaciens displayed inhibition of at least three out of the four tested microorganisms. It became obvious during this study that the production of antibiotic substances not only is strain-specific, but in many cases also depends on the media composition and growth conditions. In addition, the antimicrobial potential of bacteria associated with A. digitatum may represent a promising source for antimicrobial substances.

  12. Low copy number DNA profiling from isolated sperm using the aureka®-micromanipulation system.

    Science.gov (United States)

    Schneider, C; Müller, U; Kilper, R; Siebertz, B

    2012-07-01

    A new cell isolation technique linked to the aureka® micromanipulation system (aureka®) was used to pick sperm from mixed samples containing sperm and epithelial cells. Both cell types were stained using the HY-LITER™ high-resolution, fluorescent staining kit. To isolate a single sperm of interest under a fluorescent microscope, a specific microsphere picking technique was used. This sensitive and reliable cell identification and isolation technique enables low-copy-number (LCN) DNA profiling, as few as 20 sperm are sufficient for obtaining a full short tandem repeat (STR) profile without any allelic drop out. The presented protocol covers the whole workflow, from sample staining and cell pick up to STR analysis.

  13. Genetic diversity of Clavispora lusitaniae isolated from Agave fourcroydes Lem, as revealed by DNA fingerprinting.

    Science.gov (United States)

    Pérez-Brito, Daisy; Magaña-Alvarez, Anuar; Lappe-Oliveras, Patricia; Cortes-Velazquez, Alberto; Torres-Calzada, Claudia; Herrera-Suarez, Teófilo; Larqué-Saavedra, Alfonso; Tapia-Tussell, Raul

    2015-01-01

    This study characterized Clavispora lusitaniae strains isolated from different stages of the processing and early fermentation of a henequen (Agave fourcroydes) spirit produced in Yucatan, Mexico using a molecular technique. Sixteen strains identified based on morphological features, obtained from different substrates, were typed molecularly. Nine different versions of the divergent D1/D2 domain of the large-subunit ribosomal DNA sequence were identified among the C. lusitaniae strains. The greatest degree of polymorphism was found in the 90-bp structural motif of the D2 domain. The MSP-PCR technique was able to differentiate 100% of the isolates. This study provides significant insight into the genetic diversity of the mycobiota present during the henequen fermentation process, especially that of C. lusitaniae, for which only a few studies in plants have been published. The applied MSP-PCR markers were very efficient in revealing olymorphisms between isolates of this species.

  14. Potential of Polycyclic Aromatic Hydrocarbon-Degrading Bacterial Isolates to Contribute to Soil Fertility

    Directory of Open Access Journals (Sweden)

    Maryam Bello-Akinosho

    2016-01-01

    Full Text Available Restoration of polycyclic aromatic hydrocarbon- (PAH- polluted sites is presently a major challenge in agroforestry. Consequently, microorganisms with PAH-degradation ability and soil fertility improvement attributes are sought after in order to achieve sustainable remediation of polluted sites. This study isolated PAH-degrading bacteria from enriched cultures of spent automobile engine-oil polluted soil. Isolates’ partial 16S rRNA genes were sequenced and taxonomically classified. Isolates were further screened for their soil fertility attributes such as phosphate solubilization, atmospheric nitrogen fixation, and indoleacetic acid (IAA production. A total of 44 isolates were obtained and belong to the genera Acinetobacter, Arthrobacter, Bacillus, Flavobacterium, Microbacterium, Ochrobactrum, Pseudomonas, Pseudoxanthomonas, Rhodococcus, and Stenotrophomonas. Data analysed by principal component analysis showed the Bacillus and Ochrobactrum isolates displayed outstanding IAA production. Generalized linear modelling statistical approaches were applied to evaluate the contribution of the four most represented genera (Pseudomonas, Acinetobacter, Arthrobacter, and Rhodococcus to soil fertility. The Pseudomonas isolates were the most promising in all three soil fertility enhancement traits evaluated and all isolates showed potential for one or more of the attributes evaluated. These findings demonstrate a clear potential of the isolates to participate in restorative bioremediation of polluted soil, which will enhance sustainable agricultural production and environmental protection.

  15. DnaK as Antibiotic Target: Hot Spot Residues Analysis for Differential Inhibition of the Bacterial Protein in Comparison with the Human HSP70.

    Directory of Open Access Journals (Sweden)

    Federica Chiappori

    Full Text Available DnaK, the bacterial homolog of human Hsp70, plays an important role in pathogens survival under stress conditions, like antibiotic therapies. This chaperone sequesters protein aggregates accumulated in bacteria during antibiotic treatment reducing the effect of the cure. Although different classes of DnaK inhibitors have been already designed, they present low specificity. DnaK is highly conserved in prokaryotes (identity 50-70%, which encourages the development of a unique inhibitor for many different bacterial strains. We used the DnaK of Acinetobacter baumannii as representative for our analysis, since it is one of the most important opportunistic human pathogens, exhibits a significant drug resistance and it has the ability to survive in hospital environments. The E.coli DnaK was also included in the analysis as reference structure due to its wide diffusion. Unfortunately, bacterial DnaK and human Hsp70 have an elevated sequence similarity. Therefore, we performed a differential analysis of DnaK and Hsp70 residues to identify hot spots in bacterial proteins that are not present in the human homolog, with the aim of characterizing the key pharmacological features necessary to design selective inhibitors for DnaK. Different conformations of DnaK and Hsp70 bound to known inhibitor-peptides for DnaK, and ineffective for Hsp70, have been analysed by molecular dynamics simulations to identify residues displaying stable and selective interactions with these peptides. Results achieved in this work show that there are some residues that can be used to build selective inhibitors for DnaK, which should be ineffective for the human Hsp70.

  16. Interplay between the bacterial nucleoid protein H-NS and macromolecular crowding in compacting DNA

    NARCIS (Netherlands)

    Wintraecken, C.H.J.M.

    2012-01-01

      In this dissertation we discuss H-NS and its connection to nucleoid compaction and organization. Nucleoid formation involves a dramatic reduction in coil volume of the genomic DNA. Four factors are thought to influence coil volume: supercoiling, DNA charge neutralization, macromolecular crow

  17. The action of the bacterial toxin microcin B17. Insight into the cleavage-religation reaction of DNA gyrase.

    Science.gov (United States)

    Pierrat, Olivier A; Maxwell, Anthony

    2003-09-12

    We have examined the effects of the bacterial toxin microcin B17 (MccB17) on the reactions of Escherichia coli DNA gyrase. MccB17 slows down but does not completely inhibit the DNA supercoiling and relaxation reactions of gyrase. A kinetic analysis of the cleavage-religation equilibrium of gyrase was performed to determine the effect of the toxin on the forward (cleavage) and reverse (religation) reactions. A simple mechanism of two consecutive reversible reactions with a nicked DNA intermediate was used to simulate the kinetics of cleavage and religation. The action of MccB17 on the kinetics of cleavage and religation was compared with that of the quinolones ciprofloxacin and oxolinic acid. With relaxed DNA as substrate, only a small amount of gyrase cleavage complex is observed with MccB17 in the absence of ATP, whereas the presence of the nucleotide significantly enhances the effect of the toxin on both the cleavage and religation reactions. In contrast, ciprofloxacin, oxolinic acid, and Ca2+ show lesser dependence on ATP to stabilize the cleavage complex. MccB17 enhances the overall rate of DNA cleavage by increasing the forward rate constant (k2) of the second equilibrium. In contrast, ciprofloxacin increases the amount of cleaved DNA by a combined effect on the forward and reverse rate constants of both equilibria. Based on these results and on the observations that MccB17 only slowly inhibits the supercoiling and relaxation reactions, we suggest a model of the interaction of MccB17 with gyrase.

  18. Surface physicochemistry and ionic strength affects eDNA's role in bacterial adhesion to abiotic surfaces

    DEFF Research Database (Denmark)

    Regina, Viduthalai R.; Lokanathan, Arcot R.; Modrzynski, Jakub Jan;

    2014-01-01

    DNA-mediated adhesion depends on the physicochemical properties of the surface and surrounding liquid. We investigated eDNA alteration of cell surface hydrophobicity and zeta potential, and subsequently quantified the effect of eDNA on the adhesion of Staphylococcus xylosus to glass surfaces functionalised...... ionic strengths. No effect was seen on glass surfaces and carboxyl-functionalised surfaces at high ionic strength, and a reverse effect occurred on amine-functionalised surfaces at low ionic strength. However, eDNA promoted adhesion of cells to hydrophobic surfaces irrespective of the ionic strength...... with different chemistries resulting in variable hydrophobicity and charge. Cell adhesion experiments were carried out at three different ionic strengths. Removal of eDNA from S. xylosus cells by DNase treatment did not alter the zeta potential, but rendered the cells more hydrophilic. DNase treatment impaired...

  19. Novel bacterial isolate from Permian groundwater, capable of aggregating potential biofuel-producing microalga Nannochloropsis oceanica IMET1.

    Science.gov (United States)

    Wang, Hui; Laughinghouse, Haywood D; Anderson, Matthew A; Chen, Feng; Willliams, Ernest; Place, Allen R; Zmora, Odi; Zohar, Yonathan; Zheng, Tianling; Hill, Russell T

    2012-03-01

    Increasing petroleum costs and climate change have resulted in microalgae receiving attention as potential biofuel producers. Little information is available on the diversity and functions of bacterial communities associated with biofuel-producing algae. A potential biofuel-producing microalgal strain, Nannochloropsis oceanica IMET1, was grown in Permian groundwater. Changes in the bacterial community structure at three temperatures were monitored by two culture-independent methods, and culturable bacteria were characterized. After 9 days of incubation, N. oceanica IMET1 began to aggregate and precipitate in cultures grown at 30°C, whereas cells remained uniformly distributed at 15°C and 25°C. The bacterial communities in cultures at 30°C changed markedly. Some bacteria isolated only at 30°C were tested for their potential for aggregating microalgae. A novel bacterium designated HW001 showed a remarkable ability to aggregate N. oceanica IMET1, causing microalgal cells to aggregate after 3 days of incubation, while the total lipid content of the microalgal cells was not affected. Direct interaction of HW001 and N. oceanica is necessary for aggregation. HW001 can also aggregate the microalgae N. oceanica CT-1, Tetraselmis suecica, and T. chuii as well as the cyanobacterium Synechococcus WH8007. 16S rRNA gene sequence comparisons indicated the great novelty of this strain, which exhibited only 89% sequence similarity with any previously cultured bacteria. Specific primers targeted to HW001 revealed that the strain originated from the Permian groundwater. This study of the bacterial communities associated with potential biofuel-producing microalgae addresses a little-investigated area of microalgal biofuel research and provides a novel approach to harvest biofuel-producing microalgae by using the novel bacterium strain HW001.

  20. Evaluating robustness of a diesel-degrading bacterial consortium isolated from contaminated soil

    DEFF Research Database (Denmark)

    Sydow, Mateusz; Owsianiak, Mikolaj; Szczepaniak, Zuzanna;

    2016-01-01

    It is not known whether diesel-degrading bacterial communities are structurally and functionally robust when exposed to different hydrocarbon types. Here, we exposed a diesel-degrading consortium to model either alkanes, cycloalkanes or aromatic hydrocarbons as carbon sources to study its...... structural resistance. The structural resistance was low, with changes in relative abundances of up to four orders of magnitude, depending on hydrocarbon type and bacterial taxon. This low resistance is explained by the presence of hydrocarbon-degrading specialists in the consortium and differences in growth...... kinetics on individual hydrocarbons. However, despite this low resistance, structural and functional resilience were high, as verified by re-exposing the hydrocarbon-perturbed consortium to diesel fuel. The high resilience is either due to the short exposure time, insufficient for permanent changes...

  1. The ATRX cDNA is prone to bacterial IS10 element insertions that alter its structure.

    Science.gov (United States)

    Valle-García, David; Griffiths, Lyra M; Dyer, Michael A; Bernstein, Emily; Recillas-Targa, Félix

    2014-01-01

    The SWI/SNF-like chromatin-remodeling protein ATRX has emerged as a key factor in the regulation of α-globin gene expression, incorporation of histone variants into the chromatin template and, more recently, as a frequently mutated gene across a wide spectrum of cancers. Therefore, the availability of a functional ATRX cDNA for expression studies is a valuable tool for the scientific community. We have identified two independent transposon insertions of a bacterial IS10 element into exon 8 of ATRX isoform 2 coding sequence in two different plasmids derived from a single source. We demonstrate that these insertion events are common and there is an insertion hotspot within the ATRX cDNA. Such IS10 insertions produce a truncated form of ATRX, which significantly compromises its nuclear localization. In turn, we describe ways to prevent IS10 insertion during propagation and cloning of ATRX-containing vectors, including optimal growth conditions, bacterial strains, and suggested sequencing strategies. Finally, we have generated an insertion-free plasmid that is available to the community for expression studies of ATRX.

  2. Prevalence and antibiogram of bacterial isolates from urinary tract infections at Dessie Health Research Laboratory, Ethiopia

    Directory of Open Access Journals (Sweden)

    Mulugeta Kibret

    2014-02-01

    Conclusions: In the study area resistance rates to erythromycin, amoxycillin and tetracycline were high. Since most isolates were sensitive to nitrofurantoin and gentamicin, they are considered as appropriate antimicrobials for empirical treatment urinary tract infections.

  3. Gene expression regulation in retinal pigment epithelial cells induced by viral RNA and viral/bacterial DNA

    Science.gov (United States)

    Brosig, Anton; Kuhrt, Heidrun; Wiedemann, Peter; Kohen, Leon; Bringmann, Andreas

    2015-01-01

    Purpose The pathogenesis of age-related macular degeneration (AMD) is associated with systemic and local inflammation. Various studies suggested that viral or bacterial infection may aggravate retinal inflammation in the aged retina. We compared the effects of synthetic viral RNA (poly(I:C)) and viral/bacterial DNA (CpG-ODN) on the expression of genes known to be involved in the development of AMD in retinal pigment epithelial (RPE) cells. Methods Cultured human RPE cells were stimulated with poly(I:C; 500 µg/ml) or CpG-ODN (500 nM). Alterations in gene expression and protein secretion were determined with real-time RT–PCR and ELISA, respectively. Phosphorylation of signal transduction molecules was revealed by western blotting. Results Poly(I:C) induced gene expression of the pattern recognition receptor TLR3, transcription factors (HIF-1α, p65/NF-κB), the angiogenic factor bFGF, inflammatory factors (IL-1β, IL-6, TNFα, MCP-1, MIP-2), and complement factors (C5, C9, CFB). Poly(I:C) also induced phosphorylation of ERK1/2 and p38 MAPK proteins, and the secretion of bFGF and TNFα from the cells. CpG-ODN induced moderate gene expression of transcription factors (p65/NF-κB, NFAT5) and complement factors (C5, C9), while it had no effect on the expression of various TLR, angiogenic factor, and inflammatory factor genes. The activities of various signal transduction pathways and transcription factors were differentially involved in mediating the poly(I:C)-induced transcriptional activation of distinct genes. Conclusions The widespread effects of viral RNA, and the restricted effects of viral/bacterial DNA, on the gene expression pattern of RPE cells may suggest that viral RNA rather than viral/bacterial DNA induces physiologic alterations of RPE cells, which may aggravate inflammation in the aged retina. The data also suggest that selective inhibition of distinct signal transduction pathways or individual transcription factors may not be effective to inhibit

  4. Impact of in Situ Isolated Bacterial Strains on Nitrogen Fixation in Alfalfa

    OpenAIRE

    Carmen Dragomir; Rodica Schipor; Neculai Dragomir; Nicoleta Moraru; Claudiu Ghiocel; Darius Văcariu

    2013-01-01

    Symbiosis relationships among legumes and nitrogen fixing bacteria play a crucial role in agriculture since they provide the opportunity of converting atmospheric molecular nitrogen into an ammonia form of nitrogen that the plants can use in protein formation. To enhance this process we have selected nitrogen fixing bacterial strains commercialised under different forms depending on the cultivation technologies in legume species. In our research, we have pointed out the efficacy of in situ is...

  5. The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy

    Directory of Open Access Journals (Sweden)

    Pesta David

    2003-06-01

    Full Text Available Abstract Background DNA microarrays are a powerful tool with important applications such as global gene expression profiling. Construction of bacterial DNA microarrays from genomic sequence data using a two-stage PCR amplification approach for the production of arrayed DNA is attractive because it allows, in principal, the continued re-amplification of DNA fragments and facilitates further utilization of the DNA fragments for additional uses (e.g. over-expression of protein. We describe the successful construction and use of DNA microarrays by the two-stage amplification approach and discuss the technical challenges that were met and resolved during the project. Results Chimeric primers that contained both gene-specific and shared, universal sequence allowed the two-stage amplification of the 3,168 genes identified on the genome of Synechocystis sp. PCC6803, an important prokaryotic model organism for the study of oxygenic photosynthesis. The gene-specific component of the primer was of variable length to maintain uniform annealing temperatures during the 1st round of PCR synthesis, and situated to preserve full-length ORFs. Genes were truncated at 2 kb for efficient amplification, so that about 92% of the PCR fragments were full-length genes. The two-stage amplification had the additional advantage of normalizing the yield of PCR products and this improved the uniformity of DNA features robotically deposited onto the microarray surface. We also describe the techniques utilized to optimize hybridization conditions and signal-to-noise ratio of the transcription profile. The inter-lab transportability was demonstrated by the virtual error-free amplification of the entire genome complement of 3,168 genes using the universal primers in partner labs. The printed slides have been successfully used to identify differentially expressed genes in response to a number of environmental conditions, including salt stress. Conclusions The technique detailed

  6. Joint analysis of bacterial DNA methylation, predicted promoter and regulation motifs for biological significance

    Science.gov (United States)

    Advances in long-read, single molecule real-time sequencing technology and analysis software over the last two years has enabled the efficient production of closed bacterial genome sequences. However, consistent annotation of these genomes has lagged behind the ability to create them, while the avai...

  7. Using DNA Technology to Explore Marine Bacterial Diversity in a Coastal Georgia Salt Marsh

    Science.gov (United States)

    Dong, Yihe; Guerrero, Stella; Moran, Mary Ann

    2008-01-01

    An important aspect of teaching biology is to expose students to the concept of biodiversity. For this purpose, bacteria are excellent examples. The advanced placement (AP) biology class at Cedar Shoals High School in Athens, Georgia, learned how to explore bacterial biodiversity using molecular fingerprinting. They collected marine water samples,…

  8. Physiological and DNA fingerprinting of the bacterial community of Meloidogyne fallax egg masses

    NARCIS (Netherlands)

    Papert, A; Kok, CJ; van Elsas, JD

    2004-01-01

    Bacterial communities associated with the plant-parasitic nematode Meloidogyne fallax egg masses were compared with those present in the rhizoplane. Two agricultural soils with different nematode population dynamics were used in a glasshouse study, with either potato or tomato as host plant for the

  9. High-fat nutrition reduces hepatic damage following exposure to bacterial DNA and hemorrhagic shock.

    NARCIS (Netherlands)

    Luyer, M.D.; Derikx, J.P.; Beyaert, R.; Hadfoune, M.; Kuppevelt, A.H.M.S.M. van; Dejong, C.H.; Heineman, E.; Buurman, W.A.; Greve, J.W.

    2009-01-01

    BACKGROUND/AIMS: Bacterial infection combined with hypotension results in exacerbation of the inflammatory response with release of interferon (IFN) gamma. This excessive inflammation may lead to development of hepatic damage and liver failure. This study investigates the effect of dietary lipids on

  10. DNA sequence analysis of the triose phosphate isomerase gene from isolates of Giardia lamblia

    Institute of Scientific and Technical Information of China (English)

    卢思奇; 文建凡; 李继红; 王凤云

    2002-01-01

    Objective To confirm the genetic relation between Giardia lamblia (G. lamblia) isolates from different geographic regions of China and other countries. Methods Genomic DNA were extracted from the trophozoites or cysts of Giardia lamblia. The triose phosphate isomerase (tim) gene was amplified using polymerase chain reaction (PCR) technique. PCR products were digested with endonuclease and sequenced. The data of sequencing were analyzed with the DNAstar software and compared with that of the isolates acquired from GenBank. Results Of nine isolates of Giardia lamblia from China (C1, C2, CH2 and CH3), Cambodia (CAM), Australia (A1 and A2) and America (BP and CDC), respectively, 3 (A1, A2 and CAM) fit into Group 1 (WB), 2 (CH2 and CH3)) into Group 2, and 4 (C1, C2, BP and CDC) into Group 3 (GS). The results confirmed the genetic relatedness of G. lamblia isolates from all over the world. Conclusion Genotyping isolates of G. Lamblia provides important information for establishing the phylogenetic relationship or for the epidemiological evaluation of the spreading of this organism.

  11. [Bacterial infections as seen from the eukaryotic genome: DNA double strand breaks, inflammation and cancer].

    Science.gov (United States)

    Lemercier, Claudie

    2014-01-01

    An increasing number of studies report that infection by pathogenic bacteria alters the host genome, producing highly hazardous DNA double strand breaks for the eukaryotic cell. Even when DNA repair occurs, it often leaves "scars" on chromosomes that might generate genomic instability at the next cell division. Chronic intestinal inflammation promotes the expansion of genotoxic bacteria in the intestinal microbiote which in turn triggers tumor formation and colon carcinomas. Bacteria act at the level of the host DNA repair machinery. They also highjack the host cell cycle to allow themselves time for replication in an appropriate reservoir. However, except in the case of bacteria carrying the CDT nuclease, the molecular mechanisms responsible for DNA lesions are not well understood, even if reactive oxygen species released during infection make good candidates.

  12. Measuring the CCN and IN ability of bacterial isolates: implications for the southeastern United States and Puerto Rico

    Science.gov (United States)

    Purdue, S.; Waters, S.; Konstantinidis, K.; Nenes, A.; DeLeon-Rodriguez, N.

    2015-12-01

    Ice nucleation is an important process in the climate system as it influences global precipitation processes, and can affect the vertical distribution of clouds with effects that both cool and warm the atmosphere. Of the pathways to ice nucleation, immersion mode, which occurs when ice nuclei (IN) particles are surrounded by an aqueous phase that subsequently freezes, dominates primary ice production in mixed-phase clouds. A simple but effective method to study immersion freezing is to utilize a droplet freezing assay (DFA) that consists of an aluminum plate, precisely cooled by a continuous flow of an ethylene glycol-water mixture. Using such a system we study the immersion IN characteristics of bacterial isolates (for temperatures ranging from -15oC to 0oC) isolated from rainwater and air collected in Atlanta, GA and Puerto Rico, over storms throughout the year. Despite their relatively large size and the presence of hydrophilic groups on the outer membranes of many bacteria, it is unclear if bacteria possess an inherent ability to nucleate an aqueous phase (a requirement for immersion freezing) for the wide range of supersaturations found in clouds. For this, we measure the cloud condensation nucleation (CCN) activity of each isolate (over the 0.05% to 0.6% supersaturation range) using a Continuous Flow Streamwise Thermal Gradient CCN Counter. Initial results have shown certain isolates to be very efficient CCN, allowing them to form droplets even for the very low supersaturations found in radiation fogs. In combination, these experiments provide insight into the potential dual-ability of some bacteria, isolated from the southeastern United States and Puerto Rico, to act as both efficient CCN and IN.

  13. [Biotypes and antibiotic resistance patterns of Gardnerella vaginalis strains isolated from healthy women and women with bacterial vaginosis].

    Science.gov (United States)

    Tosun, Ilknur; Alpay Karaoğlu, Sengül; Ciftçi, Hasan; Buruk, Celal Kurtuluş; Aydin, Faruk; Kiliç, Ali Osman; Ertürk, Murat

    2007-01-01

    As Gardnerella vaginalis is accepted as a member of normal vaginal flora, it is one of the dominant species which has been related to bacterial vaginosis (BV). The aim of this study was to determine the isolation rate, biotypes and antibiotic resistance patterns of G.vaginalis from the vaginal swab samples of 408 women who were admitted to the outpatient clinics of Family Planning Center. Hippurate hydrolysis, lipase and beta-galactosidase tests were performed for biotyping the isolates, and agar dilution (for metronidazole) and disk diffusion (for clindamycin) tests were used for the detection of antibiotic resistance patterns. As a result, by Nugent's BV scoring protocol, 122 (29.9%), 20 (29.4%), 137 (33.6%), and 18 (4.4%) of the women were diagnosed as BV, intermediate form, normal vaginal flora (NVF) and mycotic vaginosis, respectively. The overall isolation rate of G.vaginalis was found as 23% (94/408). Of them, 56.4% (53/94) and 8.5% (8/94) were isolated from samples of BV cases and subjects with NVF, respectively, and the difference was statistically significant (pbiotyping results showed that the most frequently detected types were biotype 1 (44%), 5 (20%) and 4 (18%). There was no statistically significant difference between the biotype distribution of BV patients and the subjects who have NVF (p=0.687). The results of antibiotic susceptibility tests indicated that 70% and 53% of the isolates were resistant to metronidazole and clindamycin, respectively. It was of interest that MIC values for metronidazole was > or =128 microg/ml in 57% of resistant strains. The data of this study has emphasized that the metronidazole resistance is very high in our population, and the large scale studies are needed to clarify the relationship between BV and G.vaginalis biotypes, which can be found in the normal vaginal flora.

  14. Isolation, Mapping, DNA Sequence and RFLPs Studies of Random Single-Copy DNA Segments on Human X Chromosome

    Institute of Scientific and Technical Information of China (English)

    谭骏; 邱信芳; 薛京伦; 朱锡华; 纪贤文; 张冬梅; 秦世真

    1994-01-01

    Using the total human/mouse DNA as the probe, screening has been carried out three times with in situ plaque hybridization to obtain the single-copy DNA sequence from the human X chromosome genomic library. The effective rate of screening is 1. 45%. DNAs from clones containing single-copy inserts have been analyzed by a panel of hybrid cells with or without human X chromosome. Three segments, designated by DXFD52,73,75, are mapped to the X chromosome. DXFD52 has been precisely localized on Xq12-q13 with in situ chromosomal hybridization. DXFD52 has been partially sequenced. The results indicate that DXFD52 is a new isolated single-copy segment on the X chromosome. Great progress in the RFLPs study with DXFD52 has been achieved in the population of Chongqing, Sichuan Province. The results show that the DXFD52 can be used to detect the RFLP with Hind Ⅲ, Bgl Ⅱ, and Hinf Ⅰ. DXFD52 will be a potential "landmark" for the construction of the complete linkage map of human genome and the analysis of genomic s

  15. Heat degradation of eukaryotic and bacterial DNA: an experimental model for paleomicrobiology

    Directory of Open Access Journals (Sweden)

    Nguyen-Hieu Tung

    2012-09-01

    Full Text Available Abstract Background Theoretical models suggest that DNA degradation would sharply limit the PCR-based detection of both eukaryotic and prokaryotic DNA within ancient specimens. However, the relative extent of decay of eukaryote and prokaryote DNA over time is a matter of debate. In this study, the murine macrophage cell line J774, alone or infected with Mycobacterium smegmatis bacteria, were killed after exposure to 90°C dry heat for intervals ranging from 1 to 48 h in order to compare eukaryotic cells, extracellular bacteria and intracellular bacteria. The sizes of the resulting mycobacterial rpoB and murine rpb2 homologous gene fragments were then determined by real-time PCR and fluorescent probing. Findings The cycle threshold (Ct values of PCR-amplified DNA fragments from J774 cells and the M. smegmatis negative controls (without heat exposure varied from 26–33 for the J774 rpb2 gene fragments and from 24–29 for M. smegmatis rpoB fragments. After 90°C dry heat incubation for up to 48 h, the Ct values of test samples increased relative to those of the controls for each amplicon size. For each dry heat exposure time, the Ct values of the 146-149-bp fragments were lower than those of 746-747-bp fragments. During the 4- to 24-h dry heat incubation, the non-infected J774 cell DNA was degraded into 597-bp rpb2 fragments. After 48 h, however, only 450-bp rpb2 fragments of both non-infected and infected J774 cells could be amplified. In contrast, the 746-bp rpoB fragments of M. smegmatis DNA could be amplified after the 48-h dry heat exposure in all experiments. Infected and non-infected J774 cell DNA was degraded more rapidly than M. smegmatis DNA after dry heat exposure (ANOVA test, p  Conclusion In this study, mycobacterial DNA was more resistant to dry-heat stress than eukaryotic DNA. Therefore, the detection of large, experimental, ancient mycobacterial DNA fragments is a suitable approach for paleomicrobiological studies.

  16. Comparison of different methods for DNA-free RNA isolation from SK-N-MC neuroblastoma

    Directory of Open Access Journals (Sweden)

    Ferreira Ricardo B

    2011-01-01

    Full Text Available Abstract Background RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Extraction of high quality nucleic acids is difficult from neuronal cells and brain tissues as they are particularly rich in lipids. In addition, most common RNA extraction methods are phenol-based, resulting in RNA that may be incompatible with downstream applications such as gene expression. Findings In this work, a comparative analysis of the RNA quality obtained from SK-N-MC cells was performed using six commonly used RNA isolation kits: two phenol-based kits and four non-phenol based kits. The non-phenol based kits tested AxyPrep Multisource Total RNA Miniprep, RNeasy® Mini, EasySpin and Ilustra RNAspin Mini RNA Isolation, all performed well and resulted in the isolation of high quality RNA, as evaluated by A260/A280. The RNA extracted with AxyPrep Multisource Total RNA Miniprep, RNeasy® Mini and EasySpin provided the highest RNA yields. In particular, the RNA isolated by AxyPrep Multisource Total RNA Miniprep Kit did not show any detectable genomic DNA contamination even without previous DNase treatment or after RNA direct PCR amplification using universal 18S primers. Conclusions The RNA extracted from SK-N-MC cells with AxyPrep Multisource Total RNA Miniprep Kit was superior with respect to the RNA quality and concentration. This kit does not use aggressive organic solvents and RNA free of genomic DNA was isolated without the need for DNase treatment.

  17. Thermal isolation of microchip reaction chambers for rapid non-contact DNA amplification

    Science.gov (United States)

    Easley, Christopher J.; Humphrey, Joseph A. C.; Landers, James P.

    2007-09-01

    This paper describes further optimization of a non-contact, infrared-mediated system for microchip DNA amplification via the polymerase chain reaction (PCR). The optimization is focused on heat transfer modeling and subsequent fabrication of thermally isolated reaction chambers in glass devices that are uniquely compatible with non-contact thermal control. With a thermal conductivity approximately an order of magnitude higher than many plastics, glass is not the obvious substrate of choice for rapid thermal cycling in microfluidic chambers, yet it is preferable in terms of optical clarity, solvent compatibility and chemical inertness. Based on predictions of a lumped capacity heat transfer analysis, it is shown here that post-bonding, patterned etching of surrounding glass from microfluidic reaction chambers provides enhancements as high as 3.6- and 7.5-fold in cooling and heating rates, respectively, over control devices with the same chamber designs. These devices are then proven functional for rapid DNA amplification via PCR, in which 25 thermal cycles are completed in only 5 min in thermally isolated PCR chambers of 270 nL volume, representing the fastest static PCR in glass devices reported to date. Amplification of the 500-base pair fragment of λ-DNA was confirmed by capillary gel electrophoresis. In addition to rapid temperature control, the fabrication scheme presented, which is compatible with standard photolithography and wet etching techniques, provides a simple alternative for general thermal management in glass microfluidic devices without metallization.

  18. Isolation of Alcohol Dehydrogenase cDNA and Basal Regulatory Region from Metroxylon sagu.

    Science.gov (United States)

    Wee, Ching Ching; Roslan, Hairul Azman

    2012-01-01

    Alcohol dehydrogenase (Adh) is a versatile enzyme involved in many biochemical pathways in plants such as in germination and stress tolerance. Sago palm is plant with much importance to the state of Sarawak as one of the most important crops that bring revenue with the advantage of being able to withstand various biotic and abiotic stresses such as heat, pathogens, and water logging. Here we report the isolation of sago palm Adh cDNA and its putative promoter region via the use of rapid amplification of cDNA ends (RACE) and genomic walking. The isolated cDNA was characterized and determined to be 1464 bp long encoding for 380 amino acids. BLAST analysis showed that the Adh is similar to the Adh1 group with 91% and 85% homology with Elaeis guineensis and Washingtonia robusta, respectively. The putative basal msAdh1 regulatory region was further determined to contain promoter signals of TATA and AGGA boxes and predicted amino acids analyses showed several Adh-specific motifs such as the two zinc-binding domains that bind to the adenosine ribose of the coenzyme and binding to alcohol substrate. A phylogenetic tree was also constructed using the predicted amino acid showed clear separation of Adh from bacteria and clustered within the plant Adh group.

  19. Structure and partitioning of bacterial DNA: determined by a balance of competion and expansion forces?

    DEFF Research Database (Denmark)

    Woldringh, C. L.; Jensen, Peter Ruhdal; Westerhoff, H. V.

    1995-01-01

    The mechanisms that determine chromosome structure and chromosome partitioning in bacteria are largely unknown. Here we discuss two hypotheses: (i) the structure of the Escherichia coli nucleoid is determined by DNA binding proteins and DNA supercoiling, representing a compaction force on the one...... hand, and by the coupled transcription/translation/translocation of plasma membrane and cell wall proteins, representing an expansion force on the other hand; (ii) the two forces are important for the partitioning process of chromosomes....

  20. Mechanisms, molecular and sero-epidemiology of antimicrobial resistance in bacterial respiratory pathogens isolated from Japanese children

    Directory of Open Access Journals (Sweden)

    Sunakawa Keisuke

    2007-08-01

    Full Text Available Abstract Background The clinical management of community-acquired respiratory tract infections (RTIs is complicated by the increasing worldwide prevalence of antibacterial resistance, in particular, β-lactam and macrolide resistance, among the most common causative bacterial pathogens. This study aimed to determine the mechanisms and molecular- and sero-epidemiology of antibacterial resistance among the key paediatric respiratory pathogens in Japan. Methods Isolates were collected at 18 centres in Japan during 2002 and 2003 from children with RTIs as part of the PROTEKT surveillance programme. A proportion of Haemophilus influenzae isolates was subjected to sequencing analysis of the ftsI gene; phylogenetic relatedness was assessed using multilocus sequence typing. Streptococcus pneumoniae isolates were screened for macrolide-resistance genotype by polymerase chain reaction and serotyped using the capsular swelling method. Susceptibility of isolates to selected antibacterials was performed using CLSI methodology. Results and Discussion Of the 557 H. influenzae isolates collected, 30 (5.4% were β-lactamase-positive [BL+], 115 (20.6% were BL-nonproducing ampicillin-resistant (BLNAR; MIC ≥ 4 mg/L and 79 (14.2% were BL-nonproducing ampicillin-intermediate (BLNAI; MIC 2 mg/L. Dabernat Group III penicillin binding protein 3 (PBP3 amino acid substitutions in the ftsI gene were closely correlated with BLNAR status but phylogenetic analysis indicated marked clonal diversity. PBP mutations were also found among BL+ and BL-nonproducing ampicillin-sensitive isolates. Of the antibacterials tested, azithromycin and telithromycin were the most active against H. influenzae (100% and 99.3% susceptibility, respectively. A large proportion (75.2% of the 468 S. pneumoniae isolates exhibited macrolide resistance (erythromycin MIC ≥ 1 mg/L; erm(B was the most common macrolide resistance genotype (58.8%, followed by mef(A (37.2%. The most common pneumococcal

  1. Isolation of prawn ( Exopalaemon carinicauda) lipopolysaccharide and β-1, 3-glucan binding protein gene and its expression in responding to bacterial and viral infections

    Science.gov (United States)

    Ge, Qianqian; Li, Jian; Duan, Yafei; Li, Jitao; Sun, Ming; Zhao, Fazhen

    2016-04-01

    The pattern recognition proteins (PRPs) play a major role in immune response of crustacean to resist pathogens. In the present study, as one of PRPs, lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) gene in the ridge tail white prawn ( Exopalaemon carinicauda) ( EcLGBP) was isolated. The full-length cDNA of EcLGBP was 1338 bp, encoding a polypeptide of 366 amino acid residules. The deduced amino acid sequence of EcLGBP shared high similarities with LGBP and BGBP from other crustaceans. Some conservative domains were predicted in EcLGBP sequence. EcLGBP constitutively expressed in most tissues at different levels, and the highest expression was observed in hepatopancreas. With infection time, the cumulative mortality increased gradually followed by the proliferation of Vibrio parahaemolyticus and white spot syndrome virus (WSSV). The expression of EcLGBP in response to V. parahaemolyticus infection was up-regulated in hemocytes and hepatopancreas, and the up-regulation in hepatopancreas was earlier than that in hemocytes. EcLGBP expression after WSSV infection increased at 3 h, then significantly decreased in both hemocytes and hepatopancreas. The results indicated that EcLGBP was involved in the immune defense against bacterial and viral infections.

  2. Fluorescence-PCR Assays and Isolation of Luminescent Bacterial Clones Using an Automated Plate Reader

    Science.gov (United States)

    Crowley, Thomas E.

    2011-01-01

    The genes responsible for luminescence in various species of the marine microorganism "Photobacterium", have been used for many years as a tool by researchers and instructors. In particular, the "lux" operon of "Photobacterium fischeri" has been used by many instructors to teach recombinant DNA techniques. Two methods using an automated plate…

  3. DNA isolation protocols affect the detection limit of PCR approaches of bacteria in samples from the human gastrointestinal tract

    NARCIS (Netherlands)

    Zoetendal, E.G.; Ben-Amor, K.; Akkermans, A.D.L.; Abee, T.; Vos, de W.M.

    2001-01-01

    A major concern in molecular ecological studies is the lysis efficiency of different bacteria in a complex ecosystem. We used a PCR-based 16S rDNA approach to determine the effect of two DNA isolation protocols (i.e. the bead beating and Triton-X100 method) on the detection limit of seven feces-asso

  4. 18S-rDNA SEQUENCING, ENZYME PATTERNS AND MORPHOLOGICAL CHARACTERIZATION OF TRICHOPHYTON ISOLATES

    Directory of Open Access Journals (Sweden)

    Nascimento Adriana Mendes do

    2001-01-01

    Full Text Available Dermatophytes, capable to use keratin of the host for nutrition, belong to one of the major groups of pathogenic fungi. Since dermatophytes are a closely related group they share various common features, and the morphology of isolates of a given species can be atypical, making species identification and differentiation even more difficult. Many methods have been explored in attempts to distinguish dermatophytes, but the combined use of different approaches for the investigation of the intraspecific and interspecific variability of Trichophyton continues to be scarce. Some studies have shown that amplified fragments of the small ribosomal DNA subunit 18S contains variable regions which can be used to discriminate between medically relevant yeast species, indicating that these regions could also be used for differentiation between dermatophytes. In our study, sequence analysis of the 18S-rDNA gene was combined with morphological and biochemical criteria in order to detect genetic differences between seven Trichophyton isolates and estimate their phylogenetic relationships. The results show that the isolates investigated belong to the Trichophyton group, which potentially contains the Trichophyton rubrum cluster.

  5. Infectivity and complete nucleotide sequence of cucumber fruit mottle mosaic virus isolate Cm cDNA.

    Science.gov (United States)

    Rhee, Sun-Ju; Hong, Jin-Sung; Lee, Gung Pyo

    2014-07-01

    Three isolates of cucumber fruit mottle mosaic virus (CFMMV) were collected from melon, cucumber, and pumpkin plants in Korea. A full-length cDNA clone of CFMMV-Cm (melon isolate) was produced and evaluated for infectivity after T7 transcription in vitro (pT7CF-Cmflc). The complete CFMMV genome sequence of the infectious clone pT7CF-Cmflc was determined. The genome of CFMMV-Cm consisted of 6,571 nucleotides and shared high nucleotide sequence identity (98.8 %) with the Israel isolate of CFMMV. Based on the infectious clone pT7CF-Cmflc, a CaMV 35S-promoter driven cDNA clone (p35SCF-Cmflc) was subsequently constructed and sequenced. Mechanical inoculation with RNA transcripts of pT7CF-Cmflc and agro-inoculation with p35SCF-Cmflc resulted in systemic infection of cucumber and melon, producing symptoms similar to those produced by CFMMV-Cm. Progeny virus in infected plants was detected by RT-PCR, western blot assay, and transmission electron microscopy.

  6. Resistance and Inactivation Kinetics of Bacterial Strains Isolated from the Non-Chlorinated and Chlorinated Effluents of a WWTP

    Science.gov (United States)

    Martínez-Hernández, Sylvia; Vázquez-Rodríguez, Gabriela A.; Beltrán-Hernández, Rosa I.; Prieto-García, Francisco; Miranda-López, José M.; Franco-Abuín, Carlos M.; Álvarez-Hernández, Alejandro; Iturbe, Ulises; Coronel-Olivares, Claudia

    2013-01-01

    The microbiological quality of water from a wastewater treatment plant that uses sodium hypochlorite as a disinfectant was assessed. Mesophilic aerobic bacteria were not removed efficiently. This fact allowed for the isolation of several bacterial strains from the effluents. Molecular identification indicated that the strains were related to Aeromonas hydrophila, Escherichia coli (three strains), Enterobacter cloacae, Kluyvera cryocrescens (three strains), Kluyvera intermedia, Citrobacter freundii (two strains), Bacillus sp. and Enterobacter sp. The first five strains, which were isolated from the non-chlorinated effluent, were used to test resistance to chlorine disinfection using three sets of variables: disinfectant concentration (8, 20 and 30 mg·L−1), contact time (0, 15 and 30 min) and water temperature (20, 25 and 30 °C). The results demonstrated that the strains have independent responses to experimental conditions and that the most efficient treatment was an 8 mg·L−1 dose of disinfectant at a temperature of 20 °C for 30 min. The other eight strains, which were isolated from the chlorinated effluent, were used to analyze inactivation kinetics using the disinfectant at a dose of 15 mg·L−1 with various retention times (0, 10, 20, 30, 60 and 90 min). The results indicated that during the inactivation process, there was no relationship between removal percentage and retention time and that the strains have no common response to the treatments. PMID:23924881

  7. Resistance and Inactivation Kinetics of Bacterial Strains Isolated from the Non-Chlorinated and Chlorinated Effluents of a WWTP

    Directory of Open Access Journals (Sweden)

    Claudia Coronel-Olivares

    2013-08-01

    Full Text Available The microbiological quality of water from a wastewater treatment plant that uses sodium hypochlorite as a disinfectant was assessed. Mesophilic aerobic bacteria were not removed efficiently. This fact allowed for the isolation of several bacterial strains from the effluents. Molecular identification indicated that the strains were related to Aeromonas hydrophila, Escherichia coli (three strains, Enterobacter cloacae, Kluyvera cryocrescens (three strains, Kluyvera intermedia, Citrobacter freundii (two strains, Bacillus sp. and Enterobacter sp. The first five strains, which were isolated from the non-chlorinated effluent, were used to test resistance to chlorine disinfection using three sets of variables: disinfectant concentration (8, 20 and 30 mg·L−1, contact time (0, 15 and 30 min and water temperature (20, 25 and 30 °C. The results demonstrated that the strains have independent responses to experimental conditions and that the most efficient treatment was an 8 mg·L−1 dose of disinfectant at a temperature of 20 °C for 30 min. The other eight strains, which were isolated from the chlorinated effluent, were used to analyze inactivation kinetics using the disinfectant at a dose of 15 mg·L−1 with various retention times (0, 10, 20, 30, 60 and 90 min. The results indicated that during the inactivation process, there was no relationship between removal percentage and retention time and that the strains have no common response to the treatments.

  8. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

    Energy Technology Data Exchange (ETDEWEB)

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.; Baelum, Jacob; Tas, Neslihan; Elberling, Bo; Jansson, Janet K.; Semenchuk, Phillip; Prieme, Anders

    2015-04-30

    The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below -10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface.

  9. The detection of genomic DNA from bacterial cells using iron oxide nanoparticles synthesized by a hydrothermal process

    Science.gov (United States)

    Kim, Young-Sung; Kim, Ki-Chul; Hong, Tae-Whan

    2010-04-01

    We used iron oxide nanoparticles in order to extract purified DNA from bacterial cells. Magnetite (Fe3O4) and maghemite (γ-Fe2O3) are synthesized with FeSO4·7H2O via a hydrothermal process and used as a medium to detect DNA. Various characterizations were performed including X-ray powder diffraction (XRD), high resolution transmission electron microscopy (HRTEM), field emission scanning electron microscopy, vibrating sample magnetometry, and Mössbauer spectroscopy. According to the XRD results, the XRD peaks of the synthesized magnetite and maghemite nanoparticles corresponded well with JCPDS standard data, respectively. The particle size of the iron oxide nanoparticles was about 20 nm, and the particle shape was almost spherical, which was confirmed by observation of the HRTEM image. The magnetite nanoparticles have a face-centeredcubic inverse spinel structure with a space group Fd bar 3 m, as confirmed by HRTEM and Mössbauer spectroscopy analyses. An agarose gel eletrophoresis analysis was performed to confirm the extraction ability of DNA using these iron oxide nanoparticles, revealing stronger reaction of the maghemite nanoparticles than the magnetite nanoparticles.

  10. The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons.

    Science.gov (United States)

    Starke, Ingo C; Vahjen, Wilfried; Pieper, Robert; Zentek, Jürgen

    2014-01-01

    In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2 × 10(5) sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis.

  11. The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons

    Directory of Open Access Journals (Sweden)

    Ingo C. Starke

    2014-01-01

    Full Text Available In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2×105 sequences were used for analysis after processing for read length (150 bp, minimum sequence occurrence (5, and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis.

  12. Diversity of 16S ribosomal DNA-defined bacterial population in acid rock drainage from Japanese pyrite mine.

    Science.gov (United States)

    Okabayashi, Ai; Wakai, Satoshi; Kanao, Tadayoshi; Sugio, Tsuyoshi; Kamimura, Kazuo

    2005-12-01

    Four acidophilic bacteria (YARDs1-4) were isolated from an acid rock drainage (ARD) from Yanahara mine, Okayama prefecture, Japan. The physiological and 16S rDNA sequence analyses revealed that YARD1 was closely affiliated with Acidithiobacillus ferrooxidans, YARD2 was an Acidiphilium-like bacterium, and YARD3 and YARD4 were sulfur-oxidizing bacteria with a relatively close relationship to A. ferrooxidans in the phylogenetic analysis. A molecular approach based on the construction of a 16S rDNA clone library was used to investigate the microbial population of the ARD. Small-subunit rRNA genes were PCR amplified, subsequently cloned and screened for variation by a restriction fragment length polymorphism (RFLP) analysis. A total of 284 clones were grouped into 133 operational taxonomic units (OTUs) by the RFLP analysis. Among them, an OTU showing the same RFLP pattern as those of the isolates from the ARD was not detected. The phylogenetic analysis based on the 16S rDNA sequences from 10 major OTUs and their close relatives revealed that 4 OTUs containing 32.1% of the total clones were loosely affiliated with Verrucomicrobia, 2 OTUs containing 6.6% of the total clones were loosely affiliated with Chloribi, and other OTUs were affiliated with Actinobacteria, Nitrospirae, and beta-Proteobacteria.

  13. Isolated Bacterial Spores at High-velocity Survive Surface Impacts in Vacuum

    Science.gov (United States)

    Austin, Daniel; Barney, Brandon

    We present experiments in which bacterial spores were found to survive being accelerated in vacuum to velocities in the range 30-120 m/s and impacted on a dense target. In these experiments, spores of Bacillus subtilis spores were charged using electrospray at atmospheric pressure, dried, and then introduced into high vacuum. Through choice of skimmers and beam tubes, different velocity ranges were achieved. An image-charge detector observed the charged spores, providing total charge and velocity. The spores then impacted a glass target within a collection vessel. After the experiment, the collection vessel contents were extracted and cultured. Several positive and negative controls were used, including the use of antibiotic-resistant spores and antibiotic-containing (rifampicin) agar for culturing. These impact velocities are of particular interest for possible transport of bacterial spores from Mars to Phobos, and may have implications for planetary protection in a Phobos sample return mission. In addition, bacteria may reach similar velocities during a spacecraft crash (e.g., within components, or from spacecraft to surface materials during impact, etc.), raising concerns about forward contamination. The velocities of interest to transport of life between planets (panspermia) are somewhat higher, but these results complement shock-based experiments and contribute to the general discussion of impact survivability of organisms.