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Sample records for bacterial isolates dna

  1. Chemically synthesized silver nanoparticles as cell lysis agent for bacterial genomic DNA isolation

    Science.gov (United States)

    Goswami, Gunajit; Boruah, Himangshu; Gautom, Trishnamoni; Jyoti Hazarika, Dibya; Barooah, Madhumita; Boro, Robin Chandra

    2017-12-01

    Silver nanoparticles (AgNPs) have seen a recent spurt of use in varied fields of science. In this paper, we showed a novel application of AgNP as a promising microbial cell-lysis agent for genomic DNA isolation. We utilized chemically synthesized AgNPs for lysing bacterial cells to isolate their genomic DNA. The AgNPs efficiently lysed bacterial cells to yield good quality DNA that could be subsequently used for several molecular biology works.

  2. DNA fingerprinting of spore-forming bacterial isolates, using Bacillus ...

    African Journals Online (AJOL)

    User

    Bc-repetitive extragenic palindromic polymerase chain reaction (Bc-Rep PCR) analysis was conducted on seven Bacillus thuringiensis isolates accessed from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) culture collection and on five local isolates of entomopathogenic spore- forming bacteria.

  3. DNA fingerprinting of spore-forming bacterial isolates, using Bacillus ...

    African Journals Online (AJOL)

    Bc-repetitive extragenic palindromic polymerase chain reaction (Bc-Rep PCR) analysis was conducted on seven Bacillus thuringiensis isolates accessed from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) culture collection and on five local isolates of entomopathogenic spore-forming bacteria.

  4. Application of silica magnetite nanocomposites to the isolation of ultrapure plasmid DNA from bacterial cells

    Science.gov (United States)

    Chiang, Chen-Li; Sung, Ching-Shan; Chen, Chuh-Yean

    2006-10-01

    The aim of this study was to develop a simple and rapid method for purification of ultrapure plasmid DNA with high yields from bacterial cultures. Nanosized superparamagnetic nanoparticles (Fe 3O 4) were prepared by chemical precipitation method using Fe 2+, Fe 3+ salt, and ammonium hydroxide under a nitrogen atmosphere. Silica-magnetite nanocomposites were prepared by the method of acid hydrolysis of tetraethoxysilane (TEOS) to coat the silica onto magnetite nanoparticles. DNA was adsorbed to the support under high salt conditions, and recovered directly in water for immediate downstream application, without the need for precipitation. We demonstrated that a useful plasmid, pRSETB-EGFP, encoding for the green fluorescent protein with T7 promoter, could be amplified in Escherichia coli of DE3 strain. Up to approximately 43 μg of high-purity ( A260/ A280 ratio=1.75) plasmid DNA was isolated from 3 ml of an overnight bacterial culture. The eluted plasmid DNA was used directly for restriction enzyme digestion, bacterial cell transformation and polymerase chain reaction (PCR) amplification with success. The protocol, starting from the preparation of bacterial lysate and ending with purified plasmid takes less than 8 min. The silica-magnetite nanocomposites deliver significant time-savings, overall higher yields, lower RNA contamination, and better PCR amplification compared to commercial available silica-based and other methods.

  5. 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.

  6. The bacterial condensin MukB compacts DNA by sequestering supercoils and stabilizing topologically isolated loops.

    Science.gov (United States)

    Kumar, Rupesh; Grosbart, Małgorzata; Nurse, Pearl; Bahng, Soon; Wyman, Claire L; Marians, Kenneth J

    2017-10-13

    MukB is a structural maintenance of chromosome-like protein required for DNA condensation. The complete condensin is a large tripartite complex of MukB, the kleisin, MukF, and an accessory protein, MukE. As found previously, MukB DNA condensation is a stepwise process. We have defined these steps topologically. They proceed first via the formation of negative supercoils that are sequestered by the protein followed by hinge-hinge interactions between MukB dimers that stabilize topologically isolated loops in the DNA. MukB itself is sufficient to mediate both of these topological alterations; neither ATP nor MukEF is required. We show that the MukB hinge region binds DNA and that this region of the protein is involved in sequestration of supercoils. Cells carrying mutations in the MukB hinge that reduce DNA condensation in vitro exhibit nucleoid decondensation in vivo . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. (+)-(10R)-Germacrene A synthase from goldenrod, Solidago canadensis; cDNA isolation, bacterial expression and functional analysis.

    Science.gov (United States)

    Prosser, Ian; Phillips, Andy L; Gittings, Simon; Lewis, Mervyn J; Hooper, Antony M; Pickett, John A; Beale, Michael H

    2002-08-01

    Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC-MS revealed the presence of both enantiomers of germacrene D and lesser amounts of germacrene A, alpha-humulene, and beta-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC-MS analysis of products from of (3)H- and (2)H-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco.

  8. Identification of leptospiral isolates by bacterial restriction endonuclease analysis (Brenda

    Directory of Open Access Journals (Sweden)

    Venkatesha M

    2001-01-01

    Full Text Available DNA samples from 19 reference serovars belonging to 19 different serogroups of Leptospira interrogans and two serovars belonging to Leptospira biflexa were examined by bacterial restriction endonuclease analysis using EcoR I and Hae III enzymes. All the serovars gave unique restriction patterns that differed from each other. DNA from 10 local isolates digested with these enzymes produced patterns which on comparison with the standard patterns produced by reference strains could be identified to serovar level.

  9. 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.

  10. 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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. BACTERIAL CELL KILLING MEDIATED BY TOPOISOMERASE I DNA CLEAVAGE ACTIVITY

    Science.gov (United States)

    Cheng, Bokun; Shukla, Shikha; Vasunilashorn, Sarinnapha; Mukhopadhyay, Somshuvra; Tse-Dinh, Yuk-Ching

    2005-01-01

    DNA topoisomerases are important clinical targets for antibacterial and anticancer therapy. At least one type IA DNA topoisomerases can be found in every bacterium, making it a logical target for antibacterial agents that can convert the enzyme into poison by trapping its covalent complex with DNA. However, it has not been possible previously to observe the consequence of having such stabilized covalent complex of bacterial topoisomerase I in vivo. We isolated a mutant of recombinant Yersinia pestis topoisomerase I that forms a stabilized covalent complex with DNA by screening for the ability to induce the SOS response in Escherichia coli. Overexpression of this mutant topoisomerase I resulted in bacterial cell death. From sequence analysis and site-directed mutagenesis, it was determined that a single amino acid substitution in the TOPRIM domain changing a strictly conserved glycine residue to serine in either the Y. pestis or E. coli topoisomerase I can result in a mutant enzyme that has the SOS inducing and cell killing properties. Analysis of the purified mutant enzymes showed that they have no relaxation activity but retain the ability to cleave DNA and form a covalent complex. These results demonstrate that perturbation of the active site region of bacterial topoisomerase I can result in stabilization of the covalent intermediate, with the in vivo consequence of bacterial cell death. Small molecules that induce similar perturbation in the enzyme-DNA complex should be candidates as leads for novel antibacterial agents. PMID:16159875

  12. 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

  13. Germacrene C synthase from Lycopersicon esculentum cv. VFNT cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase.

    Science.gov (United States)

    Colby, S M; Crock, J; Dowdle-Rizzo, B; Lemaux, P G; Croteau, R

    1998-03-03

    Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, beta-caryophyllene, alpha-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (Mr = 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton delta-cadinene synthase (50% identity).

  14. Germacrene C synthase from Lycopersicon esculentum cv. VFNT Cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase

    Science.gov (United States)

    Colby, Sheila M.; Crock, John; Dowdle-Rizzo, Barbara; Lemaux, Peggy G.; Croteau, Rodney

    1998-01-01

    Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, β-caryophyllene, α-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (Mr = 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton δ-cadinene synthase (50% identity). PMID:9482865

  15. Hexadecane degradation by bacterial strains isolated from ...

    African Journals Online (AJOL)

    A study was undertaken to detect and monitor the degradation of hexadecane by three potential degrading bacteria (Pseudomonas putida, Rhodococcus erythroplolis and Bacillus thermoleovorans) isolated from contaminated soils in Riyadh, Saudi Arabia. The extraction of the bacterial populations from these polluted soils ...

  16. Molecular cloning of cellulase genes from indigenous bacterial isolates

    International Nuclear Information System (INIS)

    Jong Bor Chyan; Pauline Liew Woan Ying; Mat Rasol Awang

    2006-01-01

    Indigenous cellulolytic bacterial isolates having high activities in degrading carboxymethyl cellulose (CMC) were isolated from local environments. Identification of these isolates were performed by molecular techniques. By using polymerase chain reaction (PCR) techniques, PCR products encoding cellulase gene were amplified from the total genomic DNAs. Purified PCR product was successfully cloned and expressed in Escherichia coli host system. The complete nucleotide sequences of the cellulase genes determined. The analysis of amino acid sequences deduced from the genes indicated that the cloned DNA fragments show high homology to those of endoglucanase genes of family GH5. All cloned genes consist of an N-terminal signal peptide, a catalytic domain of family 5 glycosyl hydrolase and a cellulose-binding domain of family III. (Author)

  17. Isolation of Enterobacter cowanii from Eucalyptus showing symptoms of bacterial blight and dieback in Uruguay.

    Science.gov (United States)

    Brady, C L; Venter, S N; Cleenwerck, I; Engelbeen, K; de Vos, P; Wingfield, M J; Telechea, N; Coutinho, T A

    2009-10-01

    This study was performed to identify bacterial strains isolated simultaneously with Pantoea species from Eucalyptus trees showing symptoms of bacterial blight and dieback in Uruguay. Several molecular techniques including 16S rRNA and rpoB gene sequencing and DNA-DNA hybridization were used to characterize the gram-negative, facultatively anaerobic, slime-producing bacterial strains isolated along with Pantoea species from Eucalyptus. Hypersensitivity reactions (HR) and pathogenicity tests were performed on tobacco and Eucalyptus seedlings, respectively. The isolates clustered closely with the type strain of Enterobacter cowanii in both phylogenetic trees constructed. The DNA-DNA similarity between the isolates and the type strain of Ent. cowanii ranged from 88% to 92%. A positive HR was observed on the tobacco seedlings, but no disease symptoms were visible on the inoculated Eucalyptus seedlings. Enterobacter cowanii was isolated from trees with symptoms of bacterial blight although strains of this bacterial species do not appear to be the causal agent of the disease. This study provides the first report of Ent. cowanii isolated from Eucalyptus. Its presence in Eucalyptus tissue suggests that it is an endophyte in trees showing symptoms of blight.

  18. Germacrene C synthase from Lycopersicon esculentum cv. VFNT Cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase

    OpenAIRE

    Colby, Sheila M.; Crock, John; Dowdle-Rizzo, Barbara; Lemaux, Peggy G.; Croteau, Rodney

    1998-01-01

    Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, β-caryophyllene, α-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of...

  19. Biodegradation of phenol by a newly isolated marine bacterial strain ...

    African Journals Online (AJOL)

    ajl yemi

    2011-12-26

    peptone agar plates with. 1500 mg/L phenol. ... biodegradation of the strain was up to 92.0% under the optimum conditions even when the phenol ... Growth of marine bacterial isolates in various concentration of phenol. Isolate.

  20. 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.

  1. Isolation and development of bioluminescent reporter phages for bacterial dysentery.

    Science.gov (United States)

    Schofield, D A; Wray, D J; Molineux, I J

    2015-02-01

    Shigellosis is a significant cause of morbidity and mortality worldwide, most notably amongst children. Moreover, there is a global increase in the occurrence of multidrug-resistant isolates, including the epidemic and pandemic Shigella dysenteriae type 1 strain. We developed a bioluminescent reporter phage assay to facilitate detection and simultaneously determine antibiotic susceptibility. A Shigella flexneri phage (Shfl25875) was isolated from environmental wastewater and characterized by DNA sequencing. Shfl25875 is T4-like, harbors a 169,062-bp genome, and grows on most (28/29) S. flexneri strains and all 12 S. dysenteriae type 1 strains tested. The genes encoding bacterial luciferase were integrated into the Shfl25875 genome to create a "light-tagged" phage capable of transducing a bioluminescent phenotype to infected cells. Shfl25875::luxAB rapidly detects cultured isolates with high sensitivity. Specificity experiments indicate that the reporter does not respond to Shigella boydii, non-type 1 S. dysenteriae strains, and most non-Shigella Enterobacteriaceae. Shfl25875::luxAB generates ampicillin and ciprofloxacin susceptibility profiles that are similar to the standard Clinical and Laboratory Standards Institute (CLSI) growth microdilution method, but in a significantly shorter time. In addition, the reporter phage detects Shigella in mock-infected stool. This new reporter phage shows promise as a tool for the detection of cultured isolates or complex clinical samples.

  2. characterisation of bacterial isolates from patients wounds and ...

    African Journals Online (AJOL)

    2013-12-12

    Dec 12, 2013 ... resistance pattern of bacterial isolates from the patient's wounds and ward environment. Conclusion: There is a high incidence of antibiotic resistance of bacterial isolates from samples cultured from patients ... friction/ shear force, pressure or as a result of disease, such as leg ulcers or carcinomas (2,3).

  3. Utilization of chitinolytic bacterial isolates to control anthracnose of ...

    African Journals Online (AJOL)

    Colletotrichum spp. are causal agents of anthracnose in many plant species. Biological control of Colletotrichum spp. utilizing bacterial isolates and fungi has been reported. However, chitinolytic bacterial isolate utilization to control anthracnose of cocoa leaf has not seemingly been studied yet. In this study, we used ...

  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. Bacterial species determination from DNA-DNA hybridization by using genome fragments and DNA microarrays.

    Science.gov (United States)

    Cho, J C; Tiedje, J M

    2001-08-01

    Whole genomic DNA-DNA hybridization has been a cornerstone of bacterial species determination but is not widely used because it is not easily implemented. We have developed a method based on random genome fragments and DNA microarray technology that overcomes the disadvantages of whole-genome DNA-DNA hybridization. Reference genomes of four fluorescent Pseudomonas species were fragmented, and 60 to 96 genome fragments of approximately 1 kb from each strain were spotted on microarrays. Genomes from 12 well-characterized fluorescent Pseudomonas strains were labeled with Cy dyes and hybridized to the arrays. Cluster analysis of the hybridization profiles revealed taxonomic relationships between bacterial strains tested at species to strain level resolution, suggesting that this approach is useful for the identification of bacteria as well as determining the genetic distance among bacteria. Since arrays can contain thousands of DNA spots, a single array has the potential for broad identification capacity. In addition, the method does not require laborious cross-hybridizations and can provide an open database of hybridization profiles, avoiding the limitations of traditional DNA-DNA hybridization.

  6. Quinolone Resistance in Bacterial Isolates from Chicken Carcasses ...

    African Journals Online (AJOL)

    Two hundred bacterial isolates including Escherichia coli (95; 47.5%), Salmonella serotypes (78; 38.0%), Klebsiella (17; 8.5%) and Staphylococcus aureus (12; 6.0%) were isolated from chicken carcasses within the six-year period. On the overall, the isolates were resistant to ciprofloxacin (40.5%), enrofloxacin (21.0%), ...

  7. Immersion Refractometry of Isolated Bacterial Cell Walls

    Science.gov (United States)

    Marquis, Robert E.

    1973-01-01

    Immersion-refractometric and light-scattering measurements were adapted to determinations of average refractive indices and physical compactness of isolated bacterial cell walls. The structures were immersed in solutions containing various concentrations of polymer molecules that cannot penetrate into wall pores, and then an estimate was made of the polymer concentration or the refractive index of the polymer solution in which light scattering was reduced to zero. Because each wall preparation was heterogeneous, the refractive index of the medium for zero light scattering had to be estimated by extrapolation. Refractive indices for walls suspended in bovine serum albumin solutions ranged from 1.348 for walls of the rod form of Arthrobacter crystallopoietes to 1.382 for walls of the teichoic acid deficient, 52A5 strain of Staphylococcus aureus. These indices were used to calculate approximate values for solids content per milliliter, and the calculated values agreed closely with those estimated from a knowledge of dextran-impermeable volumes per gram, dry weight, of the walls. When large molecules such as dextrans or serum albumin were used for immersion refractometry, the refractive indices obtained were for entire walls, including both wall polymers and wall water. When smaller molecules that can penetrate wall pores to various extents were used with Micrococcus lysodeikticus walls, the average, apparent refractive index of the structures increased as the molecular size of probing molecules was decreased. It was possible to obtain an estimate of 1.45 to 1.46 for the refractive index of wall polymers, predominantly peptidoglycans in this case, by extrapolating the curve for refractive index versus molecular radius to a value of 0.2 nm, the approximate radius of a water molecule. This relatively low value for polymer refractive index was interpreted as evidence in favor of the amorphous, elastic model of peptidoglycan structure and against the crystalline, rigid

  8. 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.

  9. Rapid DNA extraction of bacterial genome using laundry detergents ...

    African Journals Online (AJOL)

    Genomic DNA extraction from bacterial cells involves processes normally performed in most biological laboratories. Therefore, various methods have been offered, manually and kit, but these methods may be time consuming and costly. In this paper, genomic DNA extraction of Pseudomonas aeruginosa was investigated ...

  10. Rapid DNA extraction of bacterial genome using laundry detergents ...

    African Journals Online (AJOL)

    Yomi

    2012-01-03

    Jan 3, 2012 ... Genomic DNA extraction from bacterial cells involves processes normally performed in most biological laboratories. Therefore, various methods have been offered, manually and kit, but these methods may be time consuming and costly. In this paper, genomic DNA extraction of Pseudomonas aeruginosa ...

  11. Antimicrobial resistance of bacterial strains isolated from orange ...

    African Journals Online (AJOL)

    The organisms encountered include Saccharomyces cerevisiae, Saccharomyces sp, Rhodotorula sp, Bacillus cereus, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes and Micrococcus sp. The resistances of thirty bacterial strains isolated from orange juice products to the commonly used ...

  12. (MDR) Bacterial Activity of Endophytic Fungi Isolated from the

    African Journals Online (AJOL)

    MDR) bacterial potential of culturable endophytes from A. corniculatum in Beibu Gulf, China. Methods: The plant parts were collected from healthy-looking A. corniculatum. The endophytes were isolated and identified by colonial morphology and ...

  13. Antibiotics susceptibility patterns of urine bacterial isolates in Zaria ...

    African Journals Online (AJOL)

    Methods: Urine samples collected from students of Faculty of Pharmaceutical Sciences, Ahmadu Bello University and Kaduna State Polytechnic, Zaria, Nigeria, commercial sex workers, and illiterate men and women in Zaria were cultured and bacterial isolates identified using standard microbiological procedures.

  14. 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.

  15. Antimicrobial resistance of bacterial isolates from urinary tract ...

    African Journals Online (AJOL)

    Urinary tract infections are common health problems and vary according to geography and regions. A retrospective analysis was conducted to determine the antimicrobial resistance of bacterial isolates from urine at Felege Hiwot Referral Hospital from September 2003 to June 2008. From 529 urine specimens, bacterial ...

  16. Specific amplification of bacterial DNA by optimized so-called universal bacterial primers in samples rich of plant DNA.

    Science.gov (United States)

    Dorn-In, Samart; Bassitta, Rupert; Schwaiger, Karin; Bauer, Johann; Hölzel, Christina S

    2015-06-01

    Universal primers targeting the bacterial 16S-rRNA-gene allow quantification of the total bacterial load in variable sample types by qPCR. However, many universal primer pairs also amplify DNA of plants or even of archaea and other eukaryotic cells. By using these primers, the total bacterial load might be misevaluated, whenever samples contain high amounts of non-target DNA. Thus, this study aimed to provide primer pairs which are suitable for quantification and identification of bacterial DNA in samples such as feed, spices and sample material from digesters. For 42 primers, mismatches to the sequence of chloroplasts and mitochondria of plants were evaluated. Six primer pairs were further analyzed with regard to the question whether they anneal to DNA of archaea, animal tissue and fungi. Subsequently they were tested with sample matrix such as plants, feed, feces, soil and environmental samples. To this purpose, the target DNA in the samples was quantified by qPCR. The PCR products of plant and feed samples were further processed for the Single Strand Conformation Polymorphism method followed by sequence analysis. The sequencing results revealed that primer pair 335F/769R amplified only bacterial DNA in samples such as plants and animal feed, in which the DNA of plants prevailed. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Exploration, Isolation, and Identification of Carotenoid from Bacterial Symbiont of Sponge Callyspongia vaginalis

    Directory of Open Access Journals (Sweden)

    Iqna Kamila Abfa

    2017-06-01

    Full Text Available During the past two decades research on marine bacteria has highlighted the tremendous potential of symbiotic-microorganisms as a source of bioactive secondary. One of the potential of the bacterial symbionts is producing a natural pigment, and these organisms can be used as a sustainable source of natural pigments. Carotenoid is one of the most important pigments that has important roles in physiological and molecular processes of microorganisms, as well as for human health. The objective of this study is to analyze carotenoid pigments from marine bacterial symbionts from sponge and to identify bacterial symbionts that produce carotenoid pigments. Pigment analysis was performed by a UV-VIS spectrophotometer and High Performance Liquid Chromatography (HPLC. Molecular bacterial identification was performed based on 16S rDNA sequence. The isolation of bacterial symbionts from C. vaginalison Zobell 2216E medium resulted in one bacterium, CB-SP5, positively synthesized carotenoids. By reverse phase HPLC analysis, the carotenoid pigments in the bacterial symbionts were identified as diadinoxanthin, fucoxanthin, neoxanthin, dinoxanthin, anddiadinochrome. CB-SP5 shared the highest level of 16S rDNA gene sequence similarity with Psychrobacter celer (99%.   Keywords : carotenoid, sponge, bacterial symbiont, 16S rDNA.

  18. 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

  19. Comparative study of HOCl-inflicted damage to bacterial DNA ex vivo and within cells

    Science.gov (United States)

    Suquet, Christine; Warren, Jeffrey J.; Seth, Nimulrith; Hurst, James K.

    2009-01-01

    The prospects for using bacterial DNA as an intrinsic probe for HOCl and secondary oxidants/chlorinating agents associated with it has been evaluated using both in vitro and in vivo studies. Single-strand and double-strand breaks occurred in bare plasmid DNA that had been exposed to high levels of HOCl, although these reactions were very inefficient compared to polynucleotide chain cleavage caused by the OH•-generating reagent, peroxynitrite. Plasmid nicking was not increased when intact Escherichia coli were exposed to HOCl; rather, the amount of recoverable plasmid diminished in a dose-dependent manner. At concentration levels of HOCl exceeding lethal doses, genomic bacterial DNA underwent extensive fragmentation and the amount of precipitable DNA-protein complexes increased several-fold. The 5-chlorocytosine content of plasmid and genomic DNA isolated from HOCl-exposed E. coli was also slightly elevated above controls, as measured by mass spectrometry of the deaminated product, 5-chlorouracil. However, the yields were not dose-dependent over the bactericidal concentration range. Genomic DNA recovered from E. coli that had been subjected to phagocytosis by human neutrophils occasionally showed small increases in 5-chlorocytosine content when compared to analogous cellular reactions where myeloperoxidase activity was inhibited by azide ion. Overall, the amount of isolable 5-chlorouracil from the HOCl-exposed bacterial cells was far less than the damage manifested in polynucleotide bond cleavage and cross-linking. PMID:19850004

  20. 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

    DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often <100 bp) and may persist in the environment for more than half a million years. Fragmented DNA is recognized as nutrient source f...... quantities in the environment, can be acquired by bacteria through natural transformation. Our findings open for the possibility that natural genetic exchange can occur with DNA up to several hundreds of thousands years old.......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...

  1. Pseudomonas floridensis sp. nov., a bacterial pathogen isolated from tomato.

    Science.gov (United States)

    Timilsina, Sujan; Minsavage, Gerald V; Preston, James; Newberry, Eric A; Paret, Matthews L; Goss, Erica M; Jones, Jeffrey B; Vallad, Gary E

    2018-01-01

    An unusual fluorescent pseudomonad was isolated from tomato exhibiting leaf spot symptoms similar to bacterial speck. Strains were fluorescent, oxidase- and arginine-dihydrolase-negative, elicited a hypersensitive reaction on tobacco and produced a soft rot on potato slices. However, the strains produced an unusual yellow, mucoid growth on media containing 5 % sucrose that is not typical of levan. Based on multilocus sequence analysis using 16S rRNA, gap1, gltA, gyrB and rpoD, these strains formed a distinct phylogenetic group in the genus Pseudomonas and were most closely related to Pseudomonas viridiflava within the Pseudomonassyringae complex. Whole-genome comparisons, using average nucleotide identity based on blast, of representative strain GEV388 T and publicly available genomes representing the genus Pseudomonas revealed phylogroup 7 P. viridiflava strain UASW0038 and P. viridiflava type strain ICMP 2848 T as the closest relatives with 86.59 and 86.56 % nucleotide identity, respectively. In silico DNA-DNA hybridization using the genome-to-genome distance calculation method estimated 31.1 % DNA relatedness between GEV388 T and P. viridiflava ATCC 13223 T , strongly suggesting the strains are representatives of different species. These results together with Biolog GEN III tests, fatty acid methyl ester profiles and phylogenetic analysis using 16S rRNA and multiple housekeeping gene sequences demonstrated that this group represents a novel species member of the genus Pseudomonas. The name Pseudomonas floridensis sp. nov. is proposed with GEV388 T (=LMG 30013 T =ATCC TSD-90 T ) as the type strain.

  2. Isolation and enrichment of Cryptosporidium DNA and verification of DNA purity for whole-genome sequencing.

    Science.gov (United States)

    Guo, Yaqiong; Li, Na; Lysén, Colleen; Frace, Michael; Tang, Kevin; Sammons, Scott; Roellig, Dawn M; Feng, Yaoyu; Xiao, Lihua

    2015-02-01

    Whole-genome sequencing of Cryptosporidium spp. is hampered by difficulties in obtaining sufficient, highly pure genomic DNA from clinical specimens. In this study, we developed procedures for the isolation and enrichment of Cryptosporidium genomic DNA from fecal specimens and verification of DNA purity for whole-genome sequencing. The isolation and enrichment of genomic DNA were achieved by a combination of three oocyst purification steps and whole-genome amplification (WGA) of DNA from purified oocysts. Quantitative PCR (qPCR) analysis of WGA products was used as an initial quality assessment of amplified genomic DNA. The purity of WGA products was assessed by Sanger sequencing of cloned products. Next-generation sequencing tools were used in final evaluations of genome coverage and of the extent of contamination. Altogether, 24 fecal specimens of Cryptosporidium parvum, C. hominis, C. andersoni, C. ubiquitum, C. tyzzeri, and Cryptosporidium chipmunk genotype I were processed with the procedures. As expected, WGA products with low (sequences in Sanger sequencing. The cloning-sequencing analysis, however, showed significant contamination in 5 WGA products (proportion of positive colonies derived from Cryptosporidium genomic DNA, ≤25%). Following this strategy, 20 WGA products from six Cryptosporidium species or genotypes with low (mostly sequencing, generating sequence data covering 94.5% to 99.7% of Cryptosporidium genomes, with mostly minor contamination from bacterial, fungal, and host DNA. These results suggest that the described strategy can be used effectively for the isolation and enrichment of Cryptosporidium DNA from fecal specimens for whole-genome sequencing. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  3. In vitro susceptibility of gram-negative bacterial isolates to ...

    African Journals Online (AJOL)

    In vitro susceptibility of gram-negative bacterial isolates to chlorhexidine gluconate. Y Mengistu, W Erge, B Bellete. Abstract. Objective: To investigate the susceptibility of clinical isolates of gram-negative bacteria to chlorhexidine gluconate. Design: Prospective laboratory study. Setting: Tikur Anbessa Hospital, Addis Ababa, ...

  4. Clinical bacterial isolates from hospital environment as agents of ...

    African Journals Online (AJOL)

    The relationship between bacteria isolated from the hospital environment and those from wounds of operated patients was investigated to determine the causal agents of surgical site nosocomial infections. The study was carried out on bacterial species isolated from the theatre, surgical ward and patients' surgical wounds ...

  5. Studies on hydrocarbon degradation by the bacterial isolate ...

    African Journals Online (AJOL)

    The hydrocarbon utilizing capability of Stenotrophomonas rhizophila (PM-1), isolated from oil contaminated soil composts from Western Ghats region of Karnataka was analyzed. In the bioremediation experiment, ONGC heavy crude oil and poly aromatic hydrocarbons (PAHs) utilization by the bacterial isolate was studied.

  6. Bacterial Isolates Associated with Pelvic Inflammatory Disease ...

    African Journals Online (AJOL)

    This situation is not in any way different here in Nigeria and more so in the Federal Capital Territory, Abuja where this research was conducted, there had never been any published report so far on PID. It therefore became pertinent that such studies be carried out to evaluate the bacterial organisms which may be associated ...

  7. AEROBIC BACTERIAL ISOLATES FROM INFECTED WOUNDS

    African Journals Online (AJOL)

    boaz

    Nurs. Times. 1985; 81:16-19. 5. Calvin M. Cutaneous wound repair. Wounds. 1998; 10(1): 12-32. 6. Brook I. Aerobic and anaerobic microbiology of necrotizing fasciitis in children. PediatrDermatol. 1996; 13:281-284. 7. Madsen SM, Westh H, Danielson L,. Rosadahi VT Bacterial colonization and healing of venous leg ulcers.

  8. 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

  9. Chitinolytic bactery activity isolated from the mud fields

    Directory of Open Access Journals (Sweden)

    Nuniek Herdyastuti

    2012-01-01

    Full Text Available A chitinolytic microorganism had been isolated from mud fields in Ketintang Surabaya area by using a minimal media containing 0.4% colloidal chitin. Activity assay is based on released N-acetyl-glucosamine which reacted with 3,5 dinitrosalicyclic acid. Mud fields produce 63 bacterial isolates with chitinase activity (TNH1 – TNH63 isolates. The highest activity was shown by TNH11 isolate with specific activity 1.27 U/mg. TNH11 isolates was Gram negative, rod-cocoid cell, has a colony of yellow, round shape, convex elevation, flat margin and the colony diameter 3–5 mm.

  10. Isolation and characterization of a bacterial celluloseproducing ...

    African Journals Online (AJOL)

    year ripened persimmon vinegar, which belonged to the family of Acetobacteracea based on its morphological and physiological characteristics. The phylogenetic position of the isolated strain was most closely related to Gluconacetobacter ...

  11. The bacterial DnaA-trio replication origin element specifies single-stranded DNA initiator binding.

    Science.gov (United States)

    Richardson, Tomas T; Harran, Omar; Murray, Heath

    2016-06-16

    DNA replication is tightly controlled to ensure accurate inheritance of genetic information. In all organisms, initiator proteins possessing AAA+ (ATPases associated with various cellular activities) domains bind replication origins to license new rounds of DNA synthesis. In bacteria the master initiator protein, DnaA, is highly conserved and has two crucial DNA binding activities. DnaA monomers recognize the replication origin (oriC) by binding double-stranded DNA sequences (DnaA-boxes); subsequently, DnaA filaments assemble and promote duplex unwinding by engaging and stretching a single DNA strand. While the specificity for duplex DnaA-boxes by DnaA has been appreciated for over 30 years, the sequence specificity for single-strand DNA binding has remained unknown. Here we identify a new indispensable bacterial replication origin element composed of a repeating trinucleotide motif that we term the DnaA-trio. We show that the function of the DnaA-trio is to stabilize DnaA filaments on a single DNA strand, thus providing essential precision to this binding mechanism. Bioinformatic analysis detects DnaA-trios in replication origins throughout the bacterial kingdom, indicating that this element is part of the core oriC structure. The discovery and characterization of the novel DnaA-trio extends our fundamental understanding of bacterial DNA replication initiation, and because of the conserved structure of AAA+ initiator proteins these findings raise the possibility of specific recognition motifs within replication origins of higher organisms.

  12. 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

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

    International Nuclear Information System (INIS)

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

    2013-01-01

    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. (letter)

  14. 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.

  15. Isolation of Retroelement from Plant Genomic DNA

    OpenAIRE

    sprotocols

    2014-01-01

    Author: Pat Heslop-Harrison ### Abstract: Retroelements and their derivatives are an ubiquitous and abundant component of plant genomes. From the 1990s, PCR based techniques have been developed to isolate the elements from genomic DNA of different plants, and the methods and primers used are presented here. Major classes of retroelements include the Ty1-copia, the Ty3-gypsy and the LINE (non-LTR) groups. Mixed PCR products representing the full heterogeneous pool of retrotransposo...

  16. Antibiogram of bacterial species isolated from canine pyometra

    OpenAIRE

    Madhu Swamy; Varun Bassessar; Yamini Verma

    2013-01-01

    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. ...

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

    Directory of Open Access Journals (Sweden)

    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.

  18. 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

  19. ISOLATION OF 2-METHOXYETHANOL DEGRADING BACTERIAL ...

    African Journals Online (AJOL)

    Morphological, physiological, biochemical and molecular characterization of the isolate showed that the strain, designated VB is of the genus Pseudomonas. 16S rRNA sequence analysis showed that the organism is related to Pseudomonas putida at 99.9% and Pseudomonas plecoglossicida at 99.8% similarity level.

  20. Characterization of DNA conformation inside bacterial viruses

    Science.gov (United States)

    Petrov, Anton S.; Locker, C. Rebecca; Harvey, Stephen C.

    2009-08-01

    In this study we develop a formalism to describe the organization of DNA inside bacteriophage capsids during genome packaging. We have previously shown that DNA inside bacteriophage phi29 (ϕ29) is organized into folded toroids [A. S. Petrov and S. C. Harvey, Structure 15, 21 (2007)], whereas epsilon15 (ɛ15) reveals the coaxial organization of the genetic material [A. S. Petrov, K. Lim-Hing, and S. C. Harvey, Structure 15, 807 (2007)]. We now show that each system undergoes two consecutive transitions. The first transition corresponds to the formation of global conformations and is analogous to a disorder-order conformational transition. The second transition is characterized by a significant loss of DNA mobility at the local level leading to glasslike dynamic behavior. Packing genetic material inside bacteriophages can be used as a general model to study the behavior of semiflexible chains inside confined spaces, and the proposed formalism developed here can be used to study other systems of linear polymer chains confined to closed spaces.

  1. Bacterial Endophytes Isolated from Plants in Natural Oil Seep Soils with Chronic Hydrocarbon Contamination.

    Science.gov (United States)

    Lumactud, Rhea; Shen, Shu Yi; Lau, Mimas; Fulthorpe, Roberta

    2016-01-01

    The bacterial endophytic communities of four plants growing abundantly in soils highly contaminated by hydrocarbons were analyzed through culturable and culture-independent means. Given their tolerance to the high levels of petroleum contamination at our study site, we sought evidence that Achillea millefolium, Solidago canadensis, Trifolium aureum, and Dactylis glomerata support high levels of hydrocarbon degrading endophytes. A total of 190 isolates were isolated from four plant species. The isolates were identified by partial 16S rDNA sequence analysis, with class Actinobacteria as the dominant group in all species except S. canadensis, which was dominated by Gammaproteobacteria. Microbacterium foliorum and Plantibacter flavus were present in all the plants, with M. foliorum showing predominance in D. glomerata and both endophytic bacterial species dominated T. aureum. More than 50% of the isolates demonstrated degradative capabilities for octanol, toluene, naphthalene, kerosene, or motor oil based on sole carbon source growth screens involving the reduction of tetrazolium dye. P. flavus isolates from all the sampled plants showed growth on all the petroleum hydrocarbons (PHCs) substrates tested. Mineralization of toluene and naphthalene was confirmed using gas-chromatography. 16S based terminal restriction fragment length polymorphism analysis revealed significant differences between the endophytic bacterial communities showing them to be plant host specific at this site. To our knowledge, this is the first account of the degradation potential of bacterial endophytes in these commonly occurring pioneer plants that were not previously known as phytoremediating plants.

  2. Bacterial natural transformation by highly fragmented and damaged DNA.

    Science.gov (United States)

    Overballe-Petersen, Søren; Harms, Klaus; Orlando, Ludovic A A; Mayar, J Victor Moreno; Rasmussen, Simon; Dahl, Tais W; Rosing, Minik T; Poole, Anthony M; Sicheritz-Ponten, Thomas; Brunak, Søren; Inselmann, Sabrina; de Vries, Johann; Wackernagel, Wilfried; Pybus, Oliver G; Nielsen, Rasmus; Johnsen, Pål Jarle; Nielsen, Kaare Magne; Willerslev, Eske

    2013-12-03

    DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often 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 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 generated by uptake of short DNA fragments escape mismatch repair. Moreover, double-nucleotide polymorphisms appear more common among genomes of transformable than nontransformable bacteria. Our findings reveal that short and damaged, including truly ancient, DNA molecules, which are present in large quantities in the environment, can be acquired by bacteria through natural transformation. Our findings open for the possibility that natural genetic exchange can occur with DNA up to several hundreds of thousands years old.

  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. Gene Specific Impedimetric Bacterial DNA Sensor for Rheumatic Heart Disease.

    Science.gov (United States)

    Singh, Swati; Kaushal, Ankur; Gupta, Sunil; Kumar, Ashok

    2017-03-01

    An impedimetric mga gene specific DNA sensor was developed by immobilization of single stranded DNA probe onto the screen printed modified gold-dendrimer nanohybrid composite electrode for early and rapid detection of S. pyogenes in human throat swab samples causing rheumatic heart disease. Electrochemical impedance response was measured after hybridization with bacterial single stranded genomic DNA (ssG-DNA) with probe. The sensor was found highly specific to S. pyogenes and can detect as low as 0.01 ng ssDNA in 6 µL sample only in 30 min. The nanohybrid sensor was also tested with non-specific pathogens and characterized by FTIR. An early detection of the pathogen S. pyogenes in human can save damage of mitral and aortic heart valves (rheumatic heart disease) by proper medical care.

  5. Novel bacterial consortia isolated from plastic garbage processing areas demonstrated enhanced degradation for low density polyethylene.

    Science.gov (United States)

    Skariyachan, Sinosh; Manjunatha, Vishal; Sultana, Subiya; Jois, Chandana; Bai, Vidya; Vasist, Kiran S

    2016-09-01

    This study aimed to formulate novel microbial consortia isolated from plastic garbage processing areas and thereby devise an eco-friendly approach for enhanced degradation of low-density polyethylene (LDPE). The LDPE degrading bacteria were screened and microbiologically characterized. The best isolates were formulated as bacterial consortia, and degradation efficiency was compared with the consortia formulated using known isolates obtained from the Microbial Culture Collection Centre (MTCC). The degradation products were analyzed by FTIR, GC-FID, tensile strength, and SEM. The bacterial consortia were characterized by 16S ribosomal DNA (rDNA) sequencing. The formulated bacterial consortia demonstrated 81 ± 4 and 38 ± 3 % of weight reduction for LDPE strips and LDPE pellets, respectively, over a period of 120 days. However, the consortia formulated by MTCC strains demonstrated 49 ± 4 and 20 ± 2 % of weight reduction for LDPE strips and pellets, respectively, for the same period. Furthermore, the three isolates in its individual application exhibited 70 ± 4, 68 ± 4, and 64 ± 4 % weight reduction for LDPE strips and 21 ± 2, 28 ± 2, 24 ± 2 % weight reduction for LDPE pellets over a period of 120 days (p waste management of LDPE and similar types of plastic garbage.

  6. Isolation And Characterization Of The Bacterial Flora Of Soy Milk ...

    African Journals Online (AJOL)

    A total of five bacterial isolates designated A1, A2, A3, A4 and A5 were obtained from twelve locally produced soy milk samples purchased from different locations in Nsukka town. The soy milk samples comprised seven unrefrigerated and five refrigerated samples. All samples were 24h-old according to information ...

  7. Biodegradation of orange G by a novel isolated bacterial strain ...

    African Journals Online (AJOL)

    This research article deals with biodegradation of azo dyes by a newly isolated bacterial strain from soil. Azo dyes are recalcitrant to the conventional modes of treatment due to their complex structure. This article reports decolorization of azo dye by, Gram positive, endospore forming and azo reducing, Bacillus megaterium ...

  8. Profiles of antibiotic susceptibilities of bacterial isolates and physico ...

    African Journals Online (AJOL)

    The Venda region of South Africa is predominantly rural and residents rely on untreated water sources for daily water needs. The physico-chemical quality of these water sources including antibiotic susceptibilities of enteric bacterial isolates which would guide clinicians in the empiric management of diarrhoea have ...

  9. Drug sensitivity patterns of bacterial isolates from septic post ...

    African Journals Online (AJOL)

    Drug sensitivity patterns of bacterial isolates from septic post-operative wounds in a regional referral hospital in Uganda. ... Advances in control of infections have not completely eradicated this problem because of development of drug resistance.Antimicrobial resistance can increase complications and costs associated with ...

  10. Investigation of In vitro Mineral Forming Bacterial Isolates from ...

    African Journals Online (AJOL)

    Aim: Although it is known that bacterial mechanisms are involved in dental calculus formation, which is a predisposing factor in periodontal diseases, there have been few studies of such associations, and therefore, information available is limited. The purpose of this study was to isolate and identify aerobic bacteria ...

  11. Antibiotic Sensitivity Pattern of Bacterial Isolates from the Intensive ...

    African Journals Online (AJOL)

    Antibiotic Sensitivity Pattern of Bacterial Isolates from the Intensive Care Unit of a Tertiary Care Hospital in India. ... Tropical Journal of Pharmaceutical Research ... Methods: Retrospective analysis of the indoor case papers of ICUs from January 2010 to 31st March 2011 was carried out at Department of Pharmacology, Govt.

  12. Multiple antimicrobial resistance in bacterial isolates from clinical ...

    African Journals Online (AJOL)

    A total of 545 clinical specimens (pus, blood, urine, and stool) and environmental specimens (air sample, saline solution, nasal swabs etc) were cultured for isolation and identification of aerobic bacteria and antimicrobial susceptibility testing. Out of these, 356(65%) specimens yielded one or more bacterial strains. Frequent ...

  13. Putative radioresistant bacterial isolate from sewage water

    International Nuclear Information System (INIS)

    Ang, April; Chua, Patricia; Perez, Kristine; Rey, April; Rivor Kristel; San Pablo, Czarina; Santos, Ernestine

    2001-01-01

    Sewage water was collected from a stagnant body of water in Balara, Quezon City. approximately 150 ml was aseptically transferred into eight Erlenmeyer flasks. Seven flasks were then subjected to different doses of radiation at the 60 Co irradiation facility, PNRI (Philippine Nuclear Research Institute) which are as follows: 0.01 kGy, 0.1 kGy, 0.5 kGy, 1 kGy, 5 kGy, 10 kGy, and 15 kGy. The remaining flask was used as the control. After irradiation, all the different treatments were subjected to colony count at the culture collection laboratory, NSRI. Results showed that the colonies from sewage water treatments irradiated at 0.01 kGy (treatment A), 0.10 kGy (treatment B), and 0.50 kGy (treatment C) exhibited a decreasing trend with colony counts 4.60 x 10 3 CFU/ml, and 1.30 x 10 3 CFU/ml, and 26 CFU/ml, respectively. Contrastingly, at 1 kGy (treatment D), high colony count of 2.95 x 10 3 CFU/ml was observed which is even higher compared to the control (1.02 x 10 3 CFU/ml). Treatment E that was irradiated at 5 kGy manifested low survival rate (25 CFU/ml) indicating the presence of few putative intermediate radioresistant bacteria. Radiation dose treatments higher than 5 kGy (i.e., 10 kGy and 15 kGy) exhibited no bacterial survival. (Author)

  14. Evaluating the Impact of DNA Extraction Method on the Representation of Human Oral Bacterial and Fungal Communities.

    Science.gov (United States)

    Vesty, Anna; Biswas, Kristi; Taylor, Michael W; Gear, Kim; Douglas, Richard G

    2017-01-01

    The application of high-throughput, next-generation sequencing technologies has greatly improved our understanding of the human oral microbiome. While deciphering this diverse microbial community using such approaches is more accurate than traditional culture-based methods, experimental bias introduced during critical steps such as DNA extraction may compromise the results obtained. Here, we systematically evaluate four commonly used microbial DNA extraction methods (MoBio PowerSoil® DNA Isolation Kit, QIAamp® DNA Mini Kit, Zymo Bacterial/Fungal DNA Mini PrepTM, phenol:chloroform-based DNA isolation) based on the following criteria: DNA quality and yield, and microbial community structure based on Illumina amplicon sequencing of the V3-V4 region of the 16S rRNA gene of bacteria and the internal transcribed spacer (ITS) 1 region of fungi. Our results indicate that DNA quality and yield varied significantly with DNA extraction method. Representation of bacterial genera in plaque and saliva samples did not significantly differ across DNA extraction methods and DNA extraction method showed no effect on the recovery of fungal genera from plaque. By contrast, fungal diversity from saliva was affected by DNA extraction method, suggesting that not all protocols are suitable to study the salivary mycobiome.

  15. Isolation of DNA methyltransferase from plants

    International Nuclear Information System (INIS)

    Ehrlich, K.; Malbroue, C.

    1987-01-01

    DNA methyltransferases (DMT) were isolated from nuclei of cauliflower, soybean, and pea by extraction with 0.35 M NaCl. Assays were performed on hemimethylated Micrococcus luteus DNA or on M. luteus DNA to test for maintenance or de novo methylase activity, respectively. Fully methylated DNA was used as a substrate to determine background levels of methylation. Based on these tests, yields of maintenance DMT activity in the crude extract from pea hypocotyl, soybean hypocotyl, and cauliflower inflorescence were 2.8, 0.9, and 1.6 units per g wet tissue (one unit equals 1 pmol of methyl from [ 3 H]AdoMet incorporated into acid precipitable material per h at 30 0 ). Two peaks of DMT activity were detected in the soybean nuclear extract following phosphocellulose chromatography. One eluted at 0.4 M and the other at 0.8 M KCl. With both fractions maintenance activity was approximately 2 times that of the de novo activity. Using gel filtration the DMT eluted at 220,000 Daltons. The optimal pH for activity was between 6.5 and 7.0, and the optimal temperature was 30 0

  16. Isolation of DNA methyltransferase from plants

    Energy Technology Data Exchange (ETDEWEB)

    Ehrlich, K.; Malbroue, C.

    1987-05-01

    DNA methyltransferases (DMT) were isolated from nuclei of cauliflower, soybean, and pea by extraction with 0.35 M NaCl. Assays were performed on hemimethylated Micrococcus luteus DNA or on M. luteus DNA to test for maintenance or de novo methylase activity, respectively. Fully methylated DNA was used as a substrate to determine background levels of methylation. Based on these tests, yields of maintenance DMT activity in the crude extract from pea hypocotyl, soybean hypocotyl, and cauliflower inflorescence were 2.8, 0.9, and 1.6 units per g wet tissue (one unit equals 1 pmol of methyl from (/sup 3/H)AdoMet incorporated into acid precipitable material per h at 30/sup 0/). Two peaks of DMT activity were detected in the soybean nuclear extract following phosphocellulose chromatography. One eluted at 0.4 M and the other at 0.8 M KCl. With both fractions maintenance activity was approximately 2 times that of the de novo activity. Using gel filtration the DMT eluted at 220,000 Daltons. The optimal pH for activity was between 6.5 and 7.0, and the optimal temperature was 30/sup 0/.

  17. Bacterial endophytes isolated from plants in natural oil seep soils with chronic hydrocarbon contamination

    Directory of Open Access Journals (Sweden)

    Rhea eLumactud

    2016-05-01

    Full Text Available The bacterial endophytic communities of four plants growing abundantly in soils highly contaminated by hydrocarbons were analyzed through culturable and and culture-independent means. Given their tolerance to the high levels of petroleum contamination at our study site, we sought evidence that Achillea millefolium, Solidago canadensis, Trifolium aureum and Dactylis glomerata support high levels of hydrocarbon degrading endophytes. A total of 190 isolates were isolated from four plant species. The isolates were identified by partial 16S rDNA sequence analysis, with class Actinobacteria as the dominant group in all species except Solidago canadensis, which was dominated by Gammaproteobacteria. Microbacterium foliorum and Plantibacter flavus were present in all the plants, with M. foliorum showing predominance in D. glomerata and both endophytic bacterial species dominated T. aureum. More than 50% of the isolates demonstrated degradative capabilities for octanol, toluene, naphthalene, kerosene or motor oil based on sole carbon source growth screens involving the reduction of tetrazolium dye. P. flavus isolates from all the sampled plants showed growth on all the petroleum hydrocarbons substrates tested. Mineralization of toluene and naphthalene was confirmed using gas-chromatography. 16S based terminal restriction fragment length polymorphism analysis revealed significant differences between the endophytic bacterial communities showing them to be plant host specific at this site. To our knowledge, this is the first account of the degradation potential of bacterial endophytes in these commonly occurring pioneer plants that were not previously known as phytoremediating plants.

  18. [Genetic transformation and fate of heterological DNA in bacterial cells].

    Science.gov (United States)

    Piechowska, Mirosława

    2015-01-01

    Secretion of a metabolite enabling Streptococci to undergo genetic transformation was discovered. The metabolite combined with an optimization process were applied to increase the transformation yield about 20-fold. It was observed that large amounts of DNA exert a bactericidal effect, indicating the ability of at least 70% of cells to uptake the polymer. While studying the molecular mechanism of transformation of Bacillus subtilis it was shown that the uptaken DNA forms complexes with bacterial proteins, which hinders determination of its structure. A method was found to dissociate these complexes which enabled to determine the single-stranded structure of the uptaken DNA. Donor DNA fragments incorporated into the host DNA were of about 10 Da. Non-transforming DNA can be uptaken similarly but does not undergo incorporation into the host DNA. The selectivity of Bacillus subtilis receptors was determined towards DNA of phages containing modified bases: uracil, putrescinyl-thymine and its acetylated derivative, 5'-hydroxymethylcytosine and its glycosylated derivative and also towards double-stranded RNA of f2 phage. All these modifications were tolerated by the cellular receptors, with the exception of glycosylation and the 2'-OH group in RNA.

  19. Rapid cleanup of bacterial DNA from samples containing aerosol contaminants

    Science.gov (United States)

    Menking, Darrell E.; Kracke, Suzanne K.; Emanuel, Peter A.; Valdes, James J.

    1999-01-01

    Polymerase Chain Reaction (PCR) is an in vitro enzymatic, synthetic method used to amplify specific DNA sequences from organisms. Detection of DNA using gene probes allows for absolute identification not only of specific organisms, but also of genetic material in recombinant organisms. PCR is an exquisite biological method for detecting bacteria in aerosol samples. A major challenge facing detection of DNA from field samples is that they are almost sure to contain impurities, especially impurities that inhibit amplification through PCR. DNA is being extracted from air, sewage/stool samples, food, sputum, a water and sediment; however, multi- step, time consuming methods are required to isolate the DNA from the surrounding contamination. This research focuses on developing a method for rapid cleanup of DNA which combines extraction and purification of DNA while, at the same time, removing inhibitors from 'dirty samples' to produce purified, PCR-ready DNA. GeneReleaser produces PCR-ready DNA in a rapid five-minute protocol. GeneReleaser resin was able to clean up sample contain micrograms of typical aerosol and water contaminants. The advantages of using GR are that it is rapid, inexpensive, requires one-step, uses no hazardous material and produces PCR-ready DNA.

  20. Changes In Protein Abundance Are Observed In Bacterial Isolates from a Natural Host

    Directory of Open Access Journals (Sweden)

    Megan Anne Rees

    2015-10-01

    Full Text Available Bacterial proteomic studies frequently use strains cultured in synthetic liquid media over many generations. It is uncertain whether bacterial proteins expressed under these conditions will be the same as the repertoire found in natural environments, or when bacteria are infecting a host organism. Thus, genomic and proteomic characterisation of bacteria derived from the host environment in comparison to reference strains grown in the lab, should aid understanding of pathogenesis. Isolates of Corynebacterium pseudotuberculosis were obtained from the nodes of three naturally infected sheep and compared to a laboratory reference strain using bottom-up proteomics, after whole genome sequencing of each of the field isolates. These comparisons were performed following growth in liquid media that allowed us to reach the required protein amount for proteomic analysis. Over 1350 proteins identified in the isolated strains, from which unique proteome features were revealed. Several of the identified proteins demonstrated a significant abundance difference in the field isolates compared to the reference strain even though there were no obvious differences in the DNA sequence of the corresponding gene or in nearby non-coding DNA. Higher abundance in the field isolates was observed for proteins related to hypoxia and nutrient deficiency responses as well as to thiopeptide biosynthesis.

  1. Changes in protein abundance are observed in bacterial isolates from a natural host.

    Science.gov (United States)

    Rees, Megan A; Stinear, Timothy P; Goode, Robert J A; Coppel, Ross L; Smith, Alexander I; Kleifeld, Oded

    2015-01-01

    Bacterial proteomic studies frequently use strains cultured in synthetic liquid media over many generations. It is uncertain whether bacterial proteins expressed under these conditions will be the same as the repertoire found in natural environments, or when bacteria are infecting a host organism. Thus, genomic and proteomic characterization of bacteria derived from the host environment in comparison to reference strains grown in the lab, should aid understanding of pathogenesis. Isolates of Corynebacterium pseudotuberculosis were obtained from the lymph nodes of three naturally infected sheep and compared to a laboratory reference strain using bottom-up proteomics, after whole genome sequencing of each of the field isolates. These comparisons were performed following growth in liquid media that allowed us to reach the required protein amount for proteomic analysis. Over 1350 proteins were identified in the isolated strains, from which unique proteome features were revealed. Several of the identified proteins demonstrated a significant abundance difference in the field isolates compared to the reference strain even though there were no obvious differences in the DNA sequence of the corresponding gene or in nearby non-coding DNA. Higher abundance in the field isolates was observed for proteins related to hypoxia and nutrient deficiency responses as well as to thiopeptide biosynthesis.

  2. [Bacterial endosymbiosis within the cytoplasm of Acanthamoeba lugdunensis isolated from a contact lens storage case].

    Science.gov (United States)

    Chung, D I; Kong, H H; Kim, T H; Hwang, M Y; Yu, H S; Yun, H C; Seol, S Y

    1997-06-01

    Transmission electron microscopy of an Acanthamoeba isolate (KA/L5) from a contact lens case revealed bacterial endosymbionts within cytoplasm of the amoebae. The Acanthamoeba isolate belonged to the morphological group II. Based on the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) of 18S ribosomal RNA coding DNA (rDNA), the isolate was identified as A. lugdunensis. Strain typing by isoenzyme analysis using isoelectric focusing (IEF) and mitochondrial (Mt) DNA RFLP revealed that the isolate was closely related with KA/L1, the most predominant type of isolates from contact lens storage cases, KA/E2, a clinical isolate, KA/W4, previously reported to host endosymbionts, and L3a strains of A. lugdunensis. The endosymbionts were similar to those of KA/W4 in aspects that they were randomly distributed in both trophozoites and cysts, and were rod-shaped bacteria measuring approximately 1.38 x 0.50 microns. But the number of endosymbionts per amoeba was significantly lower than that of KA/W4. They were neither limited by phagosomal membranes nor included in lacunaelike structure.

  3. Etiologic significance of bacterial isolates from rams with palpable epididymitis.

    Science.gov (United States)

    Lozano, E A

    1986-05-01

    Identified and partly identified bacterial isolates were obtained from 48 rams of various breeds that had unilateral or bilateral epididymitis. Most of the animals were approximately 1 year of age; a few were older. Brucella ovis, Actinobacillus spp, Corynebacterium spp, Haemophilus spp, Acinetobacter spp, Escherichia coli, Moraxella spp, Staphylococcus spp, Pasteurella spp, Streptococcus spp, and Chlamydia psittaci were isolated. A vaccine strain of B ovis, isolated species of bacteria, and mixtures of isolates of tissue homogenates containing all isolates except B ovis and C psittaci were inoculated via the mucous membranes of the eyes, nares, and prepuce. Palpable epididymitis was not produced by the inoculations. The vaccine strain of B ovis induced complement-fixation reaction in 11 of 20 rams.

  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. Spectrum and Sensitivity of Bacterial Keratitis Isolates in Auckland

    Directory of Open Access Journals (Sweden)

    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.

  6. Spectrum and Sensitivity of Bacterial Keratitis Isolates in Auckland.

    Science.gov (United States)

    Marasini, S; Swift, S; Dean, S J; Ormonde, S E; Craig, J P

    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.

  7. Recovery and identification of bacterial DNA from illicit drugs.

    Science.gov (United States)

    Cho, Kaymann T; Richardson, Michelle M; Kirkbride, K Paul; McNevin, Dennis; Nelson, Michelle; Pianca, Dennis; Roffey, Paul; Gahan, Michelle E

    2014-02-01

    Bacterial infections, including Bacillus anthracis (anthrax), are a common risk associated with illicit drug use, particularly among injecting drug users. There is, therefore, an urgent need to survey illicit drugs used for injection for the presence of bacteria and provide valuable information to health and forensic authorities. The objectives of this study were to develop a method for the extraction of bacterial DNA from illicit drugs and conduct a metagenomic survey of heroin and methamphetamine seized in the Australian Capital Territory during 2002-2011 for the presence of pathogens. Trends or patterns in drug contamination and their health implications for injecting drug users were also investigated. Methods based on the ChargeSwitch(®)gDNA mini kit (Invitrogen), QIAamp DNA extraction mini kit (QIAGEN) with and without bead-beating, and an organic phenol/chloroform extraction with ethanol precipitation were assessed for the recovery efficiency of both free and cellular bacterial DNA. Bacteria were identified using polymerase chain reaction and electrospray ionization-mass spectrometry (PCR/ESI-MS). An isopropanol pre-wash to remove traces of the drug and diluents, followed by a modified ChargeSwitch(®) method, was found to efficiently lyse cells and extract free and cellular DNA from Gram-positive and Gram-negative bacteria in heroin and methamphetamine which could then be identified by PCR/ESI-MS. Analysis of 12 heroin samples revealed the presence of DNA from species of Comamonas, Weissella, Bacillus, Streptococcus and Arthrobacter. No organisms were detected in the nine methamphetamine samples analysed. This study develops a method to extract and identify Gram-positive and Gram-negative bacteria from illicit drugs and demonstrates the presence of a range of bacterial pathogens in seized drug samples. These results will prove valuable for future work investigating trends or patterns in drug contamination and their health implications for injecting drug

  8. Isolated HeLa cell nuclei synthesize meaningful DNA.

    OpenAIRE

    Kristensen, T; Prydz, H

    1985-01-01

    DNA replicated at the beginning of S phase was labelled by incubating nuclei isolated from cells arrested at the G/S border with radioactive deoxyribonucleoside triphosphate in a reaction mixture sustaining DNA synthesis. By hybridization against ribosomal RNA bound to nitrocellulose, the fraction of the labelled DNA which was complementary to rRNA could be quantified, and the stability of the RNA-DNA hybrids could be estimated by sequential elution of DNA at increasing temperatures. The resu...

  9. Isolation, Characterization and Identification of Environmental Bacterial Isolates with Screening for Antagonism Against Three Bacterial Targets

    Science.gov (United States)

    2017-04-01

    making the broad-spectrum antimicrobial ineffective. When this occurs, the current strategy is to replace an ineffective antimicrobial agent with...Shlae. “Fix the Antibiotic Pipeline ”. Nature 472:32. (2011) Cotter, P.A., C. Hin, and R.P. Ross. “Bacteriocin Developing Innate Immunity for Food...and resistance. Clin. Microbiol. Rev. 12:147—179. (1999) Silver, S. “Bacterial silver resistance: molecular biology and uses and misuses of silver

  10. 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, ...Sweet MJ, Beasley SJ, Cronau SL, Hume DA. J Leukoc Biol. 1999 Oct;66(4):542-8. (.png) (.svg) (.html) (.csml) Show The... actions of bacterial DNA on murine macrophages. PubmedID 10534106 Title The actions of bacterial D

  11. Frequency of isolation and antibiotic resistance patterns of bacterial isolates from wound infections

    Directory of Open Access Journals (Sweden)

    Stojanović-Radić, Z.

    2016-12-01

    Full Text Available Six hundred and thirteen bacterial strains were isolated from wound swabs and the isolates were identified on the basis of growth on differential and selective media. In order to test the sensitivity of isolated strains to different antibiotics, the disc diffusion method, according to EUCAST protocol v 5.0 was used. The most common species isolated from wound swabs was Staphylococcus epidermidis (18.4%, followed by Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis (16.8%, 12.7% and 10.4%, respectively. The maximum resistance of Gram-positive cocci was observed to penicillin and the lowest to linezolid. Gram-negative bacteria showed the highest resistance to tetracyclines, while the same strains demonstrated the highest sensitivity to polypeptide antibiotics. Comparison of the resistance patterns of Gramnegative and Gram-positive bacterial strains showed significant difference in the tetracycline efficiency.

  12. Differentiation of some species of Neisseriaceae and other bacterial groups by DNA-DNA hybridization.

    Science.gov (United States)

    Tønjum, T; Bukholm, G; Bøvre, K

    1989-05-01

    DNA-DNA hybridization using total genomic DNA probes may represent a way of differentiating between miscellaneous bacterial species. This was studied with type and reference strains of 20 species in Moraxella, Kingella, and other selected Gram-negative groups. Both radioactive and biotin labelling were employed. Most of the species examined were easily distinguished, such as Moraxella (Branhamella) catarrhalis, M.(B.) ovis, M. atlantae, M. phenylpyruvica, M. osloensis, Neisseria elongata, N. meningitidis, Kingella kingae, K. indologenes, K. dentrificans, Oligella urethralis, Eikenella corrodens, Cardiobacterium hominis, Haemophilus aphrophilus, Actinobacillus actinomycetemcomitans, Gardnerella vaginalis, and DF-2. This reflected the extent of the genetic distances between them as a basis for identification by hybridization. There was some clustering in the Moraxella group. Especially the closely related Moraxella nonliquefaciens, M. lacunata and M. bovis showed strong hybridization affinities. This leads to potential problems in distinguishing these three species from each other by DNA-DNA hybridization with total genomic probes alone.

  13. Small and large scale genomic DNA isolation protocol for chickpea ...

    African Journals Online (AJOL)

    Small and large scale genomic DNA isolation protocol for chickpea ( Cicer arietinum L.), suitable for molecular marker and transgenic analyses. ... Chickpea is an important food legume crop with high nutritional value. Lack of appropriate DNA isolation protocol is a limiting factor for any molecular studies of this crop.

  14. Isolation and in vitro evaluation of bacteriophages against MDR-bacterial isolates from septic wound infections.

    Science.gov (United States)

    Pallavali, Roja Rani; Degati, Vijaya Lakshmi; Lomada, Dakshayani; Reddy, Madhava C; Durbaka, Vijaya Raghava Prasad

    2017-01-01

    Multi-drug resistance has become a major problem for the treatment of pathogenic bacterial infections. The use of bacteriophages is an attractive approach to overcome the problem of drug resistance in several pathogens that cause fatal diseases. Our study aimed to isolate multi drug resistant bacteria from patients with septic wounds and then isolate and apply bacteriophages in vitro as alternative therapeutic agents. Pus samples were aseptically collected from Rajiv Gandhi Institute of Medical Science (RIMS), Kadapa, A.P., and samples were analyzed by gram staining, evaluating morphological characteristics, and biochemical methods. MDR-bacterial strains were collected using the Kirby-Bauer disk diffusion method against a variety of antibiotics. Bacteriophages were collected and tested in vitro for lytic activity against MDR-bacterial isolates. Analysis of the pus swab samples revealed that the most of the isolates detected had Pseudomonas aeruginosa as the predominant bacterium, followed by Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli. Our results suggested that gram-negative bacteria were more predominant than gram-positive bacteria in septic wounds; most of these isolates were resistant to ampicillin, amoxicillin, penicillin, vancomycin and tetracycline. All the gram-positive isolates (100%) were multi-drug resistant, whereas 86% of the gram-negative isolates had a drug resistant nature. Further bacteriophages isolated from sewage demonstrated perfect lytic activity against the multi-drug resistant bacteria causing septic wounds. In vitro analysis of the isolated bacteriophages demonstrated perfect lysis against the corresponding MDR-bacteria, and these isolated phages may be promising as a first choice for prophylaxis against wound sepsis, Moreover, phage therapy does not enhance multi-drug resistance in bacteria and could work simultaneously on a wide variety of MDR-bacteria when used in a bacteriophage cocktail. Hence, our results suggest

  15. Isolation and in vitro evaluation of bacteriophages against MDR-bacterial isolates from septic wound infections.

    Directory of Open Access Journals (Sweden)

    Roja Rani Pallavali

    Full Text Available Multi-drug resistance has become a major problem for the treatment of pathogenic bacterial infections. The use of bacteriophages is an attractive approach to overcome the problem of drug resistance in several pathogens that cause fatal diseases. Our study aimed to isolate multi drug resistant bacteria from patients with septic wounds and then isolate and apply bacteriophages in vitro as alternative therapeutic agents. Pus samples were aseptically collected from Rajiv Gandhi Institute of Medical Science (RIMS, Kadapa, A.P., and samples were analyzed by gram staining, evaluating morphological characteristics, and biochemical methods. MDR-bacterial strains were collected using the Kirby-Bauer disk diffusion method against a variety of antibiotics. Bacteriophages were collected and tested in vitro for lytic activity against MDR-bacterial isolates. Analysis of the pus swab samples revealed that the most of the isolates detected had Pseudomonas aeruginosa as the predominant bacterium, followed by Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli. Our results suggested that gram-negative bacteria were more predominant than gram-positive bacteria in septic wounds; most of these isolates were resistant to ampicillin, amoxicillin, penicillin, vancomycin and tetracycline. All the gram-positive isolates (100% were multi-drug resistant, whereas 86% of the gram-negative isolates had a drug resistant nature. Further bacteriophages isolated from sewage demonstrated perfect lytic activity against the multi-drug resistant bacteria causing septic wounds. In vitro analysis of the isolated bacteriophages demonstrated perfect lysis against the corresponding MDR-bacteria, and these isolated phages may be promising as a first choice for prophylaxis against wound sepsis, Moreover, phage therapy does not enhance multi-drug resistance in bacteria and could work simultaneously on a wide variety of MDR-bacteria when used in a bacteriophage cocktail. Hence

  16. Isolation of cell-free bacterial inclusion bodies.

    Science.gov (United States)

    Rodríguez-Carmona, Escarlata; Cano-Garrido, Olivia; Seras-Franzoso, Joaquin; Villaverde, Antonio; García-Fruitós, Elena

    2010-09-17

    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. 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. The application of the developed protocol allows obtaining bacterial free inclusion bodies suitable for use in mammalian cell cultures and other biological interfaces.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Isolating silkworm genomic DNA without liquid nitrogen suitable for ...

    African Journals Online (AJOL)

    Genomic DNA was isolated from posterior silk gland of silkworms, Antheraea assama. Absolute alcohol was used as tissue fixing solution instead of grinding in liquid nitrogen, which yielded high molecular weight DNA (>40 kb). Samples yielded similar amount of DNA when fixed in absolute alcohol (400 μmg/g of silk gland ...

  19. An efficient DNA isolation method for tropical plants

    African Journals Online (AJOL)

    walkinnet

    2013-05-08

    May 8, 2013 ... Due to interfering components such as polysacharrides, polyphenols, etc, DNA isolation from tropical plants had been challenging. We developed a safe, universal and efficient DNA extraction method, which yielded high-quality DNA from 10 tropical plants including cassava, rubber tree, banana, etc. In the.

  20. An efficient DNA isolation method for tropical plants | Huang | African ...

    African Journals Online (AJOL)

    Due to interfering components such as polysacharrides, polyphenols, etc, DNA isolation from tropical plants had been challenging. We developed a safe, universal and efficient DNA extraction method, which yielded high-quality DNA from 10 tropical plants including cassava, rubber tree, banana, etc. In the extraction buffer, ...

  1. Antibiotic resistance pattern of bacterial isolates in neonatal care unit

    Directory of Open Access Journals (Sweden)

    S Shrestha

    2010-12-01

    Full Text Available INTRODUCTION: Bacterial infections account for a huge proportion of neonatal deaths worldwide. The problem of antibiotic resistance among common bacterial pathogens mainly the gram negative bacteria is emerging globally which is of more serious concern in developing countries like Nepal. METHODS: A one year retrospective hospital based study was carried out to analyze the results of neonatal blood, cerebrospinal fluid, urine, stool and surface cultures and to look into the sensitivity pattern of the commonly used antibiotics. RESULTS: The positive yield of blood, urine, eye swab and CSF cultures were 19.56%, 38.5%, 60% and 0.36% respectively. The most common isolates in the blood culture were coagulase negative Staphylococcus, Acinetobacter, Enterobacter and non-haemolytic Streptococcus. A significant percent of the isolates were resistant to the first line antibiotics. Among the gram negative isolates more than 30% are resistant to cefotaxime and more than 50% are resistant to gentamicin. During the one year period we had Nursery outbreaks of methicillin resistant Staphylococcus aureus and Salmonella infections. With the help of environmental cultures we were able to trace the source and intervene appropriately. CONCLUSIONS: Continuous surveillance for antibiotic susceptibility, rational use of antibiotics and the strategy of antibiotic cycling can provide some answers to the emerging problem of antibiotic resistance.

  2. Antibiotic resistance pattern of bacterial isolates in neonatal care unit.

    Science.gov (United States)

    Shrestha, S; Adhikari, N; Rai, B K; Shreepaili, A

    2010-01-01

    Bacterial infections account for a huge proportion of neonatal deaths worldwide. The problem of antibiotic resistance among common bacterial pathogens mainly the gram negative bacteria is emerging globally which is of more serious concern in developing countries like Nepal. A one year retrospective hospital based study was carried out to analyze the results of neonatal blood, cerebrospinal fluid, urine, stool and surface cultures and to look into the sensitivity pattern of the commonly used antibiotics. The positive yield of blood, urine, eye swab and CSF cultures were 19.56%, 38.5%, 60% and 0.36% respectively. The most common isolates in the blood culture were coagulase negative Staphylococcus, Acinetobacter, Enterobacter and non-haemolytic Streptococcus. A significant percent of the isolates were resistant to the first line antibiotics. Among the gram negative isolates more than 30% are resistant to cefotaxime and more than 50% are resistant to gentamicin. During the one year period we had Nursery outbreaks of methicillin resistant Staphylococcus aureus and Salmonella infections. With the help of environmental cultures we were able to trace the source and intervene appropriately. Continuous surveillance for antibiotic susceptibility, rational use of antibiotics and the strategy of antibiotic cycling can provide some answers to the emerging problem of antibiotic resistance.

  3. 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. Copyright © 2016 Elsevier GmbH. All rights reserved.

  4. Stimulation of bacterial DNA synthesis by algal exudates in attached algal-bacterial consortia

    International Nuclear Information System (INIS)

    Murray, R.E.; Cooksey, K.E.; Priscu, J.C.

    1986-01-01

    Algal-bacterial consortia attached to polystyrene surfaces were prepared in the laboratory by using the marine diatom Amphora coffeaeformis and the marine bacterium Vibrio proteolytica (the approved name of this bacterium is Vibrio proteolyticus. The organisms were attached to the surfaces at cell densities of approximately 5 x 10 4 cells cm -2 (diatoms) and 5 x 10 6 cells cm -2 (bacteria). The algal-bacterial consortia consistently exhibited higher rates of [ 3 H]thymidine incorporation than did biofilms composed solely of bacteria. The rates of [ 3 H]thymidine incorporation by the algal-bacterial consortia were fourfold greater than the rates of incorporation by monobacterial biofilms 16 h after biofilm formation and were 16-fold greater 70 h after biofilm formation. Extracellular material released from the attached Amphora cells supported rates of bacterial activity (0.8 x 10 -21 mol to 17.9 x 10 -21 mol of [ 3 H]thymidine incorporated cell -1 h -1 ) and growth (doubling time, 29.5 to 1.4 days) comparable to values reported for a wide variety of marine and freshwater ecosystems. In the presence of sessile diatom populations, DNA synthesis by attached V. proteolytica cells was light dependent and increased with increasing algal abundance. The metabolic activity of diatoms thus appears to be the rate-limiting process in biofilm development on illuminated surfaces under conditions of low bulk-water dissolved organic carbon

  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. 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

  7. 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.

  8. Effect of irradiation on the detection of bacterial DNA in contaminated food samples by DNA hybridization

    International Nuclear Information System (INIS)

    Rowe, T.F.; Towner, K.J.

    1994-01-01

    A membrane-based DNA hybridization technique was used in a model system to examine the effect of irradiation treatment on the detection of bacterial contamination in foodstuffs. Although hybridization signals were reduced compared with otherwise identical unirradiated food samples, artificial contamination levels in excess of 10 5 cfu per test could be distinguished in 12 of the 13 foods examined following the irradiation process. In no case were viable bacteria detected following irradiation treatment. (Author)

  9. 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.

  10. DNA immunization with a herpes simplex virus 2 bacterial artificial chromosome

    International Nuclear Information System (INIS)

    Meseda, Clement A.; Schmeisser, Falko; Pedersen, Robin; Woerner, Amy; Weir, Jerry P.

    2004-01-01

    Construction of a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) is described. BAC vector sequences were inserted into the thymidine kinase gene of HSV-2 by homologous recombination. DNA from cells infected with the resulting recombinant virus was transformed into E. coli, and colonies containing the HSV-2 BAC (HSV2-BAC) were isolated and analyzed for the expected genotype. HSV2-BAC DNA was infectious when transfected back into mammalian cells and the resulting virus was thymidine kinase negative. When used to immunize mice, the HSV2-BAC DNA elicited a strong HSV-2 specific antibody response that was equal to or greater than live virus immunization. Further, HSV2-BAC immunization was protective when animals were challenged with a lethal dose of virus. The utility of the HSV2-BAC for construction of recombinant virus genomes was demonstrated by elimination of the HSV-2 glycoprotein D (gD) gene. A recombinant HSV-2 BAC with the gD gene deleted was isolated and shown to be incapable of producing infectious virus following transfection unless an HSV gD gene was expressed in a complementing cell line. Immunization of mice with the HSV2 gD-BAC also elicited an HSV-2 specific antibody response and was protective. The results demonstrate the feasibility of DNA immunization with HSV-2 bacterial artificial chromosomes for replicating and nonreplicating candidate HSV-2 vaccines, as well as the utility of BAC technology for construction and maintenance of novel HSV-2 vaccines. The results further suggest that such technology will be a powerful tool for dissecting the immune response to HSV-2

  11. Antimicrobial resistance of bacterial enteropathogens isolated from stools in Madagascar.

    Science.gov (United States)

    Randrianirina, Frederique; Ratsima, Elisoa Hariniana; Ramparany, Lova; Randremanana, Rindra; Rakotonirina, Hanitra Clara; Andriamanantena, Tahiry; Rakotomanana, Fanjasoa; Rajatonirina, Soatiana; Richard, Vincent; Talarmin, Antoine

    2014-02-25

    Diarrheal diseases are a major public health problem in developing countries, and are one of the main causes of hospital admissions in Madagascar. The Pasteur Institute of Madagascar undertook a study to determine the prevalence and the pathogenicity of bacterial, viral and protozoal enteropathogens in diarrheal and non-diarrheal stools of children aged less than 5 years in Madagascar. We present here the results of the analysis of antimicrobial susceptibility of the bacteria isolated during this study. The study was conducted in the community setting in 14 districts of Madagascar from October 2008 to May 2009. Conventional methods and PCR were used to identify the bacteria; antimicrobial susceptibility was determined using an agar diffusion method for enterobacteriaceae and MICs were measured by an agar dilution method for Campylobacter sp. In addition to the strains isolated during this study, Salmonella sp and Shigella sp isolated at the Pasteur Institute of Madagascar from 2005 to 2009 were included in the analysis to increase the power of the study. Twenty-nine strains of Salmonella sp, 35 strains of Shigella sp, 195 strains of diarrheagenic E. coli, 203 strains of C. jejuni and 71 strains of C. coli isolated in the community setting were tested for antibiotic resistance. Fifty-five strains of Salmonella sp and 129 strains of Shigella sp isolated from patients referred to the Pasteur Institute of Madagascar were also included in the study. Many E. coli and Shigella isolates (around 80%) but fewer Salmonella isolates were resistant to ampicillin and trimethoprim/sulfamethoxazole. A small proportion of strains of each species were resistant to ciprofloxacin and only 3% of E. coli strains presented a resistance to third generation cephalosporins due to the production of extended-spectrum beta-lactamases. The resistance of Campylobacter sp to ampicillin was the most prevalent, whereas less than 5% of isolates were resistant to each of the other antibiotics. The

  12. Identification of airborne bacterial and fungal species in the clinical microbiology laboratory of a university teaching hospital employing ribosomal DNA (rDNA) PCR and gene sequencing techniques.

    Science.gov (United States)

    Nagano, Yuriko; Walker, Jim; Loughrey, Anne; Millar, Cherie; Goldsmith, Colin; Rooney, Paul; Elborn, Stuart; Moore, John

    2009-06-01

    Universal or "broad-range" PCR-based ribosomal DNA (rDNA) was performed on a collection of 58 isolates (n = 30 bacteria + 28 fungi), originating from environmental air from several locations within a busy clinical microbiology laboratory, supporting a university teaching hospital. A total of 10 bacterial genera were identified including both Gram-positive and Gram-negative genera. Gram-positive organisms accounted for 27/30 (90%) of total bacterial species, consisting of seven genera and included (in descending order of frequency) Staphylococcus, Micrococcus, Corynebacterium, Paenibacillus, Arthrobacter, Janibacter and Rothia. Gram-negative organisms were less frequently isolated 3/30 (10%) and comprised three genera, including Moraxella, Psychrobacter and Haloanella. Eight fungal genera were identified among the 28 fungal organisms isolated, including (in descending order of frequency) Cladosporium, Penicillium, Aspergillus, Thanatephorus, Absidia, Eurotium, Paraphaeosphaeria and Tritirachium, with Cladosporium accounting for 10/28 (35.7%) of the total fungal isolates. In conclusion, this study identified the presence of 10 bacterial and eight fungal genera in the air within the laboratory sampled. Although this reflected diversity of the microorganisms present, none of these organisms have been described previously as having an inhalational route of laboratory-acquired infection. Therefore, we believe that the species of organisms identified and the concentration levels of these airborne contaminants determined, do not pose a significant health and safety threat for immunocompotent laboratory personnel and visitors.

  13. 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...... of a microbial community able to receive an introduced plasmid at both quantitative and phylogenetic levels. In this chapter, we describe a protocol for simultaneous quantification of plasmid transfer frequency to and high-throughput isolation of transconjugants from a mixed bacterial community after introducing...

  14. 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)

  15. Optimization of DNA isolation and PCR protocol for RAPD analysis ...

    African Journals Online (AJOL)

    hope&shola

    Genetic analysis of plants relies on high yields of pure DNA samples. Here we present the optimization of DNA isolation and PCR conditions for RAPD analysis of selected medicinal and aromatic plants of conservation concern from Peninsular India containing high levels of polysaccharides, polyphenols and secondary ...

  16. Isolation of human spontaneous killer lymphocytes by bacterial adherence.

    Science.gov (United States)

    Kleinman, R; De Boer, K P; Teodorescu, M

    1980-01-01

    Human lymphocyte subpopulations (B, T1, T2, T3, and T4 our denomination) have been identified previously by bacterial adherence and differences between them in mitogen responses and specific cytotoxic activity have been found. In this study another aspect has been investigated in order to find functions associated with these subpopulations, namely the spontaneous killing (SK) ability. Freshly isolated human peripheral blood lymphocytes (PBL) were separated into adherent and non-adherent cells following centrifugation against various bact:rial monolayers. The PBL and the resulting subpopulations of PBL were tested alone or in combination as effector cells in a 4 hr cytotoxicity assay against human lymphoblastoid cel- lines of B or T cell origin. The T3 + T4 cells or T4 cells alone showed a significantly higher SK activity against both B and T target cell lines when compared with unseparated PBL, T1 + T2, or T3 cells alone. Whe Fc portion of IgG, contain the lymphocytes responsible for SK activity and that SK cells can be purified by negative selection using bacterial adherence. PMID:7389207

  17. Isolation of amoebic-bacterial consortia capable of degrading trichloroethylene

    International Nuclear Information System (INIS)

    Tyndall, R.L.; Ironside, K.; Little, C.D.; Katz, S.; Kennedy, J.

    1990-01-01

    Groundwater from a waste disposal site contaminated with chlorinated alkenes was examined for the presence of amoebic-bacterial consortia capable of degrading the suspected carcinogen, trichloroethylene (TCE). Consortia were readily isolated from all of four test wells. They contained free-living amoebae, and heterotrophic and methylotrophic bacteria. Electron microscopic examination showed bacteria localized throughout the amoebic cytoplasm and an abundance of hyphomicrobium, but not Type I methanotrophs. The presence of Type II methanotrophs was indirectly indicated by lipid analysis of one consortium. The consortia have been passaged for over two years on mineral salts media in a methane atmosphere, which would not be expected to maintain the heterotrophs or amoebae separately. The methanotrophic bacteria apparently provided a stable nutrient source, allowing the persistence of the various genera. By use of 14 C-radiotracer techniques, the degradation of TCE by the consortia was observed with 14 C eventuating predominantly in CO 2 and water-soluble products. In a more detailed examination of one consortia, the amoebae and heterotrohic components did not degrade TCE, while a mixed culture of heterotrophs and methanotrophs did degrade TCE, suggesting the latter component was the primary cause for the consortium's ability to degrade TCE. Amoebic-bacterial consortia may play a role in stabilizing and preserving methylotrophic bacteria in hostile environments

  18. 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.

  19. 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.

  20. Occurrence, isolation and DNA identification of involved in Algerian ...

    African Journals Online (AJOL)

    A total of 12 strains were tested by DNA identification analysis and these indigenous isolates were unambiguously characterised by their housekeeping gene profiles. Finally, four genotypes were recognised. To our knowledge, this is the first report on the isolation and molecular characterization of S. thermophilus strains ...

  1. Identification of Campylobacter pyloridis isolates by restriction endonuclease DNA analysis

    NARCIS (Netherlands)

    Langenberg, W.; Rauws, E. A.; Widjojokusumo, A.; Tytgat, G. N.; Zanen, H. C.

    1986-01-01

    Campylobacter pyloridis isolates recovered from gastric biopsy specimens of 16 patients were examined by restriction endonuclease DNA analysis with HindIII. For 8 of these 16 patients two different isolates were compared to study the persistence of the colonizing strains and the stability of their

  2. 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.

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

    Science.gov (United States)

    Nangia, Yogesh; Wangoo, Nishima; Goyal, Nisha; Shekhawat, G; Suri, C Raman

    2009-07-20

    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. 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. 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.

  4. 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

  5. Orbital abscess bacterial isolates and in vitro antimicrobial susceptibility patterns in dogs and cats.

    Science.gov (United States)

    Wang, Annie L; Ledbetter, Eric C; Kern, Thomas J

    2009-01-01

    To determine bacterial populations, in vitro antimicrobial susceptibility patterns, and sources of microorganisms for dogs and cats with orbital abscess. In total, 34 dogs and 7 cats with orbital abscess participated in the study. Medical records of dogs and cats with a clinical diagnosis of orbital abscess, confirmed by cytologic or histopathologic evaluation of orbital specimens, were reviewed from the years 1990 to 2007. Animal signalment, presumptive source of microorganisms and mechanism of orbital introduction, bacterial isolates, and aerobic bacterial in vitro antimicrobial susceptibility test results were recorded. Percentages of susceptible aerobic bacterial isolates were compared among antimicrobials. Twenty dogs and five cats had positive culture results. The most frequent bacterial genera isolated from dogs were Staphylococcus, Escherichia, Bacteroides, Clostridium and Pasteurella. The most frequent bacterial genera isolated from cats were Pasteurella and Bacteroides. Aerobic bacterial isolates from dogs had the highest percentage of susceptibility to amikacin, ceftiofur, gentamicin, imipenem, ticarcillin and trimethoprim-sulfamethoxazole. Aerobic bacterial isolates from dogs had the lowest percentage of susceptibility to ampicillin, clindamycin, erythromycin and penicillin. Antimicrobial resistance was uncommon among feline aerobic bacterial isolates. The most commonly identified routes of orbital bacteria introduction were extension from adjacent anatomical structures, penetrating exogenous trauma, and foreign bodies. Mixed aerobic and anaerobic bacterial infections of the orbit occur commonly in dogs and cats. On the basis of aerobic and anaerobic bacterial isolates and in vitro susceptibility testing of aerobic bacterial isolates, cephalosporins, extended-spectrum penicillins, potentiated-penicillins and carbapenems are recommended for initial antimicrobial therapy of orbital abscess in dogs and cats.

  6. 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.)

  7. Antibiotic resistance profile of bacterial isolates from food sold on a ...

    African Journals Online (AJOL)

    The antibiotic resistance profile of bacterial isolates from cooked food samples sold in different eateries on the campus of the University of Ado-Ekiti was investigated. A total of seventy-eight bacterial isolates belonging to six genera were encountered in the following proportion: Escherichia coli (29.5%), Klebsiella spp.

  8. Bacterial isolates from equine infections in western Canada (1998–2003)

    OpenAIRE

    Clark, Chris; Greenwood, Sarah; Boison, Joe O.; Chirino-Trejo, Manuel; Dowling, Patricia M.

    2008-01-01

    All bacterial samples of equine origin submitted to the diagnostic laboratory at the Western College of Veterinary Medicine from January 1998 to December 2003 from either “in-clinic” or Field Service cases were accessed (1323 submissions). The most common bacterial isolates from specific presenting signs were identified, along with their in vitro antimicrobial susceptibility patterns. The most common site from which significant bacterial isolates were recovered was the respiratory tract, foll...

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

    Directory of Open Access Journals (Sweden)

    Ming-Wei Shao

    Full Text Available 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.

  10. Occurrence, isolation and DNA identification of Streptococcus ...

    African Journals Online (AJOL)

    ajl yemi

    2011-11-28

    Nov 28, 2011 ... Streptococcus thermophilus isolates from traditional butter 'Smen', a fermented product from cow's ... temperature of milk. The fresh butter is removed manually in a single lump called 'Zebda' which is trans- formed into 'Smen' by washing with salt ... Egyptian artisanal butter 'zabady' was reported recently.

  11. Metabolic fingerprinting of bacterial strains isolated from northern areas of Pakistan

    International Nuclear Information System (INIS)

    Zaheer, A.; Latif, Z.

    2017-01-01

    The diversity of Plant Growth Promoting Rhizobacteria (PGPR) in the rhizosphere plays a key role in the maintenance of sustainable agricultural system. In this study, samples were obtained from northern areas of Pakistan. Thirty bacterial strains were isolated, purified, characterized biochemically and subjected to the metabolic fingerprinting by performing nitrogen fixation, phosphate solubilization, protease, indole acetic acid (IAA) production, antibiotic susceptibility and heavy metal resistance test, lead acetate assay for the H2S production. Strains showing distinct characteristics were further characterized by 16S rDNA sequencing and characterized as Bacillus pumilus (KT273321), Acinetobacter baumanii (KT273323), Acinetobacter junii (KT273324), Pseudomonas aeruginosa (KT273325), Bacillus circulans (KT273326) and Bacillus cereus (KT273327). As most of the strains show positive results for resistance against heavy metals, phosphate solubilization, nitrogen fixation, IAA production, and so these strains might be utilized for the removal of heavy metals from the ecosystem as well as biofertilizer in agriculture lands of northern areas. (author)

  12. Drug resistance analysis of bacterial strains isolated from burn patients.

    Science.gov (United States)

    Wang, L F; Li, J L; Ma, W H; Li, J Y

    2014-01-22

    This study aimed to analyze the spectrum and drug resistance of bacteria isolated from burn patients to provide a reference for rational clinical use of antibiotics. Up to 1914 bacterial strain specimens isolated from burn patients admitted to hospital between 2001 and 2010 were subjected to resistance monitoring by using the K-B paper disk method. Retrospective analysis was performed on drug resistance analysis of burn patients. The top eight bacterium strains according to detection rate. A total of 1355 strains of Gram-negative (G(-)) bacteria and 559 strains of Gram-positive (G(+)) bacteria were detected. The top eight bacterium strains, according to detection rate, were Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae, Enterobacter cloacae, and Enterococcus. Drug resistance rates were higher than 90% in A. baumannii, P. aeruginosa, S. epidermidis, and S. aureus, which accounted for 52.2, 21.7, 27.8, and 33.3%, respectively, of the entire sample. Those with drug resistance rates lower than 30% accounted for 4.3, 30.4, 16.7, and 16.7%, respectively. Multidrug-resistant S. aureus (MRSA) and methicillin-resistant S. epidermidis (MRSE) accounted for 49.2 and 76.4% of the S. epidermis and S. aureus resistance, respectively. Antibacterial drugs that had drug resistance rates to MRSE and MRSA higher than 90% accounted for 38.9 and 72.2%, respectively, whereas those with lower than 30% drug resistance rates accounted for 11.1 and 16.7%, respectively. The burn patients enrolled in the study were mainly infected with G(-) bacteria. These results strongly suggest that clinicians should practice rational use of antibiotics based on drug susceptibility test results.

  13. PROTOCOL FOR EXTRACTION OF BACTERIAL METAGENOME DNA TO PRAWN Macrobrachium carcinus L

    Directory of Open Access Journals (Sweden)

    J U González de la Cruz

    2011-07-01

    Full Text Available In this work we adapted a protocol for the extraction of metagenomic DNA (ADNmg bacteria in the digestive system (intestines, stomach and hepatopancreas of Macrobrachium carcinus L., with reference to the method of extracting bacterial DNA from soils and sediments (Rojas-Herrera et al., 2008. This methodology consisted of enzymatic, physics, mechanics and chemistry after a series of tests was abolished enzymatic lysis. However, the success ADNmg extraction was influenced mainly by the preparation of the samples, in particular the hepatopancreas, where it was necessary to remove the fat by thermal shock temperature and phase separation by centrifugation with the sample frozen.The effectiveness of isolated DNA fragmentation was verified by gel electrophoresis in denaturing gradient (DGGE after amplification with universal primers. In general, it had a low diversity (19 phylotypes between the different organs analyzed of 13.5 ± 1 (intestines to 11.7 ± 0.96 (stomach. The Shannon-Weaver index (2.45, Simpsons (10.88 and equity (0972 obtained from the digitization of the image of the gel, suggested that the phylotypes that form the gut microflora M. carcinus, is distributed unevenly between the different organs analyzed.

  14. Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency.

    Science.gov (United States)

    Tu, Qiang; Yin, Jia; Fu, Jun; Herrmann, Jennifer; Li, Yuezhong; Yin, Yulong; Stewart, A Francis; Müller, Rolf; Zhang, Youming

    2016-04-20

    Bacterial competent cells are essential for cloning, construction of DNA libraries, and mutagenesis in every molecular biology laboratory. Among various transformation methods, electroporation is found to own the best transformation efficiency. Previous electroporation methods are based on washing and electroporating the bacterial cells in ice-cold condition that make them fragile and prone to death. Here we present simple temperature shift based methods that improve DNA transformation and recombineering efficiency in E. coli and several other gram-negative bacteria thereby economizing time and cost. Increased transformation efficiency of large DNA molecules is a significant advantage that might facilitate the cloning of large fragments from genomic DNA preparations and metagenomics samples.

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

    International Nuclear Information System (INIS)

    Yu, L.D.; Sangwijit, K.; Prakrajang, K.; Phanchaisri, B.; Thongkumkoon, P.; Thopan, P.; Singkarat, S.; Anuntalabhochai, S.

    2014-01-01

    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

  16. Identification, Characterization and Antibiotic Resistance of Bacterial Isolates Obtained from Waterpipe Device Hoses

    Directory of Open Access Journals (Sweden)

    Majed M. Masadeh

    2015-05-01

    Full Text Available The general lack of knowledge about the health effects of waterpipe smoking is among the reasons for its global spread. In this study, bacterial contamination of waterpipe hoses was investigated. Twenty hoses were collected from waterpipe cafés and screened for bacterial pathogens using standard culture and isolation techniques. Additionally, resistance of isolated bacteria to common antibiotics was determined by identifying the minimum inhibitory concentration (MIC of each isolate. Forty eight bacterial isolates were detected. Isolates included both Gram-positive and Gram-negative pathogens from species that included Micrococcus (12, Corynebacterium (13 and Bacillus (9. In addition, some of the detected pathogens were found to be resistant to aztreonam (79%, cefixime (79%, norfloxacin, amoxicillin (47%, clarithromycin (46% and enrofloxacin (38%. In conclusion, the hose of the waterpipe device is a good environment for the growth of bacterial pathogens, which can then be transmitted to users.

  17. Identification, characterization and antibiotic resistance of bacterial isolates obtained from waterpipe device hoses.

    Science.gov (United States)

    Masadeh, Majed M; Hussein, Emad I; Alzoubi, Karem H; Khabour, Omar; Shakhatreh, Muhamad Ali K; Gharaibeh, Mahmoud

    2015-05-13

    The general lack of knowledge about the health effects of waterpipe smoking is among the reasons for its global spread. In this study, bacterial contamination of waterpipe hoses was investigated. Twenty hoses were collected from waterpipe cafés and screened for bacterial pathogens using standard culture and isolation techniques. Additionally, resistance of isolated bacteria to common antibiotics was determined by identifying the minimum inhibitory concentration (MIC) of each isolate. Forty eight bacterial isolates were detected. Isolates included both Gram-positive and Gram-negative pathogens from species that included Micrococcus (12), Corynebacterium (13) and Bacillus (9). In addition, some of the detected pathogens were found to be resistant to aztreonam (79%), cefixime (79%), norfloxacin, amoxicillin (47%), clarithromycin (46%) and enrofloxacin (38%). In conclusion, the hose of the waterpipe device is a good environment for the growth of bacterial pathogens, which can then be transmitted to users.

  18. Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency.

    OpenAIRE

    Tu, Qiang; Yin, Jia; Fu, Jun; Herrmann, Jennifer; Li, Yuezhong; Yin, Yulong; Stewart, A Francis; Müller, Rolf; Zhang, Youming

    2016-01-01

    Bacterial competent cells are essential for cloning, construction of DNA libraries, and mutagenesis in every molecular biology laboratory. Among various transformation methods, electroporation is found to own the best transformation efficiency. Previous electroporation methods are based on washing and electroporating the bacterial cells in ice-cold condition that make them fragile and prone to death. Here we present simple temperature shift based methods that improve DNA transformation and re...

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

    International Nuclear Information System (INIS)

    Sangwijit, K.; Yu, L.D.; Sarapirom, S.; Pitakrattananukool, S.; Anuntalabhochai, S.

    2015-01-01

    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 12 to 1 × 10 17 ions/cm 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.

  20. Characterization of Botrytis cinerea isolates from chickpea: DNA ...

    African Journals Online (AJOL)

    Administrator

    2010-11-15

    Nov 15, 2010 ... Characterization of Botrytis cinerea isolates from chickpea: DNA polymorphisms, cultural, morphological and virulence characteristics. Suresh Pande1, Mamta Sharma1*, G. Krishna Kishore1, L. Shivram2 and U. Naga Mangala1. 1International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), ...

  1. Rapid isolation of high molecular weight DNA from single dried ...

    African Journals Online (AJOL)

    ANAND

    For studying genetic diversity in populations of predatory coccinellid, Cryptolaemus montrouzieri. Mulsant (Coccinellidae: Coleoptera), our attempts to isolate high quality DNA from individual adult beetle using several previously reported protocols and even modifications were quite unsuccessful as the insect size was small ...

  2. Rapid isolation of high molecular weight DNA from single dry ...

    African Journals Online (AJOL)

    For studying genetic diversity in populations of predatory coccinellid, Cryptolaemus montrouzieri Mulsant (Coccinellidae: Coleoptera), our attempts to isolate high quality DNA from individual adult beetle using several previously reported protocols and even modifications were quite unsuccessful as the insect size was small ...

  3. One-Tube Isolation of DNA from Mouse Tails.

    Science.gov (United States)

    Green, Michael R; Sambrook, Joseph

    2018-02-01

    This protocol describes a variation of a simple method for isolation of DNA from mouse tails that uses commercially available gel-barrier tubes to eliminate the tedious transfer of samples during serial extractions with organic solvents. This variant is useful when processing very large numbers of samples. © 2018 Cold Spring Harbor Laboratory Press.

  4. On the fate of exogenously supplied bacterial DNA in soybean

    International Nuclear Information System (INIS)

    Luyindula Ndiku

    1980-01-01

    The fate of exogenously supplied radiolabelled DNA from agrobacterium tumefaciens and micrococcus lysodeikticus was investigated in soybean tissues growing under various physiological conditions. The following observations are made: a) Rapid degradation and reutilization of the donor DNA was observed in callus tissue culture. b) Germinating seeds and five-day old seedlings were shown to degrade DNA in the incubation medium and to ultilize these degradation products for their own DNA synthesis. Reutilization could be almost totally suppressed the addition of unlabelled thymidine as a competitor. This allowed a detection of significant amounts of residuel donor closely but transiently associated with the plant tissues. c) In soybean shoots dipped into a solution of donor DNA, partly this DNA was found to first migrate to the leaves where mostly labelled endogenous DNA was later found. Very large amounts of polymerized exogenous DNA were found in the regenerated roots after 12 days of culture. (author)

  5. 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.

  6. Comparison of two methods of bacterial DNA extraction from human fecal samples contaminated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni.

    Science.gov (United States)

    Kawase, Jun; Kurosaki, Morito; Kawakami, Yuta; Kashimoto, Takashi; Tsunomori, Yoshie; Sato, Koji; Ikeda, Tetsuya; Yamaguchi, Keiji; Watahiki, Masanori; Shima, Tomoko; Kameyama, Mitsuhiro; Etoh, Yoshiki; Horikawa, Kazumi; Fukushima, Hiroshi; Goto, Ryoichi; Shirabe, Komei

    2014-01-01

    In this study, 2 methods of DNA extraction were evaluated for use in conjunction with the screening system Rapid Foodborne Bacterial Screening 24 (RFBS24), which employs multiplex real-time SYBR Green polymerase chain reaction (SG-PCR) and can simultaneously detect 24 target genes of foodborne pathogens in fecal DNA samples. The QIAamp DNA Stool mini kit (Qkit) and Ultra Clean Fecal DNA Isolation Kit (Ukit) were used for bacterial DNA extraction from fecal samples artificially inoculated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni. SG-PCR and simplex real-time quantitative PCR (S-qPCR) analyses revealed higher copy numbers (8-234 times) of DNA in samples obtained using Ukit compared with those obtained using Qkit, resulting in lower cycle threshold values for the Ukit samples of the 4 bacteria on SG-PCR analysis. Fecal DNA samples from patients infected during foodborne outbreaks of Salmonella and Campylobacter were also prepared by Qkit and Ukit methods and subjected to RFBS24 analyses. Higher numbers of RFBS24 bacterial target genes were detected in DNA samples obtained using Ukit compared with those obtained using Qkit. Thus, the higher DNA extraction efficiency of the Ukit method compared with Qkit renders the former more useful in achieving improved detection rates of these 4 bacteria in fecal samples using SG-PCR.

  7. 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.

  8. A role for the weak DnaA binding sites in bacterial replication origins

    DEFF Research Database (Denmark)

    Charbon, Godefroid; Løbner-Olesen, Anders

    2011-01-01

    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...... that bacterial origins might be more alike than previously thought....

  9. Quantification of total phosphorothioate in bacterial DNA by a bromoimane-based fluorescent method.

    Science.gov (United States)

    Xiao, Lu; Xiang, Yu

    2016-06-01

    The discovery of phosphorothioate (PT) modifications in bacterial DNA has challenged our understanding of conserved phosphodiester backbone structure of cellular DNA. This exclusive DNA modification in bacteria is not found in animal cells yet, and its biological function in bacteria is still poorly understood. Quantitative information about the bacterial PT modifications is thus important for the investigation of their possible biological functions. In this study, we have developed a simple fluorescence method for selective quantification of total PTs in bacterial DNA, based on fluorescent labeling of PTs and subsequent release of the labeled fluorophores for absolute quantification. The method was highly selective to PTs and not interfered by the presence of reactive small molecules or proteins. The quantification of PTs in an E. coli DNA sample was successfully achieved using our method and gave a result of about 455 PTs per million DNA nucleotides, while almost no detectable PTs were found in a mammalian calf thymus DNA. With this new method, the content of phosphorothioate in bacterial DNA could be successfully quantified, serving as a simple method suitable for routine use in biological phosphorothioate related studies. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Microbial Observatory (ISS-MO): Study of BSL-2 bacterial isolates from the International Space Station

    Data.gov (United States)

    National Aeronautics and Space Administration — In an on-going Microbial Observatory experimental investigation on the International Space Station (ISS) multiple bacterial isolates of Biosafety Level 2 (BSL-2)...

  11. 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.

  12. Antibacterial activity of some disinfectants, essential oils and radiation against some bacterial isolates

    International Nuclear Information System (INIS)

    Ramadan, A.B.; Abo-State, M.A.M.; Ghaly, M.F.; Ezzat, S.M.; Hefni, H.M.I.

    2006-01-01

    Nosocomial bacteria has been considered problems for all hospitals. Bacterial isolates of the present study were isolated from Sharkia Hospitals, Egypt. The isolates were S. aureus, P. aeruginosa, E. coli, Klebsiella spp., Citrobacter spp., Enterobacter spp., Proteus spp., Serratia spp. and S. epidermidis. Two disinfectants (savlon and phenolics) were examined against all the bacterial isolates at the hospitals recommended concentrations to determine the most resistance bacterial isolates. Twelve essential oils, ultraviolet radiation, Gamma radiation and laser were tested against the most resistant bacterial isolates to disinfectants and antiseptics. These bacterial isolates were S. aureus and P. aeruginosa. The most effective concentration of savlon was 2%, while that of phenolics were 4 and 5% . Fumigation of essential oils of Eugenia caryophyllata L., Marjorum hortensis L., Foeniculum vulgare L., Rosmarinus officinalis L. and Thymus vulgaris L. had high effects against these bacterial isolates. Rosmarinus officinalis L., Marjorum hortensis L., Eugenia caryophyllata L. and Thymus vulgaris L. essential oils had high effects on bacterial isolates by disc diffusion method. Matricaria chamomilla L. and Eucalyptus spp. oils had no effects against P. aeruginosa. Both essential oils of Jasminum gradiflocum L. and Jasminum sambac L. had no effects by fumigation and diffused essential oils. Ultraviolet irradiation had lethal effect on S. aureus when it exposed to ultraviolet for 10 minutes at a distance of 20 cm, while it had a lethal effect on P. aeruginosa when exposed to the rays for 7 minutes at the same distance. Gamma irradiation had lethal effect on P. aeruginosa and S. aureus at 2.5 and 3 KGy, respectively. Laser had a lethal effect on P. aeruginosa and S. aureus after 14 and 15 minutes of exposure, respectively

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

    Science.gov (United States)

    Hartwich, Heiner; Nothwang, Hans Gerd

    2012-03-20

    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. 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. 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. © 2011 Hartwich et al; licensee BioMed Central Ltd.

  14. 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.

  15. Necessity of purification during bacterial DNA extraction with environmental soils

    Directory of Open Access Journals (Sweden)

    Hyun Jeong Lim

    2017-08-01

    Full Text Available Complexity and heterogeneity of soil samples have often implied the inclusion of purification steps in conventional DNA extraction for polymerase chain reaction (PCR assays. Unfortunately the purification steps are also time and labor intensive. Therefore the necessity of DNA purification was re-visited and investigated for a variety of environmental soil samples that contained various amounts of PCR inhibitors. Bead beating and centrifugation was used as the baseline (without purification method for DNA extraction. Its performance was compared with that of conventional DNA extraction kit (with purification. The necessity criteria for DNA purification were established with environmental soil samples. Using lysis conditions at 3000 rpm for 3 minutes with 0.1 mm glass beads, centrifugation time of 10 minutes and 1:10 dilution ratio, the baseline method outperformed conventional DNA extraction on cell seeded sand samples. Further investigation with PCR inhibitors (i.e., humic acids, clay, and magnesium [Mg] showed that sand samples containing less than 10 μg/g humic acids and 70% clay may not require purifications. Interestingly, the inhibition pattern of Mg ion was different from other inhibitors due to the complexation interaction of Mg ion with DNA fragments. It was concluded that DNA extraction method without purification is suitable for soil samples that have less than 10 μg/g of humic acids, less than 70% clay content and less than 0.01% Mg ion content.

  16. 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 CO 2 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.

  17. 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 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. Copyright © 2012. Published by Elsevier Ltd.

  18. The role indigenous bacterial isolates for bioremediation agent in the uranium contaminated aquatic environment

    International Nuclear Information System (INIS)

    Mochd Yazid

    2014-01-01

    A Research on the role of indigenous bacterial isolates for bio-remediation agent of the uranium contaminated in the aquatic environment has been conducted. The objective of the research is to study the role of Pseudomonas sp and Bacillus sp. have been isolated from low level uranium waste for bioremediation agent in their environment, such as the determination of efficiency of the uranium binding compared by the non indigenous bacterial, location of these binding and the influences of added acethyl acid stimulant. The uranium reduction studied was measured by weighting bacterial biomass and uranium concentration was measured by spectrophotometer. The acethyl acid stimulant addition has been done with the variation of concentration and volume. The efficiency of the uranium reduction by indigenous bacterial isolate such as Pseudomonas sp were 84.99 % and Bacillus sp were 52.70 %, so the reduction efficiency by non indigenous bacterial such as Pseudomonas aerogenes were 78.47 % and Bacillus subtilis were 45.22 % for 54 hours incubation time. The result of this research can be concluded that Pseudomonas sp and Bacillus sp. Indigenous bacterial have been isolates from the liquid uranium waste can contributed in bioremediation agent for uranium radionuclide in the environment for 60 ppm concentration with reduction efficiency 52.70 %-84.99 %, that is higher non indigenous bacterial for 54 hours incubation time, the stimulant addition of acethyl acid, the efficiency can be increased up to 99.8 %. (author)

  19. Isolation of fetal DNA from nucleated erythrocytes in maternal blood

    Energy Technology Data Exchange (ETDEWEB)

    Bianchi, D.W.; Knoll, J.H.M. (Children' s Hospital, Boston, MA (USA) Harvard Medical School, Boston, MA (USA)); Flint, A.F. (Howard Hughes Medical Institute, Boston, MA (USA)); Pizzimenti, M.F. (Brigham and Women' s Hospital, Boston, MA (USA)); Latt, S.A. (Children' s Hospital, Boston, MA (USA) Harvard Medical School, Boston, MA (USA))

    1990-05-01

    Fetal nucleated cells within maternal blood represent a potential source of fetal genes obtainable by venipuncture. The authors used monoclonal antibody against the transferrin receptor (TIR) to identify nucleated erythrocytes in the peripheral blood of pregnant women. Candidate fetal cells from 19 pregnancies were isolated by flow sorting at 12 1/2-17 weeks gestation. The DNA in these cells was amplified for a 222-base-pair (bp) sequence present on the short arm of the Y chromosome as proof that the cells were derived from the fetus. The amplified DNA was compared with standardized DNA concentrations. In the case of the female fetus, DNA prepared from samples at 32 weeks of gestation and cord blood at delivery also showed the presence of the Y chromosomal sequence, suggesting Y sequence mosaicism or translocation. In 10/12 cases where the 222-bp band was absent, the fetuses were female. Thus, they were successful in detecting the Y chromosomal sequence in 75% of the male-bearing pregnancies, demonstrating that it is possible to isolate fetal gene sequences from cells in maternal blood. Further refinement in methodology should increase sensitivity and facilitate noninvasive screening for fetal gene mutations.

  20. Bacterial Profile and Antimicrobial Susceptibility Pattern of Isolates ...

    African Journals Online (AJOL)

    Frequent isolates were S. aureus, (34.04%), and P. aeruginosa, (31.8%). About 82.16% of the isolates showed multiple resistances. In light of our findings, regular antibiotic resistance test has to be done for each patient in order to select an appropriate antimicrobial agent. Keywords: Bacteraemia, Burn, Sepsis, Thermal ...

  1. Degradation of petroleum hydrocarbons by oil field isolated bacterial ...

    African Journals Online (AJOL)

    A mixed consortium was prepared with 15 bacteria isolated by enrichment technique from the sample collected from an oil contaminated site. This consortium was incubated with crude oil to investigate the metabolic capability of bacteria. The degradation efficiency of the isolates in consortium was checked with 2% crude oil ...

  2. DNA marker characterization for allele mining of blast and bacterial ...

    African Journals Online (AJOL)

    admiistrator

    2013-05-01

    May 1, 2013 ... Marker assisted selection of bacterial blight resistance genes in rice. Biochem Genet. 39 (7): 261-278. Du PV, Loan, LC, Sang ND (2007). Blast research in Mekong river delta of Vietnam. In: JIRCAS (Japan International Research Center for Agricultural Sciences, Tsukuba, Japan) working report No. 53,.

  3. An isolated bacterial consortium for crude oil biodegradation

    African Journals Online (AJOL)

    GREGORY

    2011-12-16

    Dec 16, 2011 ... designed using DesignExpert 6.0.8 software by optimizing the amount of crude oil, microbial inoculum and sludge which are initially ... Key words: Crude oil, bacterial consortium, kinetics, bioremediation, biostimulation, natural attenuation. ... For the preparation of the consortiums, colonies were plated on.

  4. Drug resistance patterns of bacterial isolates from infected wounds ...

    African Journals Online (AJOL)

    unhcc

    Conclusions: High frequency of mono and multi-drug resistant bacterial pathogens were documented. Thus, an alternative method to the causative agent and antimicrobial susceptibility testing surveillance in areas where there is no culture facility is needed to assist health professionals for the selection of appropriate ...

  5. Bacterial Isolates andAntibiotic Sensitivity in Community Acquired ...

    African Journals Online (AJOL)

    Objective: The objective of the studywas to determine bacterial causes of community acquired pneumonia and their antibiotic sensitivity pattern amongst patients admitted intomedicalwards inAminu Kano Teaching Hospital, Kano, Nigeria Methods: The study incorporated patients aged fifteen years and above admitted into ...

  6. Sensitivity pattern of bacterial isolates in childhood sepsis in clinical ...

    African Journals Online (AJOL)

    To determine the commonest organism cultured from the blood of children suspected to have bacterial sepsis and their antibiogram sensitivity using the commonly used antibiotics in a private practice and see if there are differences from established data from other areas. Children beyond neonatal age presenting to a ...

  7. Antibiogram of bacterial pathogens isolated from subclinical mastitis ...

    African Journals Online (AJOL)

    The present investigation was carried out to study the prevalence of bacterial pathogens responsible for subclinical mastitis in cattle and their antibiogram pattern to selected antibiotics. The study was carried out on lactating cows in small holder dairy farms in and around Kombolcha, South Wollo, Amhara region, Ethiopia.

  8. Comparison of protease production from newly isolated bacterial ...

    African Journals Online (AJOL)

    Fermentation medium (by using sub-merged fermentation technique) was incubated for 48 h at 37°C temperature and agitation speed of 200 rpm. The protease was partially purified with 70% ammonium sulphate. Four different supports were used for the immobilization of the bacterial protease by physical adsorption ...

  9. Investigation of In vitro Mineral Forming Bacterial Isolates from ...

    African Journals Online (AJOL)

    2018-01-30

    Jan 30, 2018 ... bacterial colony in the oral cavity is important in calculus formation because bacteria have a direct effect on the pH ... Department of Biology,. Faculty of Science, Atatürk. University, 1Department of ... performed with oral bacteria in environments with high ion levels, and thus, their findings have very limited.

  10. Antibiotic Activity Assessment of Bacterial Strains Isolated from Urine ...

    African Journals Online (AJOL)

    Urinary tract infections (UTI) are common worldwide and affect all sexes and age groups. An estimated 20% or more of the female population suffers from some form of UTIs in their lifetime. Although antibiotics are the first choice of treatment for many urinary tract infections, antibiotic-resistant strains of bacterial species ...

  11. Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP system.

    Science.gov (United States)

    Dunbar, Sherry A; Vander Zee, Coe A; Oliver, Kerry G; Karem, Kevin L; Jacobson, James W

    2003-05-01

    Escherichia coli, Salmonella, Listeria monocytogenes and Campylobacter jejuni are bacterial pathogens commonly implicated in foodborne illnesses. Generally used detection methods (i.e., culture, biochemical testing, ELISA and nucleic acid amplification) can be laborious, time-consuming and require multiple tests to detect all of the pathogens. Our objective was to develop rapid assays to simultaneously detect these four organisms through the presence of antigen or DNA using the Luminex LabMAP system. For nucleic acid detection, organism-specific capture probes corresponding to the 23S ribosomal RNA gene (rrl) were coupled covalently to LabMAP microspheres. Target molecules included synthetic complementary oligonucleotides and genomic DNA isolated from ATCC type strains or other well-characterized strains of each organism. Universal PCR primers were designed to amplify variable regions of bacterial 23S ribosomal DNA, yielding biotinylated amplicons of 86 to 109 bp in length. Varying quantities of targets were hybridized to the combined microsphere sets, labeled with streptavidin-R-phycoerythrin and analyzed on the Luminex(100) system. Results of nucleic acid detection assays, obtained in 30 to 40 min following amplification, correctly and specifically identified each bacterial species with a detection sensitivity of 10(3) to 10(5) genome copies. Capture-sandwich immunoassays were developed with organism-specific antibodies coupled to different microsphere sets. Microspheres were incubated with organism-specific standards and reactivity was assessed with biotinylated detection antibodies and streptavidin-R-phycoerythrin. In the immunoassays, microsphere-associated fluorescence was organism concentration dependent with detectable response at detection of pathogens. The practical significance of this multiplexing approach would be to provide more timely, economical and comprehensive information than is available with conventional isolation and identification

  12. Isolate PM1 populations are dominant and novel methyl tert-butyl ether-degrading bacterial in compost biofilter enrichments.

    Science.gov (United States)

    Bruns, M A; Hanson, J R; Mefford, J; Scow, K M

    2001-03-01

    The gasoline additive MTBE, methyl tert-butyl ether, is a widespread and persistent groundwater contaminant. MTBE undergoes rapid mineralization as the sole carbon and energy source of bacterial strain PM1, isolated from an enrichment culture of compost biofilter material. In this report, we describe the results of microbial community DNA profiling to assess the relative dominance of isolate PM1 and other bacterial strains cultured from the compost enrichment. Three polymerase chain reaction (PCR)-based profiling approaches were evaluated: denaturing gradient gel electrophoresis (DGGE) analysis of 230 bp 16S rDNA fragments; thermal gradient gel electrophoresis (TGGE) analysis of 575 bp 16S rDNA fragments; and non-denaturing polyacrylamide gel electrophoresis of 300-1,500 bp fragments containing 16S/23S ribosomal intergenic transcribed spacer (ITS) regions. Whereas all three DNA profiling approaches indicated that PM1-like bands predominated in mixtures from MTBE-grown enrichments, ITS profiling provided the most abundant and specific sequence data to confirm strain PM1's presence in the enrichment. Moreover, ITS profiling did not produce non-specific PCR products that were observed with T/DGGE. A further advantage of ITS community profiling over other methods requiring restriction digestion (e.g. terminal restriction fragment length polymorphisms) was that it did not require an additional digestion step or the use of automated sequencing equipment. ITS bands, excised from similar locations in profiles of the enrichment and PM1 pure culture, were 99.9% identical across 750 16S rDNA positions and 100% identical across 691 spacer positions. BLAST comparisons of nearly full-length 16S rDNA sequences showed 96% similarity between isolate PM1 and representatives of at least four different genera in the Leptothrix subgroup of the beta-Proteobacteria (Aquabacterium, Leptothrix, Rubrivivax and Ideonella). Maximum likelihood and parsimony analyses of 1,249 nucleotide

  13. 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.

  14. 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.

  15. DNA recognition by an RNA-guided bacterial Argonaute

    Science.gov (United States)

    Doudna, Jennifer A.

    2017-01-01

    Argonaute (Ago) proteins are widespread in prokaryotes and eukaryotes and share a four-domain architecture capable of RNA- or DNA-guided nucleic acid recognition. Previous studies identified a prokaryotic Argonaute protein from the eubacterium Marinitoga piezophila (MpAgo), which binds preferentially to 5′-hydroxylated guide RNAs and cleaves single-stranded RNA (ssRNA) and DNA (ssDNA) targets. Here we present a 3.2 Å resolution crystal structure of MpAgo bound to a 21-nucleotide RNA guide and a complementary 21-nucleotide ssDNA substrate. Comparison of this ternary complex to other target-bound Argonaute structures reveals a unique orientation of the N-terminal domain, resulting in a straight helical axis of the entire RNA-DNA heteroduplex through the central cleft of the protein. Additionally, mismatches introduced into the heteroduplex reduce MpAgo cleavage efficiency with a symmetric profile centered around the middle of the helix. This pattern differs from the canonical mismatch tolerance of other Argonautes, which display decreased cleavage efficiency for substrates bearing sequence mismatches to the 5′ region of the guide strand. This structural analysis of MpAgo bound to a hybrid helix advances our understanding of the diversity of target recognition mechanisms by Argonaute proteins. PMID:28520746

  16. A primary assessment of the endophytic bacterial community in a xerophilous moss (Grimmia montana using molecular method and cultivated isolates

    Directory of Open Access Journals (Sweden)

    Xiao Lei Liu

    2014-01-01

    Full Text Available Investigating the endophytic bacterial community in special moss species is fundamental to understanding the microbial-plant interactions and discovering the bacteria with stresses tolerance. Thus, the community structure of endophytic bacteria in the xerophilous moss Grimmia montana were estimated using a 16S rDNA library and traditional cultivation methods. In total, 212 sequences derived from the 16S rDNA library were used to assess the bacterial diversity. Sequence alignment showed that the endophytes were assigned to 54 genera in 4 phyla (Proteobacteria, Firmicutes, Actinobacteria and Cytophaga/Flexibacter/Bacteroids. Of them, the dominant phyla were Proteobacteria (45.9% and Firmicutes (27.6%, the most abundant genera included Acinetobacter, Aeromonas, Enterobacter, Leclercia, Microvirga, Pseudomonas, Rhizobium, Planococcus, Paenisporosarcina and Planomicrobium. In addition, a total of 14 species belonging to 8 genera in 3 phyla (Proteobacteria, Firmicutes, Actinobacteria were isolated, Curtobacterium, Massilia, Pseudomonas and Sphingomonas were the dominant genera. Although some of the genera isolated were inconsistent with those detected by molecular method, both of two methods proved that many different endophytic bacteria coexist in G. montana. According to the potential functional analyses of these bacteria, some species are known to have possible beneficial effects on hosts, but whether this is the case in G. montana needs to be confirmed.

  17. An isolated bacterial consortium for crude oil biodegradation

    African Journals Online (AJOL)

    GREGORY

    2011-12-16

    ) and tested for their ability to degrade COCS. ... Environmental pollution, especially with hydrocarbons is a major environmental ..... Rhodochrous Isolated from Soil and Marine Environments in Kuwait. Environ. Pollut. 65: 1-17 ...

  18. Mode of action of closthioamide: the first member of the polythioamide class of bacterial DNA gyrase inhibitors.

    Science.gov (United States)

    Chiriac, Alina Iulia; Kloss, Florian; Krämer, Jonas; Vuong, Cuong; Hertweck, Christian; Sahl, Hans-Georg

    2015-09-01

    The spread of MDR bacteria represents a serious threat to human society and novel antibiotic drugs, preferably from new chemical classes, are urgently needed. Closthioamide was isolated from the strictly anaerobic bacterium Clostridium cellulolyticum and belongs to a new class of natural products, the polythioamides. Here, we investigated the antimicrobial activity and mechanism of action of closthioamide. For assessing the antimicrobial activity of closthioamide, MIC values and killing kinetics were determined. To identify its target pathway, whole-cell-based assays were used including analysis of macromolecular synthesis and recording the susceptibility profile of a library of clones with down-regulated potential target genes. Subsequently, the inhibitory effect of closthioamide on the activity of isolated target enzymes, e.g. DNA gyrase and topoisomerase IV, was evaluated. Closthioamide had broad-spectrum activity against Gram-positive bacteria. Notably, closthioamide was very potent against MRSA and VRE strains. Closthioamide impaired DNA replication and inhibited DNA gyrase activity, in particular the ATPase function of gyrase and of topoisomerase IV, whereas there was little effect on the cleavage-rejoining function. Closthioamide also inhibited the relaxation activity of DNA gyrase, which does not require ATP hydrolysis, and thus may allosterically rather than directly interfere with the ATPase activity of gyrase. Cross-resistance to ciprofloxacin and novobiocin could not be detected in experimental mutants and clinical isolates. Closthioamide, a member of an unprecedented class of antibiotics, is a potent inhibitor of bacterial DNA gyrase; however, its molecular mechanism differs from that of the quinolones and aminocoumarins. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  19. Phylogenetic distribution of virulence-associated genes among Escherichia coli isolates associated with neonatal bacterial meningitis in the Netherlands

    NARCIS (Netherlands)

    Johnson, James R.; Oswald, Eric; O'Bryan, Timothy T.; Kuskowski, Michael A.; Spanjaard, Lodewijk

    2002-01-01

    Seventy cerebrospinal fluid Escherichia coli isolates from infants with neonatal bacterial meningitis (NBM), as submitted to the Netherlands Reference Laboratory for Bacterial Meningitis from 1989 through 1997, were assessed for phylogenetic background and extended virulence genotypes, in comparison

  20. DNA supercoiling is a fundamental regulatory principle in the control of bacterial gene expression.

    Science.gov (United States)

    Dorman, Charles J; Dorman, Matthew J

    2016-11-01

    Although it has become routine to consider DNA in terms of its role as a carrier of genetic information, it is also an important contributor to the control of gene expression. This regulatory principle arises from its structural properties. DNA is maintained in an underwound state in most bacterial cells and this has important implications both for DNA storage in the nucleoid and for the expression of genetic information. Underwinding of the DNA through reduction in its linking number potentially imparts energy to the duplex that is available to drive DNA transactions, such as transcription, replication and recombination. The topological state of DNA also influences its affinity for some DNA binding proteins, especially in DNA sequences that have a high A + T base content. The underwinding of DNA by the ATP-dependent topoisomerase DNA gyrase creates a continuum between metabolic flux, DNA topology and gene expression that underpins the global response of the genome to changes in the intracellular and external environments. These connections describe a fundamental and generalised mechanism affecting global gene expression that underlies the specific control of transcription operating through conventional transcription factors. This mechanism also provides a basal level of control for genes acquired by horizontal DNA transfer, assisting microbial evolution, including the evolution of pathogenic bacteria.

  1. 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…

  2. Isolation, Screening and Development of Local Bacterial Consortia With Azo Dyes Decolourising Capability

    Directory of Open Access Journals (Sweden)

    Khadijah, O.

    2009-01-01

    Full Text Available A total of 1540 bacterial isolates were isolated and screened for their ability to degrade selected azo dyes. Of these, nine isolates were chosen for further studies based on their ability to degrade a wide spectrum of dyes efficiently and rapidly. Several microbial consortia were developed and tested for their effectiveness. Overall the consortia were able to degrade 70 - 100% colour within 72 hours compared to 60 – 97% colour removed by individual isolates. A microbial consortium labelled C15 showed good growth in agitation culture but the colour removal was best in static culture with 80 - 100% colour removed in less than 72 hours. Based on the 16S rRNA sequencing, two of the bacterial isolates in C15 belong to the Chryseobacterium genus while the other one belongs to Flavobacterium genus.

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

    OpenAIRE

    Zhang, Yongwei; Werling, Uwe; Edelmann, Winfried

    2012-01-01

    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 s...

  4. Characterization of a Campylobacter fetus-like strain isolated from the faeces of a sick leopard tortoise (Stigmochelys pardalis) using matrix-assisted laser desorption/ionization time of flight as an alternative to bacterial 16S rDNA phylogeny.

    Science.gov (United States)

    Benejat, L; Gravet, A; Sifré, E; Ben Amor, S; Quintard, B; Mégraud, F; Lehours, P

    2014-04-01

    This article describes the isolation and characterization of a Campylobacter-like isolate originating from the faeces of a sick leopard tortoise. Molecular as well as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) characterization suggests that it could correspond to a new Campylobacter species. The major impact of this work is the demonstration that proteomics and especially MALDI-TOF typing can be used as an alternative method to 16S rDNA sequencing for phylogeny and can lead to the discovery of new Campylobacters. © 2013 The Society for Applied Microbiology.

  5. Bacterial promoter prediction: Selection of dynamic and static physical properties of DNA for reliable sequence classification.

    Science.gov (United States)

    Ryasik, Artem; Orlov, Mikhail; Zykova, Evgenia; Ermak, Timofei; Sorokin, Anatoly

    2018-01-30

    Predicting promoter activity of DNA fragment is an important task for computational biology. Approaches using physical properties of DNA to predict bacterial promoters have recently gained a lot of attention. To select an adequate set of physical properties for training a classifier, various characteristics of DNA molecule should be taken into consideration. Here, we present a systematic approach that allows us to select less correlated properties for classification by means of both correlation and cophenetic coefficients as well as concordance matrices. To prove this concept, we have developed the first classifier that uses not only sequence and static physical properties of DNA fragment, but also dynamic properties of DNA open states. Therefore, the best performing models with accuracy values up to 90% for all types of sequences were obtained. Furthermore, we have demonstrated that the classifier can serve as a reliable tool enabling promoter DNA fragments to be distinguished from promoter islands despite the similarity of their nucleotide sequences.

  6. Bacterial DNA sifted from the Trichoplax adhaerens (Animalia: Placozoa) genome project reveals a putative rickettsial endosymbiont.

    Science.gov (United States)

    Driscoll, Timothy; Gillespie, Joseph J; Nordberg, Eric K; Azad, Abdu F; Sobral, Bruno W

    2013-01-01

    Eukaryotic genome sequencing projects often yield bacterial DNA sequences, data typically considered as microbial contamination. However, these sequences may also indicate either symbiont genes or lateral gene transfer (LGT) to host genomes. These bacterial sequences can provide clues about eukaryote-microbe interactions. Here, we used the genome of the primitive animal Trichoplax adhaerens (Metazoa: Placozoa), which is known to harbor an uncharacterized Gram-negative endosymbiont, to search for the presence of bacterial DNA sequences. Bioinformatic and phylogenomic analyses of extracted data from the genome assembly (181 bacterial coding sequences [CDS]) and trace read archive (16S rDNA) revealed a dominant proteobacterial profile strongly skewed to Rickettsiales (Alphaproteobacteria) genomes. By way of phylogenetic analysis of 16S rDNA and 113 proteins conserved across proteobacterial genomes, as well as identification of 27 rickettsial signature genes, we propose a Rickettsiales endosymbiont of T. adhaerens (RETA). The majority (93%) of the identified bacterial CDS belongs to small scaffolds containing prokaryotic-like genes; however, 12 CDS were identified on large scaffolds comprised of eukaryotic-like genes, suggesting that T. adhaerens might have recently acquired bacterial genes. These putative LGTs may coincide with the placozoan's aquatic niche and symbiosis with RETA. This work underscores the rich, and relatively untapped, resource of eukaryotic genome projects for harboring data pertinent to host-microbial interactions. The nature of unknown (or poorly characterized) bacterial species may only emerge via analysis of host genome sequencing projects, particularly if these species are resistant to cell culturing, as are many obligate intracellular microbes. Our work provides methodological insight for such an approach.

  7. Studies on antibiotic resistance of some bacterial isolates from ...

    African Journals Online (AJOL)

    Sachet water samples of different brands were collected from several retail outlets in Nsukka, Nigeria and analysed microbiologically to determine their bacteriological quality and antimicrobial susceptibility of the isolates. A brand of the sachet water samples tested negative for the presence of bacteria while a total of 61 ...

  8. Microbiological Assessment of Bacterial Isolates from Ear, Nose And ...

    African Journals Online (AJOL)

    Samples from patients who reported to in-patient ENT unit of Aminu Kano Teaching Hospital, Kano were isolated for further microbiological assessment. One hundred (100) from both male and female patients comprising 55 ear swabs, 30 and 15 throat and nose swabs respectively were screened between February and ...

  9. Antibiotics susceptibility patterns of urine bacterial isolates in Zaria ...

    African Journals Online (AJOL)

    Purpose: The prevalence of E. coli, Ps. aeruginosa and Staph aureus isolates from urine of selected residents in Zaria was investigated. This was an attempt to elucidate the antibiotic susceptibility profiles of these bacteria commonly implicated in urinary tact infection. Methods: Urine samples collected from students of ...

  10. Manganese oxidation by bacterial isolates from the Indian Ridge System

    Digital Repository Service at National Institute of Oceanography (India)

    Fernandes, S.O.; Krishnan, K.P.; Khedekar, V.D; LokaBharathi, P.A.

    ) observations of both isolates revealed free-living cells in clustered matrices apprrox. 2 Mu diameter. Energy dispersive spectrum of the cell matrix of CR35 cultured in 1 mM Mn detected 30%Mn, while the cell aggregates of CR48 harbored 7 -10% Mn. The relatively...

  11. Incidence and Antibiotic Susceptibility Pattern of Bacterial Isolates ...

    African Journals Online (AJOL)

    Purpose: To investigate the incidence of different bacteria isolates in 150 wound infections in Aminu Kano Teaching Hospital, Kano, Nigeria and their antibacterial susceptibility patterns. Methods: Wound swab samples were collected from general culture bench of the Microbiology Department, after obtaining consent from ...

  12. Bacterial isolates of tonsillitis and pharyngitis in a paediatric casualty ...

    African Journals Online (AJOL)

    BHS and S. aureus showed 100% sensitivity to cefuroxine, azithromycin, ceftazidine and genticin. All the isolates had little or no sensitivity to ampicillin and cotrimoxazole. BHS is a significant cause of pharyngitis and tonsillitis in our environment and therefore poses a potential danger of rheumatic fever and rheumatic heart ...

  13. Incidence and Antibiotic Susceptibility Pattern of Bacterial Isolates ...

    African Journals Online (AJOL)

    Purpose: To investigate the incidence of different bacteria isolates in 150 wound infections in Aminu Kano Teaching Hospital, Kano, ... antibiotic-resistant bacteria are present in the wound sites. Keywords: Wound infection , Antibiotic, ... and injections, but post-operative wound infections in hospital are most common. Some.

  14. Increasing incidence of bacterial resistance to antibiotics by isolates ...

    African Journals Online (AJOL)

    Antibiotic sensitivity testing was performed on sensitivity test agar (Biotec, UK) using the disc diffusion method in accordance with the National Committee for Clinical Laboratory Standards (5). The results were compared with a previous study in 1993. A total of 336 urinary isolates were identified, with the coliforms being the ...

  15. The Prevalent Bacterial Isolates Of Dental Caries In School Age ...

    African Journals Online (AJOL)

    The study was conducted at the dental clinic of Obafemi Awolowo University Teaching Hospital Complex, Ile-Ife. A total of 100 carious samples were collected from children of varying age and sexes. The bacteria isolated were S. mutans: 45.6%, Lactobacillus spp: 41.2% and S. aureus: 13.2%. Out of the 100 samples, 88(5) ...

  16. EFFECT OT KUNNU-ZAKI ON CLINICAL BACTERIAL ISOLATES

    African Journals Online (AJOL)

    omidiji

    2012-03-08

    Mar 8, 2012 ... antimicrobial effect represents another property for Kunun-zaki and obviates the possibility of public health risk in its consumption. Key words: Kunnu-zaki, clinical isolates, antibiotic sensitivity. INTRODUCTION. Kunnu-zaki is a cereal food drink that has become a popular refreshing non-alcoholic beverage ...

  17. Production of bacterial amylase by Bacillus species isolated from ...

    African Journals Online (AJOL)

    Optimum pH activity was obtained at 4.0 with a concentration of 0.376 mg/ml. Bacillus licheniformis has the greatest potential for producing amylase than the other isolates and rice husk can be exploited for amylase production. The B. licheniformis strain produced thermostable alpha-amylase with characteristics suitable for ...

  18. Environmental monitoring of bacterial contamination and antibiotic resistance patterns of the fecal coliforms isolated from Cauvery River, a major drinking water source in Karnataka, India.

    Science.gov (United States)

    Skariyachan, Sinosh; Mahajanakatti, Arpitha Badarinath; Grandhi, Nisha Jayaprakash; Prasanna, Akshatha; Sen, Ballari; Sharma, Narasimha; Vasist, Kiran S; Narayanappa, Rajeswari

    2015-05-01

    The present study focuses prudent elucidation of microbial pollution and antibiotic sensitivity profiling of the fecal coliforms isolated from River Cauvery, a major drinking water source in Karnataka, India. Water samples were collected from ten hotspots during the year 2011-2012. The physiochemical characteristics and microbial count of water samples collected from most of the hotspots exhibited greater biological oxygen demand and bacterial count especially coliforms in comparison with control samples (p ≤ 0.01). The antibiotic sensitivity testing was performed using 48 antibiotics against the bacterial isolates by disk-diffusion assay. The current study showed that out of 848 bacterial isolates, 93.51% (n = 793) of the isolates were found to be multidrug-resistant to most of the current generation antibiotics. Among the major isolates, 96.46% (n = 273) of the isolates were found to be multidrug-resistant to 30 antibiotics and they were identified to be Escherichia coli by 16S rDNA gene sequencing. Similarly, 93.85% (n = 107), 94.49% (n = 103), and 90.22% (n = 157) of the isolates exhibited multiple drug resistance to 32, 40, and 37 antibiotics, and they were identified to be Enterobacter cloacae, Pseudomonas trivialis, and Shigella sonnei, respectively. The molecular studies suggested the prevalence of bla TEM genes in all the four isolates and dhfr gene in Escherichia coli and Sh. sonnei. Analogously, most of the other Gram-negative bacteria were found to be multidrug-resistant and the Gram-positive bacteria, Staphylococcus spp. isolated from the water samples were found to be methicillin and vancomycin-resistant Staphylococcus aureus. This is probably the first study elucidating the bacterial pollution and antibiotic sensitivity profiling of fecal coliforms isolated from River Cauvery, Karnataka, India.

  19. Diversity of Clinical and Environmental Isolates of Vibrio cholerae in Natural Transformation and Contact-Dependent Bacterial Killing Indicative of Type VI Secretion System Activity.

    Science.gov (United States)

    Bernardy, Eryn E; Turnsek, Maryann A; Wilson, Sarah K; Tarr, Cheryl L; Hammer, Brian K

    2016-05-01

    The bacterial pathogen Vibrio cholerae can occupy both the human gut and aquatic reservoirs, where it may colonize chitinous surfaces that induce the expression of factors for three phenotypes: chitin utilization, DNA uptake by natural transformation, and contact-dependent bacterial killing via a type VI secretion system (T6SS). In this study, we surveyed a diverse set of 53 isolates from different geographic locales collected over the past century from human clinical and environmental specimens for each phenotype outlined above. The set included pandemic isolates of serogroup O1, as well as several serogroup O139 and non-O1/non-O139 strains. We found that while chitin utilization was common, only 22.6% of the isolates tested were proficient at chitin-induced natural transformation, suggesting that transformation is expendable. Constitutive contact-dependent killing of Escherichia coli prey, which is indicative of a functional T6SS, was rare among clinical isolates (only 4 of 29) but common among environmental isolates (22 of 24). These results bolster the pathoadaptive model in which tight regulation of T6SS-mediated bacterial killing is beneficial in a human host, whereas constitutive killing by environmental isolates may give a competitive advantage in natural settings. Future sequence analysis of this set of diverse isolates may identify previously unknown regulators and structural components for both natural transformation and T6SS. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  20. Diversity of Clinical and Environmental Isolates of Vibrio cholerae in Natural Transformation and Contact-Dependent Bacterial Killing Indicative of Type VI Secretion System Activity

    Science.gov (United States)

    Bernardy, Eryn E.; Turnsek, Maryann A.; Wilson, Sarah K.; Tarr, Cheryl L.

    2016-01-01

    The bacterial pathogen Vibrio cholerae can occupy both the human gut and aquatic reservoirs, where it may colonize chitinous surfaces that induce the expression of factors for three phenotypes: chitin utilization, DNA uptake by natural transformation, and contact-dependent bacterial killing via a type VI secretion system (T6SS). In this study, we surveyed a diverse set of 53 isolates from different geographic locales collected over the past century from human clinical and environmental specimens for each phenotype outlined above. The set included pandemic isolates of serogroup O1, as well as several serogroup O139 and non-O1/non-O139 strains. We found that while chitin utilization was common, only 22.6% of the isolates tested were proficient at chitin-induced natural transformation, suggesting that transformation is expendable. Constitutive contact-dependent killing of Escherichia coli prey, which is indicative of a functional T6SS, was rare among clinical isolates (only 4 of 29) but common among environmental isolates (22 of 24). These results bolster the pathoadaptive model in which tight regulation of T6SS-mediated bacterial killing is beneficial in a human host, whereas constitutive killing by environmental isolates may give a competitive advantage in natural settings. Future sequence analysis of this set of diverse isolates may identify previously unknown regulators and structural components for both natural transformation and T6SS. PMID:26944842

  1. 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)). © 2015 Fundacion MEDINA, Centro de Excelencia en Investigacion de Medicamentos Innovadores en Andalucia.

  2. 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 deficient...

  3. 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.

  4. Bacterial isolates from Nsukka meat market: a zoonotic appraisal.

    Science.gov (United States)

    Uche, U E; Agbo, J A

    1985-06-01

    Swabs collected from butchers hands, knives, tables and meat displayed for sale at Nsukka meat market showed that every item screened was contaminated. Bacteria isolated include Streptococcus faecalis, coagulase positive Staphylococcus, Clostridium perfringens, Brucella species, Corynebacterium haemolyticum, Proteus mirabilis, P. vulgaris, Escherichia coli, Salmonella enteritidis, Enterobacter aerogenes, Pseudomonas multophilia, P. rubescens and beta-haemolytic Streptococci. The zoonotic or public health implications of such contaminations, their probable sources and possible ways of minimizing them are discussed.

  5. Preliminary screening of bacterial isolates from mining wastes

    Directory of Open Access Journals (Sweden)

    Rodino S.,

    2016-05-01

    Full Text Available Developing innovative biotechnology for obtaining new resources of high tech critical metals is strongly influenced by the need to reduce the potential risk of shortages, to support the development of industry at European level. To set up these new technologies is essential to isolate strains with high potential in bioleaching of ore, tailings and mine wastes and bioaccumulation of high tech critical metals. Microorganisms are capable of mediating metal and mineral bioprecipitation. In this paper are presented preliminary studies performed for the isolation of strains existing in mining residues containing high tech critical metals. Were used samples collected from various depths in an area of mining wastes containing high tech critical metals. The samples were fine grounded and the powder was washed with sterile saline water. Exact quantities of samples were dispersed in sterile saline water, shaken for a period of 60 minutes, diluted and plated in triplicate on selective agar. After several steps were isolated 3 strains of gram negative bacteria.

  6. 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.

  7. DNA polymorphisms and biocontrol of Bacillus antagonistic to citrus bacterial canker with indication of the interference of phyllosphere biofilms.

    Directory of Open Access Journals (Sweden)

    Tzu-Pi Huang

    Full Text Available Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri is a devastating disease resulting in significant crop losses in various citrus cultivars worldwide. A biocontrol agent has not been recommended for this disease. To explore the potential of bacilli native to Taiwan to control this disease, Bacillus species with a broad spectrum of antagonistic activity against various phytopathogens were isolated from plant potting mixes, organic compost and the rhizosphere soil. Seven strains TKS1-1, OF3-16, SP4-17, HSP1, WG6-14, TLB7-7, and WP8-12 showing superior antagonistic activity were chosen for biopesticide development. The genetic identity based on 16S rDNA sequences indicated that all seven native strains were close relatives of the B. subtilis group and appeared to be discrete from the B. cereus group. DNA polymorphisms in strains WG6-14, SP4-17, TKS1-1, and WP8-12, as revealed by repetitive sequence-based PCR with the BOXA1R primers were similar to each other, but different from those of the respective Bacillus type strains. However, molecular typing of the strains using either tDNA-intergenic spacer regions or 16S-23S intergenic transcribed spacer regions was unable to differentiate the strains at the species level. Strains TKS1-1 and WG6-14 attenuated symptom development of citrus bacterial canker, which was found to be correlated with a reduction in colonization and biofilm formation by X. axonopodis pv. citri on leaf surfaces. The application of a Bacillus strain TKS1-1 endospore formulation to the leaf surfaces of citrus reduced the incidence of citrus bacterial canker and could prevent development of the disease.

  8. 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.

  9. [Effects of UV-induced DNA damage on vector ligation and transformation into bacterial cells].

    Science.gov (United States)

    Huang, Wan-ling; Li, Chang-zheng; Chen, Zhen-rui; He, Wei; Zhou, Ye; Zhou, Zhi-gang; Liu, Shu-wen; Zhou, Chen

    2010-01-01

    To study the effects of UV irradiation on DNA ligation and transformation efficiency of the expression vector into competent bacterial cells. The expression vector was digested with the restriction enzyme SfiI, and the purified target DNA fragments were exposed to UV light at different wavelengths. Ligation and transformation experiments with the exposed fragments were carried out and the colony number and transformation efficiency were assessed. The transformation efficiency of the DNA with a 5-min exposure to 302 nm UV was 60 colonies per nanogram of the DNA, as compared with 20400 for the DNA exposed to 365 nm UV. The time course experiment showed that prolonged DNA exposure to 365 nm UV light was associated with lowered transformation efficiency. DNA exposure for 30 min caused a reduction of the transformation efficiency to lower than 50% compared to that of DNA without UV exposure. But with a 15 min exposure, the DNA maintained a transformation efficiency more than 70%, which was sufficient for most molecular biology experiments. In construction of the expression vector, it is advisable to prevent the target DNA from UV exposure. When UV exposure is essential, we suggest that 365 nm UV be used and the exposure time controlled within 15 min.

  10. 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.

  11. Kinetic analysis of reverse transcriptase activity of bacterial family A DNA polymerases.

    Science.gov (United States)

    Yasukawa, Kiyoshi; Konishi, Atsushi; Shinomura, Mayu; Nagaoka, Eriko; Fujiwara, Shinsuke

    2012-10-26

    Some bacterial thermostable, wild-type or genetically engineered family A DNA polymerases have reverse transcriptase activity. However, difference in reverse transcriptase activities of family A DNA polymerases and retroviral reverse transcriptases (RTs) is unclear. In this study, comparative kinetic analysis was performed for the reverse transcriptase activities of the wild-type enzyme of family A DNA polymerase (M1pol(WT)) from Thermus thermophilus M1 and the variant enzyme of family A DNA polymerase (K4pol(L329A)), in which the mutation of Leu329→Ala is undertaken, from Thermotoga petrophila K4. In the incorporation of dTTP into poly(rA)-p(dT)(45), the reaction rates of K4pol(L329A) and M1pol(WT) exhibited a saturated profile of the Michaelis-Menten kinetics for dTTP concentrations but a substrate inhibition profile for poly(rA)-p(dT)(45) concentrations. In contrast, the reaction rates of Moloney murine leukemia virus (MMLV) RT exhibited saturated profiles for both dTTP and poly(rA)-p(dT)(45) concentrations. This suggests that high concentrations of DNA-primed RNA template decrease the efficiency of cDNA synthesis with bacterial family A DNA polymerases. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. 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.

  13. Oviposition responses of Aedes mosquitoes to bacterial isolates from attractive bamboo infusions.

    Science.gov (United States)

    Ponnusamy, Loganathan; Schal, Coby; Wesson, Dawn M; Arellano, Consuelo; Apperson, Charles S

    2015-09-23

    The mosquitoes Aedes aegypti and Aedes albopictus are vectors of pathogenic viruses that cause major human illnesses including dengue, yellow fever and chikungunya. Both mosquito species are expanding their geographic distributions and now occur worldwide in temperate and tropical climates. Collection of eggs in oviposition traps (ovitraps) is commonly used for monitoring and surveillance of container-inhabiting Aedes populations by public health agencies charged with managing mosquito-transmitted illness. Addition of an organic infusion in these traps increases the number of eggs deposited. Gravid females are guided to ovitraps by volatile chemicals produced from the breakdown of organic matter by microbes. We previously isolated and cultured 14 species of bacteria from attractive experimental infusions, made from the senescent leaves of canebrake bamboo (Arundinaria gigantea). Cultures were grown for 24 h at 28 °C with constant shaking (120 rpm) and cell densities were determined with a hemocytometer. Behavioral responses to single bacterial isolates and to a mix of isolates at different cell densities were evaluated using two-choice sticky-screen bioassay methods with gravid Ae. aegypti and Ae. albopictus. In behavioral assays of a mix of 14 bacterial isolates, significantly greater attraction responses were exhibited by Ae. aegypti and Ae. albopictus to bacterial densities of 10(7) and 10(8) cells/mL than to the control medium. When we tested single bacterial isolates, seven isolates (B1, B2, B3, B5, B12, B13 and B14) were significantly attractive to Ae. aegypti, and six isolates (B1, B5, B7, B10, B13 and B14) significantly attracted Ae. albopictus. Among all the isolates tested at three different cell densities, bacterial isolates B1, B5, B13 and B14 were highly attractive to both Aedes species. Our results show that at specific cell densities, some bacteria significantly influence the attraction of gravid Ae. aegypti and Ae. albopictus females to

  14. 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.

  15. Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories.

    Science.gov (United States)

    Woo, P C Y; Lau, S K P; Teng, J L L; Tse, H; Yuen, K-Y

    2008-10-01

    In the last decade, as a result of the widespread use of PCR and DNA sequencing, 16S rDNA sequencing has played a pivotal role in the accurate identification of bacterial isolates and the discovery of novel bacteria in clinical microbiology laboratories. For bacterial identification, 16S rDNA sequencing is particularly important in the case of bacteria with unusual phenotypic profiles, rare bacteria, slow-growing bacteria, uncultivable bacteria and culture-negative infections. Not only has it provided insights into aetiologies of infectious disease, but it also helps clinicians in choosing antibiotics and in determining the duration of treatment and infection control procedures. With the use of 16S rDNA sequencing, 215 novel bacterial species, 29 of which belong to novel genera, have been discovered from human specimens in the past 7 years of the 21st century (2001-2007). One hundred of the 215 novel species, 15 belonging to novel genera, have been found in four or more subjects. The largest number of novel species discovered were of the genera Mycobacterium (n = 12) and Nocardia (n = 6). The oral cavity/dental-related specimens (n = 19) and the gastrointestinal tract (n = 26) were the most important sites for discovery and/or reservoirs of novel species. Among the 100 novel species, Streptococcus sinensis, Laribacter hongkongensis, Clostridium hathewayi and Borrelia spielmanii have been most thoroughly characterized, with the reservoirs and routes of transmission documented, and S. sinensis, L. hongkongensis and C. hathewayi have been found globally. One of the greatest hurdles in putting 16S rDNA sequencing into routine use in clinical microbiology laboratories is automation of the technology. The only step that can be automated at the moment is input of the 16S rDNA sequence of the bacterial isolate for identification into one of the software packages that will generate the result of the identity of the isolate on the basis of its sequence database. However

  16. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Automated DNA extraction from genetically modified maize using aminosilane-modified bacterial magnetic particles.

    Science.gov (United States)

    Ota, Hiroyuki; Lim, Tae-Kyu; Tanaka, Tsuyoshi; Yoshino, Tomoko; Harada, Manabu; Matsunaga, Tadashi

    2006-09-18

    A novel, automated system, PNE-1080, equipped with eight automated pestle units and a spectrophotometer was developed for genomic DNA extraction from maize using aminosilane-modified bacterial magnetic particles (BMPs). The use of aminosilane-modified BMPs allowed highly accurate DNA recovery. The (A(260)-A(320)):(A(280)-A(320)) ratio of the extracted DNA was 1.9+/-0.1. The DNA quality was sufficiently pure for PCR analysis. The PNE-1080 offered rapid assay completion (30 min) with high accuracy. Furthermore, the results of real-time PCR confirmed that our proposed method permitted the accurate determination of genetically modified DNA composition and correlated well with results obtained by conventional cetyltrimethylammonium bromide (CTAB)-based methods.

  18. Bacterial Endophytes Isolated from Plants in Natural Oil Seep Soils with Chronic Hydrocarbon Contamination

    OpenAIRE

    Lumactud, Rhea; Shen, Shu Yi; Lau, Mimas; Fulthorpe, Roberta

    2016-01-01

    The bacterial endophytic communities of four plants growing abundantly in soils highly contaminated by hydrocarbons were analyzed through culturable and and culture-independent means. Given their tolerance to the high levels of petroleum contamination at our study site, we sought evidence that Achillea millefolium, Solidago canadensis, Trifolium aureum and Dactylis glomerata support high levels of hydrocarbon degrading endophytes. A total of 190 isolates were isolated from four plant species....

  19. 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.

  20. Isolation and Purification of Bacterial Strains from Treatment Plants for Effective and Efficient Bioconversion of Domestic Wastewater Sludge

    OpenAIRE

    K. C.A. Jalal; Md. Z.   Alam; Suleyman A.   Muyibi; P. Jamal

    2006-01-01

    Forty six bacterial strains were isolated from nine different sources in four treatment plants namely Indah Water Konsortium (IWK) sewage treatment plant, International Islamic University Malaysia (IIUM) treatment plant-1,-2 and 3 to evaluate the bioconversion process in terms of efficient biodegradation and bioseparation. The bacterial strains isolated were found to be 52.2% (24 isolates) and 47.8% (22 isolates) in the IWK and IIUM treatment plants respectively. The results showed that the h...

  1. Identification of bacterial strains isolated from the Mediterranean Sea exhibiting different abilities of biofilm formation.

    Science.gov (United States)

    Brian-Jaisson, Florence; Ortalo-Magné, Annick; Guentas-Dombrowsky, Linda; Armougom, Fabrice; Blache, Yves; Molmeret, Maëlle

    2014-07-01

    The Mediterranean Sea has rarely been investigated for the characterization of marine bacteria as compared to other marine environments such as the Atlantic or Pacific Ocean. Bacteria recovered from inert surfaces are poorly studied in these environments, when it has been shown that the community structure of attached bacteria can be dissimilar from that of planktonic bacteria present in the water column. The objectives of this study were to identify and characterize marine bacteria isolated from biofilms developed on inert surfaces immersed in the Mediterranean Sea and to evaluate their capacity to form a biofilm in vitro. Here, 13 marine bacterial strains have been isolated from different supports immersed in seawater in the Bay of Toulon (France). Phylogenetic analysis and different biological and physico-chemical properties have been investigated. Among the 13 strains recovered, 8 different genera and 12 different species were identified including 2 isolates of a novel bacterial species that we named Persicivirga mediterranea and whose genus had never been isolated from the Mediterranean Sea. Shewanella sp. and Pseudoalteromonas sp. were the most preponderant genera recovered in our conditions. The phenotypical characterization revealed that one isolate belonging to the Polaribacter genus differed from all the other ones by its hydrophobic properties and poor ability to form biofilms in vitro. Identifying and characterizing species isolated from seawater including from Mediterranean ecosystems could be helpful for example, to understand some aspects of bacterial biodiversity and to further study the mechanisms of biofilm (and biofouling) development in conditions approaching those of the marine environment.

  2. 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.

  3. 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.

  4. 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.

  5. A standard bacterial isolate set for research on contemporary dairy spoilage.

    Science.gov (United States)

    Trmčić, A; Martin, N H; Boor, K J; Wiedmann, M

    2015-08-01

    Food spoilage is an ongoing issue that could be dealt with more efficiently if some standardization and unification was introduced in this field of research. For example, research and development efforts to understand and reduce food spoilage can greatly be enhanced through availability and use of standardized isolate sets. To address this critical issue, we have assembled a standard isolate set of dairy spoilers and other selected nonpathogenic organisms frequently associated with dairy products. This publicly available bacterial set consists of (1) 35 gram-positive isolates including 9 Bacillus and 15 Paenibacillus isolates and (2) 16 gram-negative isolates including 4 Pseudomonas and 8 coliform isolates. The set includes isolates obtained from samples of pasteurized milk (n=43), pasteurized chocolate milk (n=1), raw milk (n=1), cheese (n=2), as well as isolates obtained from samples obtained from dairy-powder production (n=4). Analysis of growth characteristics in skim milk broth identified 16 gram-positive and 13 gram-negative isolates as psychrotolerant. Additional phenotypic characterization of isolates included testing for activity of β-galactosidase and lipolytic and proteolytic enzymes. All groups of isolates included in the isolate set exhibited diversity in growth and enzyme activity. Source data for all isolates in this isolate set are publicly available in the FoodMicrobeTracker database (http://www.foodmicrobetracker.com), which allows for continuous updating of information and advancement of knowledge on dairy-spoilage representatives included in this isolate set. This isolate set along with publicly available isolate data provide a unique resource that will help advance knowledge of dairy-spoilage organisms as well as aid industry in development and validation of new control strategies. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  6. Looping and clustering model for the organization of protein-DNA complexes on the bacterial genome

    Science.gov (United States)

    Walter, Jean-Charles; Walliser, Nils-Ole; David, Gabriel; Dorignac, Jérôme; Geniet, Frédéric; Palmeri, John; Parmeggiani, Andrea; Wingreen, Ned S.; Broedersz, Chase P.

    2018-03-01

    The bacterial genome is organized by a variety of associated proteins inside a structure called the nucleoid. These proteins can form complexes on DNA that play a central role in various biological processes, including chromosome segregation. A prominent example is the large ParB-DNA complex, which forms an essential component of the segregation machinery in many bacteria. ChIP-Seq experiments show that ParB proteins localize around centromere-like parS sites on the DNA to which ParB binds specifically, and spreads from there over large sections of the chromosome. Recent theoretical and experimental studies suggest that DNA-bound ParB proteins can interact with each other to condense into a coherent 3D complex on the DNA. However, the structural organization of this protein-DNA complex remains unclear, and a predictive quantitative theory for the distribution of ParB proteins on DNA is lacking. Here, we propose the looping and clustering model, which employs a statistical physics approach to describe protein-DNA complexes. The looping and clustering model accounts for the extrusion of DNA loops from a cluster of interacting DNA-bound proteins that is organized around a single high-affinity binding site. Conceptually, the structure of the protein-DNA complex is determined by a competition between attractive protein interactions and loop closure entropy of this protein-DNA cluster on the one hand, and the positional entropy for placing loops within the cluster on the other. Indeed, we show that the protein interaction strength determines the ‘tightness’ of the loopy protein-DNA complex. Thus, our model provides a theoretical framework for quantitatively computing the binding profiles of ParB-like proteins around a cognate (parS) binding site.

  7. 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.

  8. ‘Tidjanibacter massiliensis’ gen. nov., sp. nov., a new bacterial species isolated from human colon

    Directory of Open Access Journals (Sweden)

    M. Mailhe

    2017-05-01

    Full Text Available We report the summary of main characteristics of Tidjanibacter massiliensis strain Marseille-P3084T, a new bacterial species isolated from the liquid sample of the colon of a patient with a history of irritable bowel syndrome.

  9. Antibiotic resistance profile of bacterial isolates from food sold on a ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-11-02

    Nov 2, 2009 ... Table 1. Distribution and proportion of bacterial contaminants in food. Isolates Identified. Prevalence. E. coli. 23 (29.4%). Klebsiella spp. 20 (25.64%). Proteus spp. 14 (17.95%). Pseudomonas spp. 9 (11.54%). Salmonella spp. 19 (12.82%) analysed from February to July 2005. The food items sampled were.

  10. Bacterial Isolates from the Urine of Women in Ilorin and their ...

    African Journals Online (AJOL)

    Bacterial Isolates from the Urine of Women in Ilorin and their Antibiotic Susceptibility Patterns. ... Methods: Urine samples of women suspected to have UTI were sent for microscopy, culture and sensitivity tests. The results were analyzed and the differences between the results of pregnant and non-pregnant patients were ...

  11. Endophytic and rhizospheric bacterial communities isolated from the medicinal plants Echinacea purpurea and Echinacea angustifolia.

    Science.gov (United States)

    Chiellini, Carolina; Maida, Isabel; Emiliani, Giovanni; Mengoni, Alessio; Mocali, Stefano; Fabiani, Arturo; Biffi, Sauro; Maggini, Valentina; Gori, Luigi; Vannacci, Alfredo; Gallo, Eugenia; Firenzuoli, Fabio; Fani, Renato

    2014-09-01

    In this work we analyzed the composition and structure of cultivable bacterial communities isolated from the stem/leaf and root compartments of two medicinal plants, Echinacea purpurea (L.) Moench and Echinacea angustifolia (DC.) Hell, grown in the same soil, as well as the bacterial community from their rhizospheric soils. Molecular PCR-based techniques were applied to cultivable bacteria isolated from the three compartments of the two plants. The results showed that the two plants and their respective compartments were characterized by different communities, indicating a low degree of strain sharing and a strong selective pressure within plant tissues. Pseudomonas was the most highly represented genus, together with Actinobacteria and Bacillus spp. The presence of distinct bacterial communities in different plant species and among compartments of the same plant species could account for the differences in the medicinal properties of the two plants. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.

  12. Isolation of high molecular weight DNA from marine sponge bacteria for BAC library construction.

    Science.gov (United States)

    Ouyang, Yongchang; Dai, Shikun; Xie, Lianwu; Ravi Kumar, M S; Sun, Wei; Sun, Huimin; Tang, Danling; Li, Xiang

    2010-06-01

    Metagenomics is a powerful tool for mining the genetic repositories from environmental microorganisms. Bacteria associated with marine sponges (phylum Porifera) are rich sources of biologically active natural products. However, to date, few compounds are discovered from the sponge metagenomic libraries, and the main reason might be the difficulties in recovery of high molecular weight (HMW) DNA from sponge symbionts to construct large insert libraries. Here, we describe a method to recover HMW bacterial DNA from diverse sponges with high quality for bacterial artificial chromosome (BAC) library construction. Microorganisms concentrated from sponges by differential centrifugation were embedded in agarose plugs to lyse out the HMW DNA for recovery. DNA fragments over 436 kb size were recovered from three different types of sponges, Halichondria sp., Haliclona sp., and Xestospongia sp. To evaluate the recovered DNA quality, the diversity of bacterial DNA comprised in the HMW DNA derived from sponge Halichondria sp. was analyzed, and this HMW DNA sample was also cloned into a shuttle BAC vector between Escherichia coli and Streptomyces sp. The results showed that more than five types of bacterial DNA, i.e., Proteobacteria, Nitrospirae, Cyanobacteria, Planctomycetes, and unidentified bacteria, had been recovered by this method, and an average 100 kb size insert DNA in a constructed BAC library demonstrated that the recovered HMW DNA is suitable for metagenomic library construction.

  13. Isolation and characterization of sixty sequences of cot-1 DNA from ...

    African Journals Online (AJOL)

    It is well known that cot-1 DNA is enriched for repetitive DNA elements with high and moderately high copy numbers within the genome. In this small-scale study, cot-1 DNA from U. thibetanus was first isolated and characterized using DNA reassociation kinetics, then a Southern blotting probe was used to indicate the ...

  14. DNA thermodynamic stability and supercoil dynamics determine the gene expression program during the bacterial growth cycle.

    Science.gov (United States)

    Sobetzko, Patrick; Glinkowska, Monika; Travers, Andrew; Muskhelishvili, Georgi

    2013-07-01

    The chromosomal DNA polymer constituting the cellular genetic material is primarily a device for coding information. Whilst the gene sequences comprise the digital (discontinuous) linear code, physiological alterations of the DNA superhelical density generate in addition analog (continuous) three-dimensional information essential for regulation of both chromosome compaction and gene expression. Insight into the relationship between the DNA analog information and the digital linear code is of fundamental importance for understanding genetic regulation. Our previous study in the model organism Escherichia coli suggested that the chromosomal gene order and a spatiotemporal gradient of DNA superhelicity associated with DNA replication determine the growth phase-dependent gene transcription. In this study we reveal a general gradient of DNA thermodynamic stability correlated with the polarity of chromosomal replication and manifest in the spatiotemporal pattern of gene transcription during the bacterial growth cycle. Furthermore, by integrating the physical and dynamic features of the transcribed sequences with their functional content we identify spatiotemporal domains of gene expression encompassing different functions. We thus provide both an insight into the organisational principle of the bacterial growth program and a novel holistic methodology for exploring chromosomal dynamics.

  15. Isolation and functional characterization of bacterial endophytes from Carica papaya fruits.

    Science.gov (United States)

    Krishnan, P; Bhat, R; Kush, A; Ravikumar, P

    2012-08-01

    To isolate and characterize the endophytes from papaya fruits and to determine the fermentative potential of the strains. Endophytes provide potential sources for novel natural products for the use in agriculture and nutrition. There is very limited information on isolation and characterization of bacterial endophytes from papaya. We describe isolation and characterization of eighteen endophytes of papaya fruit from four economically important papaya varieties viz 'Red lady', 'Solo', 'Coorg Honey' and 'Bangalore'. The phylogenetic analysis based on the 16S rRNA sequence revealed that isolated endophytes are genetically distinct and cluster as discrete clades in the dendrogram. The Bacillus species is a predominant bacterial endophyte across papaya varieties. The seeds and the endocarp of papaya fruits harbour Kocuria, Acinetobacter and Enterobacter species. The Staphylococcus species were detected in the fruit mesocarp of two papaya varieties used in the study. The endophytes isolated from papaya fruits were capable of producing extracellular enzymes like amylase, cellulase, pectinase and xylanase. Three isolates, Bacillus (PE-LR-1 and PE-LR-3) and Kocuria (PE-LR-2), were selected for fruit fermentation, and antioxidant potential of the fermented product was evaluated. PE-LR-3 fermented product has the free radical scavenging activity of 61·2% and a microbial cocktail of PE-LR-3 with Saccharomyces cerevisiae MTCC 2918 enhances the antioxidant potential to 75·7%. These findings suggest that different parts of papaya fruits harbour an array of bacterial endophytes that could be important agents in attributing the high nutritive status to the fruit and can serve as potent microbial cocktails for developing value-added fermented products of this important fruit. This study describes isolation of a bacterial endophyte from papaya fruit that is capable of improving the antioxidant potential of raw papaya after fermentation. No claim to Indian Government works Journal

  16. Polyphenolics free DNA isolation and optimization of PCR-RAPD for ...

    African Journals Online (AJOL)

    , and good quality high molecular weight DNA samples. DNA was isolated from the mature and fresh young tender leaves obtained from various Italian wild populations of fennel. We performed a modified cetyl trimethyl ammonium bromide ...

  17. Isolation and identification of bacterial pathogen from mastitis milk in Central Java Indonesia

    Science.gov (United States)

    Harjanti, D. W.; Ciptaningtyas, R.; Wahyono, F.; Setiatin, ET

    2018-01-01

    Mastitis is a multi-etiologic disease of the mammary gland characterized mainly by reduction in milk production and milk quality due to intramammary infection by pathogenic bacteria. Nearly 83% of lactating dairy cows in Indonesia are infected with mastitis in various inflammation degrees. This study was conducted to isolate and identify the pathogen in milk collected from mastitis-infected dairy cows. The study was carried out in ten smallholder dairy farms in Central Java Indonesia based on animal examination, California mastitis test, isolation bacterial pathogens, Gram staining, Catalase and Coagulase test, and identification of bacteria species using Vitek. Bacteriological examination of milk samples revealed 15 isolates where Streptococcus was predominant species (73.3%) and the coagulase negative Staphylococcus species was identified at the least bacteria (26.7%). The Streptococcus bacteria found were Streptococcus uberis (2 isolates), Streptococcus sanguinis(6 isolates), Streptococcus dysgalactiaessp dysgalactiae(1 isolate) , Streptococcus mitis (1 isolate) and Streptococcus agalactiae (1 isolate). The Staphylococcus isolates comprising of Staphylococcus simulans (1 isolate) and Staphylococcus chromogens (3 isolates). Contamination of raw milkwith pathogenic bacteria can cause outbreaks of human disease (milk borne disease). Thus, proper milk processing method that couldinhibit the growth or kill these pathogenic bacteria is important to ensure the safety of milk and milk products.

  18. Biases during DNA extraction affect bacterial and archaeal community profile of anaerobic digestion samples.

    Science.gov (United States)

    Roopnarain, Ashira; Mukhuba, Mashudu; Adeleke, Rasheed; Moeletsi, Mokhele

    2017-12-01

    The anaerobic digestion (AD) of organic waste for biogas production has received much attention in recent years due to the increasing need for renewable energy and environmentally friendly waste management systems. Identification of the microbial community involved in AD aids in better understanding and optimising of the process. The choice of DNA extraction method is an integral step in any molecular biodiversity study. In the present study, potential biases introduced by DNA extraction methods were examined by comparing quality, quantity and representability of DNA extracted from AD samples using various extraction methods. In spite of the non-kit based method (cetyltrimethylammonium bromide) yielding the largest quantity of DNA (approximately 44 µg DNA per gram dry weight), the extracted DNA contained PCR inhibitors. Furthermore, the quantity of extracted DNA was not proportional to species diversity. Diversity, determined using denaturing gradient gel electrophoresis (DGGE), was strongly linked to the type of extraction method used. The spin-column filter-based kit that incorporated mechanical and chemical lysis (Macherey-Nagel kit) gave the best results in terms of bacterial and archaeal diversity (Shannon-Wiener indices: average 2.5 and 2.6, respectively). Furthermore, this kit was the most effective at lysing hard-to-lyse bacterial and archaeal cells. The choice of DNA extraction method significantly influences the reliability and comparability of results obtained during AD microbial ecology investigations. Moreover, the careful selection of the DNA extraction method is of particular importance when analysing AD samples since these samples are rich in PCR inhibitors and hard-to-lyse cells such as archaea and gram-positive bacteria.

  19. Isolation of a bacterial consortium able to degrade the fungicide thiabendazole: the key role of a Sphingomonas phylotype.

    Science.gov (United States)

    Perruchon, Chiara; Chatzinotas, Antonis; Omirou, Michalis; Vasileiadis, Sotirios; Menkissoglou-Spiroudi, Urania; Karpouzas, Dimitrios G

    2017-05-01

    Thiabendazole (TBZ) is a fungicide used in fruit-packaging plants. Its application leads to the production of wastewaters requiring detoxification. In the absence of efficient treatment methods, biological depuration of these effluents could be a viable alternative. However, nothing is known regarding the microbial degradation of the recalcitrant and toxic to aquatics TBZ. We report the isolation, via enrichment cultures from a polluted soil, of the first bacterial consortium able to rapidly degrade TBZ and use it as a carbon source. Repeated efforts using various culture-dependent approaches failed to isolate TBZ-degrading bacteria in axenic cultures. Denaturating gradient gel electrophoresis (DGGE) and cloning showed that the consortium was composed of α-, β- and γ-Proteobacteria. Culture-independent methods including antibiotics-driven selection with DNA/RNA-DGGE, q-PCR and stable isotope probing (SIP)-DGGE identified a Sphingomonas phylotype (B13) as the key degrading member. Cross-feeding studies with structurally related chemicals showed that ring substituents of the benzimidazole moiety (thiazole or furan rings) favoured the cleavage of the imidazole moiety. LC-MS/MS analysis verified that TBZ degradation proceeds via cleavage of the imidazole moiety releasing thiazole-4-carboxamidine, which was not further transformed, and the benzoyl moiety, possibly as catechol, which was eventually consumed by the bacterial consortium as suggested by SIP-DGGE.

  20. [Presence of bacterial DNA in valvular tissue of patients with chronic rheumatic heart disease].

    Science.gov (United States)

    Figueroa, Fernando E; Carrión A, Flavio; Valenzuela M, Sylvia; Turner G, Eduardo; Aceitón E, Cristian; Hirigoyen P, Carolina; Bogdanic W, Katherine; Solís D, Claudia; Mansilla A, Karina; Urra G, Soledad

    2007-08-01

    Rheumatic heart disease (RHD) is a delayed consequence of a pharyngeal infection with Group A streptococcus (GAS), usually ascribed to a cross-reactive immune response to the host cardiac tissues. Acute rheumatic fever (ARF) and its ensuing valvular sequelae are thus considered the prototype of a post-infectious autoimmune disease, with no direct evidence of residual streptococcal antigen in diseased valvular tissues. However, recent studies concerning the antigenic specificity and clonality of intralesional lymphocytes have revealed oligoclonal expansions characteristic of an antigen specific response, that might be related to GAS. To search for bacterial DNA in valvular tissue from RHD patients and controls. We extracted DNA from surgically excised valve specimens from 15 RHD patients and 6 non RHD controls and tested for the presence of bacterial DNA by Polymerase Chain Reaction (PCR) with primers for 16S rRNA. Eighty percent (12/15) of valve specimens from RHD patients were positive for bacterial DNA, as opposed to none of the valves (n =6) from non RHD controls. These results suggest that GAS might persist in valvular tissue in patients with ARF and contribute to the inflammatory scarring lesion that leads to cardiovascular sequelae.

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

    Directory of Open Access Journals (Sweden)

    Michael Politis

    Full Text Available 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.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.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 = <0.001. None of the pathogens had developed any resistance to Vancomycin. (P = 0.88.Coagulase negative staphylococci were the predominant bacteria isolated from patients with 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.

  2. 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.

  3. Evaluation of rapid protocols for DNA isolation from Cercospora beticola Sacc.

    Directory of Open Access Journals (Sweden)

    Budakov Dragana

    2012-01-01

    Full Text Available The most fungal DNA isolation protocols are designed to obtain high amounts of very pure DNA, requiring large fungal cultures and extraction procedures with many purification steps. Since the PCR does not require high purity DNA, the aim of this investigation was to evaluate three fast and simple fungal DNA isolation protocols for further use in Cercospora PCR based research. The purity and quantity of isolated DNAs were determined spectrophotometrically, electrophoretically and by PCR reaction with universal primers. The amounts of DNA evaluated on agarose gels, isolated by protocols A and C, did not correspond to the spectrophotometrical values, probably due to RNA impurities. In samples isolated by protocol B these impurities were not detected and the DNA concentrations were more similar. Neither protocol eliminated impurities such as carbohydrates and phenol. The average DNA yield of protocol A was 1.04 μg/μl, protocol B 0.88 μg/μl, and protocol C 0.55 μg/μl. The DNA quality most suitable for PCR analysis was obtained by protocol A, where amplification product with universal primers was detected in all DNA samples. The amplification product was detected in 87% of samples isolated by protocol C and in only 60% of samples isolated by protocol B. Although DNA obtained by protocol A had the highest yield and best quality, the isolation protocol C should be also recommended, for it does not require phenol, chlorophorm or liquid nitrogen.

  4. Assessing genetic structure and diversity of airborne bacterial communities by DNA fingerprinting and 16S rDNA clone library

    Science.gov (United States)

    Maron, Pierre-Alain; Lejon, David P. H.; Carvalho, Esmeralda; Bizet, Karine; Lemanceau, Philippe; Ranjard, Lionel; Mougel, Christophe

    The density, genetic structure and diversity of airborne bacterial communities were assessed in the outdoor atmosphere. Two air samples were collected on the same location (north of France) at two dates (March 2003 (sample1) and May 2003 (sample 2)). Molecular culture -independent methods were used to characterise airborne bacterial communities regardless of the cell culturability. The automated-ribosomal intergenic spacer analysis (A-RISA) was performed to characterise the community structure in each sample. For both sampling dates, complex A-RISA patterns were observed suggesting a highly diverse community structure, comparable to those found in soil, water or sediment environments. Furthermore, differences in the genetic structure of airborne bacterial communities were observed between samples 1 and 2 suggesting an important variability in time. A clone library of 16S rDNA directly amplified from air DNA of sample 1 was constructed and sequenced to analyse the community composition and diversity. The Proteobacteria group had the greatest representation (60%), with bacteria belonging to the different subdivisions α- (19%), β-(21%), γ-(12%) and δ-(8%). Firmicute and Actinobacteria were also well represented with 14% and 12%, respectively. Most of the identified bacteria are known to be commonly associated with soil or plant environments suggesting that the atmosphere is mainly colonised transiently by microorganisms from local sources, depending on air fluxes.

  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. 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

  7. Molecular characterization of Pasteurella multocida isolates from swine lungs by Randomly Amplified Polymorphic DNA

    Directory of Open Access Journals (Sweden)

    Cristiane Silva Chitarra

    2016-01-01

    Full Text Available ABSTRACT: Swine respiratory diseases such as atrophic rhinitis and bronchopneumonia caused by Pasteurella (P. multocida cause important economic losses to the modern swine industry. The purpose of this study was to characterize P. multocida strains isolated from swine lungs by RAPD (Randomly Amplified Polymorphic DNA to demonstrate their genetic diversity. Ninety-four samples of fragments from lungs with pneumonia and sixty one samples without pneumonia were collected in slaughterhouses in Mato Grosso during the period from December 2009 to March 2010. Clinical cases in 2012 and 2013 were also included in this study. Among the lung fragments with macroscopic lesions, without macroscopic lesions and clinical samples, 40.42%, 4.49% and 100% were positive for P. multocida, respectively. Bacterial identification culturing was confirmed by PCR (polymerase chain reaction by means of the amplification of the gene kmt1. RAPD technique was performed for 46 isolates, and in every isolate, a total of 7 to 11 amplification bands were detected, composed of 8 clusters based on genetic similarity. Thus, treatment, control and preventive measures should consider the genetic diversity of P. multocida populations in swine herds in order to improve the development of new protocols to produce antimicrobials and vaccines.

  8. The BER necessities: the repair of DNA damage in human-adapted bacterial pathogens.

    Science.gov (United States)

    van der Veen, Stijn; Tang, Christoph M

    2015-02-01

    During colonization and disease, bacterial pathogens must survive the onslaught of the host immune system. A key component of the innate immune response is the generation of reactive oxygen and nitrogen species by phagocytic cells, which target and disrupt pathogen molecules, particularly DNA, and the base excision repair (BER) pathway is the most important mechanism for the repair of such oxidative DNA damage. In this Review, we discuss how the human-specific pathogens Mycobacterium tuberculosis, Helicobacter pylori and Neisseria meningitidis have evolved specialized mechanisms of DNA repair, particularly their BER pathways, compared with model organisms such as Escherichia coli. This specialization in DNA repair is likely to reflect the distinct niches occupied by these important human pathogens in the host.

  9. Origin of spatial organization of DNA-polymer in bacterial chromosomes

    Science.gov (United States)

    Agarwal, Tejal; Manjunath, G. P.; Habib, Farhat; Chatterji, Apratim

    2018-01-01

    In vivo DNA organization at large length scales (∼100 \\text{nm}) is highly debated and polymer models have proved useful to understand the principles of DNA organization. Here, we show that extracted from contact maps of bacterial DNA. We are able to predict the structure of 2 DNAs (E. coli and Caulobacter crescentus) using Monte Carlo simulations of the bead-spring polymer with cross-links at these special positions. Simulations with cross-links at random positions along the chain show that the organization of the polymer is different in nature from the previous case. We provide some direct and some indirect experimental validation for our predicted organization of DNA-polymers.

  10. Bacterial fumarase and L-malic acid are evolutionary ancient components of the DNA damage response

    Science.gov (United States)

    Singer, Esti; Silas, Yardena BH; Ben-Yehuda, Sigal

    2017-01-01

    Fumarase is distributed between two compartments of the eukaryotic cell. The enzyme catalyses the reversible conversion of fumaric to L-malic acid in mitochondria as part of the tricarboxylic acid (TCA) cycle, and in the cytosol/nucleus as part of the DNA damage response (DDR). Here, we show that fumarase of the model prokaryote Bacillus subtilis (Fum-bc) is induced upon DNA damage, co-localized with the bacterial DNA and is required for the DDR. Fum-bc can substitute for both eukaryotic functions in yeast. Furthermore, we found that the fumarase-dependent intracellular signaling of the B. subtilis DDR is achieved via production of L-malic acid, which affects the translation of RecN, the first protein recruited to DNA damage sites. This study provides a different evolutionary scenario in which the dual function of the ancient prokaryotic fumarase, led to its subsequent distribution into different cellular compartments in eukaryotes. PMID:29140245

  11. Degradation of nicosulfuron by a novel isolated bacterial strain Klebsiella sp. Y1: condition optimization, kinetics and degradation pathway.

    Science.gov (United States)

    Wang, Lin; Zhang, Xiaolin; Li, Yongmei

    2016-01-01

    A novel bacterial strain Klebsiella sp. Y1 was isolated from the soil of a constructed wetland, and it was identified based on the 16S rDNA sequence analysis. The co-metabolic degradation of nicosulfuron with glucose by Klebsiella sp. Y1 was investigated. The response surface methodology analysis indicated that the optimal pH and temperature were 7.0 and 35 °C, respectively, for the degradation of nicosulfuron. Under the optimal conditions, the degradation of nicosulfuron fitted Haldane kinetics model well. The removal of nicosulfuron was triggered by the acidification of glucose, which accelerated the hydrolysis of nicosulfuron. Then, the C-N bond of the sulfonylurea bridge was attacked and cleaved. Finally, the detected intermediate 2-amino-4,6-dimethoxypyrimidine was further biodegraded.

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

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    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

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

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    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.

  14. Viral terminal protein directs early organization of phage DNA replication at the bacterial nucleoid.

    Science.gov (United States)

    Muñoz-Espín, Daniel; Holguera, Isabel; Ballesteros-Plaza, David; Carballido-López, Rut; Salas, Margarita

    2010-09-21

    The mechanism leading to protein-primed DNA replication has been studied extensively in vitro. However, little is known about the in vivo organization of the proteins involved in this fundamental process. Here we show that the terminal proteins (TPs) of phages ϕ29 and PRD1, infecting the distantly related bacteria Bacillus subtilis and Escherichia coli, respectively, associate with the host bacterial nucleoid independently of other viral-encoded proteins. Analyses of phage ϕ29 revealed that the TP N-terminal domain (residues 1-73) possesses sequence-independent DNA-binding capacity and is responsible for its nucleoid association. Importantly, we show that in the absence of the TP N-terminal domain the efficiency of ϕ29 DNA replication is severely affected. Moreover, the TP recruits the phage DNA polymerase to the bacterial nucleoid, and both proteins later are redistributed to enlarged helix-like structures in an MreB cytoskeleton-dependent way. These data disclose a key function for the TP in vivo: organizing the early viral DNA replication machinery at the cell nucleoid.

  15. [Studies on the repair of damaged DNA in bacteriophage, bacterial and mammalian systems]: Final report

    International Nuclear Information System (INIS)

    Friedberg, E.C.

    1987-08-01

    This study sought to exploit the use of uv radiation as a source of genomic damage. We explored the molecular mechanism of the repair of DNA damage at a number of different levels of biological organization, by investigating bacteriophage, bacterial, yeast and mammalian cells. Not only have observations obtained in one biological system suggested specific experimental approaches in others, but we have also learned that some biochemical pathways for DNA repair are unique to specific organisms. Our studies are summarized in terms of 4 major areas of research activity that span the past 16 years. 86 refs

  16. 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

  17. Extraction of DNA from soil for analysis of bacterial diversity in transgenic and nontransgenic papaya sites.

    Science.gov (United States)

    Sheu, Ceshing; Wu, Chung-Yi; Chen, Shu-Chuan; Lo, Chi-Chu

    2008-12-24

    The influence of transgenic crops on the soil diversity of microorganisms is one of the major risk assessments being conducted in Taiwan since 2007, and a reliable soil DNA extraction method for denaturing gradient gel electrophoresis (DGGE) is required. Six soils of different type, organic matter content, cation exchange capacity, and pH were tested, and four previously reported soil DNA extraction methods were applied to these soils. Soil DNA extracts by Zhou's CS method plus QIAquick gel was recommended in our laboratory for DGGE to monitor the microbial diversity in soil. There were some differences on the bacterial diversity based on DGGE patterns at the beginning of planting, and the difference decreased after six months. The results also indicated that clay content (10.8-25.0%) and pH (4.4-6.9) of different soil samples we tested did not affect the DNA extraction efficiencies, but positive correlations were found between the organic matter content (1.2-3.9%) of soils and the DNA yields in Widmer's GS method (r = 0.93, p = 0.005) and the MoBio UC method (r = 0.92, p = 0.007). Coefficient of determinations between organic matter content and DNA yield were higher than those between clay content, CEC, and pH, indicating that organic matter content was more correlated with DNA yield than that clay content, CEC, and pH in our soil samples tested.

  18. Conjugative DNA transfer induces the bacterial SOS response and promotes antibiotic resistance development through integron activation.

    Directory of Open Access Journals (Sweden)

    Zeynep Baharoglu

    2010-10-01

    Full Text Available Conjugation is one mechanism for intra- and inter-species horizontal gene transfer among bacteria. Conjugative elements have been instrumental in many bacterial species to face the threat of antibiotics, by allowing them to evolve and adapt to these hostile conditions. Conjugative plasmids are transferred to plasmidless recipient cells as single-stranded DNA. We used lacZ and gfp fusions to address whether conjugation induces the SOS response and the integron integrase. The SOS response controls a series of genes responsible for DNA damage repair, which can lead to recombination and mutagenesis. In this manuscript, we show that conjugative transfer of ssDNA induces the bacterial SOS stress response, unless an anti-SOS factor is present to alleviate this response. We also show that integron integrases are up-regulated during this process, resulting in increased cassette rearrangements. Moreover, the data we obtained using broad and narrow host range plasmids strongly suggests that plasmid transfer, even abortive, can trigger chromosomal gene rearrangements and transcriptional switches in the recipient cell. Our results highlight the importance of environments concentrating disparate bacterial communities as reactors for extensive genetic adaptation of bacteria.

  19. 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.

  20. Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins.

    Science.gov (United States)

    Zdżalik, Daria; Domańska, Anna; Prorok, Paulina; Kosicki, Konrad; van den Born, Erwin; Falnes, Pål Ø; Rizzo, Carmelo J; Guengerich, F Peter; Tudek, Barbara

    2015-06-01

    AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1,N(6)-ethenoadenine (ɛA), 3,N(4)-ethenocytosine (ɛC) and 1,N(2)-ethenoguanine (1,N(2)-ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N(2)-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N(2)-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins. Copyright © 2015 Elsevier B

  1. Initial nitrogen enrichment conditions determines variations in nitrogen substrate utilization by heterotrophic bacterial isolates.

    Science.gov (United States)

    Ghosh, Suchismita; Ayayee, Paul A; Valverde-Barrantes, Oscar J; Blackwood, Christopher B; Royer, Todd V; Leff, Laura G

    2017-04-04

    The nitrogen (N) cycle consists of complex microbe-mediated transformations driven by a variety of factors, including diversity and concentrations of N compounds. In this study, we examined taxonomic diversity and N substrate utilization by heterotrophic bacteria isolated from streams under complex and simple N-enrichment conditions. Diversity estimates differed among isolates from the enrichments, but no significant composition were detected. Substrate utilization and substrate range of bacterial assemblages differed within and among enrichments types, and not simply between simple and complex N-enrichments. N substrate use patterns differed between isolates from some complex and simple N-enrichments while others were unexpectedly similar. Taxonomic composition of isolates did not differ among enrichments and was unrelated to N use suggesting strong functional redundancy. Ultimately, our results imply that the available N pool influences physiology and selects for bacteria with various abilities that are unrelated to their taxonomic affiliation.

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

    OpenAIRE

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

    2016-01-01

    The isolation and analysis of single prokaryotic cells down to 1??m 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??m in size....

  3. 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.

  4. DNA replication proteins as potential targets for antimicrobials in drug-resistant bacterial pathogens.

    Science.gov (United States)

    van Eijk, Erika; Wittekoek, Bert; Kuijper, Ed J; Smits, Wiep Klaas

    2017-05-01

    With the impending crisis of antimicrobial resistance, there is an urgent need to develop novel antimicrobials to combat difficult infections and MDR pathogenic microorganisms. DNA replication is essential for cell viability and is therefore an attractive target for antimicrobials. Although several antimicrobials targeting DNA replication proteins have been developed to date, gyrase/topoisomerase inhibitors are the only class widely used in the clinic. Given the numerous essential proteins in the bacterial replisome that may serve as a potential target for inhibitors and the relative paucity of suitable compounds, it is evident that antimicrobials targeting the replisome are underdeveloped so far. In this review, we report on the diversity of antimicrobial compounds targeting DNA replication and highlight some of the challenges in developing new drugs that target this process. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

  5. Isolation of Bacterial Strain for Biodegradation of Fats, Oil and Grease

    International Nuclear Information System (INIS)

    Alkhatib, M.F.; Mohd Zahangir Alam; Shabana, H.F.M.

    2015-01-01

    Fat, oil and grease (FOG) deposition is one of the major problems that harm the environment and cause dissatisfaction for human. Uncontrolled and un-pre-treated FOG removal from the kitchen could lead to its accumulation in the piping system. Problems include the interference of fat with the aerobic microorganisms that are responsible in treating the wastewater by reducing oxygen transfer rates and for anaerobic microorganisms; their efficiency could also be reduced due to the reduction of the transport of soluble substrates to the bacterial biomass. Biodegradation could be one of the effective means to treat FOG. The main objective of this study is to isolate bacterial strains from the FOG waste and identify the strains that are capable in biodegrading FOG waste. FOG sample was collected from a sewer manhole. Enrichment technique was applied, followed by isolation of bacterial strains to determine which strain is able to degrade the FOG deposition. Some morphology for the bacterial strain was done to determine its characteristics. (author)

  6. MICROBIAL PROFILE AND ANTIBIOTIC RESISTANCE PATTERN OF THE BACTERIAL ISOLATES IN A TERTIARY CARE PSYCHIATRY HOSPITAL

    Directory of Open Access Journals (Sweden)

    Jyoti

    2015-11-01

    Full Text Available BACKGROUND: Antibiotic resistance is a challenge for effective management of infections as it increases the morbidity, mortality and costs of treating infectious diseases. AIMS: This study was aimed to obtain the profile of the bacterial isolates and their antibiotic resistance pattern. SETTINGS AND DESIGN: It is a cross sectional study carried out in a tertiary care psychiatry hospital in India. MATERIALS AND METHODS: Isolation and identification of the isolates were done by standard methods. Susceptibility patterns were checked by Kirby Bauer disc diffusion method. STATISTICAL ANALYSIS USED: Statistical analysis was done by using SPSS 16.0 version to calculate the frequencies as well as for cross tabulation. RESULTS: Significant bacterial growth observed in 43(25.6% samples, of which 39(90.7% showed resistant to at least one of the antibiotics used and 36(83.7% were multi-drug resistant. Gram negative organism accounted for the 25(58.14% of total significant isolates, Escherichia coli being the highest (76% in this group. Among multi-drug resistant (MDR isolates E.coli was the highest (44.4% and imipenem resistance was also observed in 1(5.3% of 19 E.coli isolates. Among the 43 isolates 18(41.86% were Gram positive with Streptococcus spp. showing incidence of 41.7% among the total MDR isolates. CONCLUSION: Increasing incidence of MDR strains seen in the population requires continuous monitoring and a restricted use of antibiotics to keep a check on resistance pattern, for effective treatment plan.

  7. Leaf storage conditions and genomic DNA isolation efficiency in ...

    African Journals Online (AJOL)

    Storage of plant tissues for DNA is important to avoid degradation of DNA. Preliminary studies were conducted on Ocimum gratissimum L. in order to establish the storage conditions for the collected samples before DNA extraction. Secondly, the aim was to determine the best protocol for the extraction of high quality DNA, ...

  8. Culturable bacterial endophytes isolated from Mangrove tree (Rhizophora apiculata Blume) enhance seedling growth in Rice.

    Science.gov (United States)

    Deivanai, Subramanian; Bindusara, Amitraghata Santhanam; Prabhakaran, Guruswamy; Bhore, Subhash Janardhan

    2014-07-01

    Endophytic bacteria do have several potential applications in medicine and in other various sectors of biotechnology including agriculture. Bacterial endophytes need to be explored for their potential applications in agricultural biotechnology. One of the potential applications of bacterial endophytes in agricultural is to enhance the growth of the agricultural crops. Hence, this study was undertaken to explore the plant growth promoting potential application of bacterial endophytes. The objective of this study was to examine the effect of endophytic bacteria from mangrove tree (Rhizophora apiculata Blume) for their efficacy in promoting seedling growth in rice. Eight endophytic bacterial isolates (EBIs) isolated from twig and petiole tissues of the mangrove were identified based on their 16S ribosomal ribonucleic acid (rRNA) gene sequence homology. Separately, surface sterilized paddy seeds were treated with cell-free broth and cell suspension of the EBIs. Rice seedlings were analyzed by various bioassays and data was recorded. The gene sequences of the isolates were closely related to two genera namely, Bacillus and Pantoea. Inoculation of EBIs from R. apiculata with rice seeds resulted in accelerated root and shoot growth with significant increase in chlorophyll content. Among the isolates, Pantoea ananatis (1MSE1) and Bacillus amyloliquefaciens (3MPE1) had shown predominance of activity. Endophytic invasion was recognized by the non-host by rapid accumulation of reactive oxygen species (ROS) and was counteracted by the production of hydrogen peroxide (H2O2) and lipid peroxide. The results demonstrated that EBIs from mangrove tree can increase the fitness of the rice seedlings under controlled conditions. These research findings could be useful to enhance the seedling growth and could serve as foundation in further research on enhancing the growth of the rice crop using endophytic bacteria.

  9. Isolation and characterization of a Coffea canephora ERF-like c-DNA

    African Journals Online (AJOL)

    A cDNA corresponding to an ERF gene has been isolated from a Coffea canephora fruit cDNA library. The cDNA was 1,317 nucleotides long and has an open reading frame of 987 bp. The predicted polypeptide showed a great similitude with equivalent proteins from others plant species. The binding domain shows 98.3% ...

  10. 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.

  11. General and specialized media routinely employed for primary isolation of bacterial pathogens of fishes

    Science.gov (United States)

    Starliper, C.E.

    2008-01-01

    There are a number of significant diseases among cultured and free-ranging freshwater fishes that have a bacterial etiology; these represent a variety of gram-negative and gram-positive genera. Confirmatory diagnosis of these diseases involves primary isolation of the causative bacterium on bacteriologic media. Frequently used "general" bacteriologic media simply provide the essential nutrients for growth. For most of the major pathogens, however, there are differential and/or selective media that facilitate primary recovery. Some specialized media are available as "ready-to-use" from suppliers, while others must be prepared. Differential media employ various types of indicator systems, such as pH indicators, that allow diagnosticians to observe assimilation of selected substrates. An advantage to the use of differential media for primary isolation is that they hasten bacterial characterization by yielding the appropriate positive or negative result for a particular substrate, often leading to a presumptive identification. Selective media also incorporate agent(s) that inhibit the growth of contaminants typically encountered with samples from aquatic environments. Media that incorporate differential and/or selective components are ideally based on characters that are unique to the targeted bacterium, and their use can reduce the time associated with diagnosis and facilitate early intervention in affected fish populations. In this review, the concepts of general and differential/selective bacteriologic media and their use and development for fish pathogens are discussed. The media routinely employed for primary isolation of the significant bacterial pathogens of fishes are presented. ?? Wildlife Disease Association 2008.

  12. [Isolation and partial characterization of DNA topoisomerase I from the nucleoids of white mustard chloroplasts].

    Science.gov (United States)

    Belkina, G G; Pogul'skaia, E V; Iurina, N P

    2004-01-01

    DNA topoisomerase was isolated for the first time from nucleoids of white mustard (Sinapis alba L.) chloroplasts. The enzyme had a molecular weight of 70 kDa; it was ATP-independent, required the presence of mono- (K+) and bivalent (Mg2+) cations, and was capable of relaxing both negatively and positively supercoiled DNA. These results suggest that the enzyme isolated belongs to type IB DNA topoisomerases.

  13. 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

    2018-02-01

    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.

  14. 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.

  15. Glycerol Monolaurate Inhibits Lipase Production by Clinical Ocular Isolates Without Affecting Bacterial Cell Viability.

    Science.gov (United States)

    Flanagan, Judith Louise; Khandekar, Neeta; Zhu, Hua; Watanabe, Keizo; Markoulli, Maria; Flanagan, John Terence; Papas, Eric

    2016-02-01

    We sought to determine the relative lipase production of a range of ocular bacterial isolates and to assess the efficacy of glycerol monolaurate (GML) in inhibiting this lipase production in high lipase-producing bacteria without affecting bacterial cell growth. Staphylococcus aureus,Staphylococcus epidermidis,Propionibacterium acnes, and Corynebacterium spp. were inoculated at a density of 10(6)/mL in varying concentrations of GML up to 25 μg/mL for 24 hours at 37 °C with constant shaking. Bacterial suspensions were centrifuged, bacterial cell density was determined, and production of bacterial lipase was quantified using a commercial lipase assay kit. Staphylococcus spp. produced high levels of lipase activity compared with P. acnes and Corynebacterium spp. GML inhibited lipase production by Staphylococcal spp. in a dose-dependent manner, with S. epidermidis lipase production consistently more sensitive to GML than S. aureus. Glycerol monolaurate showed significant (P lipase inhibition above concentrations of 15 μg/mL in S. aureus and was not cytotoxic up to 25 μg/mL. For S. epidermidis, GML showed significant (P lipase inhibition above 7.5 μg/mL. Lipase activity varied between species and between strains. Staphylococcal spp. produced higher lipase activity compared with P. acnes and Corynebacterium spp. Glycerol monolaurate inhibited lipase production by S. aureus and S. epidermidis at concentrations that did not adversely affect bacterial cell growth. GML can be used to inhibit ocular bacterial lipase production without proving detrimental to commensal bacteria viability.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. Structural and Biochemical Analysis of DNA Helix Invasion by the Bacterial 8-Oxoguanine DNA Glycosylase MutM*

    Science.gov (United States)

    Sung, Rou-Jia; Zhang, Michael; Qi, Yan; Verdine, Gregory L.

    2013-01-01

    MutM is a bacterial DNA glycosylase that serves as the first line of defense against the highly mutagenic 8-oxoguanine (oxoG) lesion, catalyzing glycosidic bond cleavage of oxoG to initiate base excision DNA repair. Previous work has shown that MutM actively interrogates DNA for the presence of an intrahelical oxoG lesion. This interrogation process involves significant buckling and bending of the DNA to promote extrusion of oxoG from the duplex. Structural snapshots have revealed several different highly conserved residues that are prominently inserted into the duplex in the vicinity of the target oxoG before and after base extrusion has occurred. However, the roles of these helix-invading residues during the lesion recognition and base extrusion process remain unclear. In this study, we set out to probe the function of residues Phe114 and Met77 in oxoG recognition and repair. Here we report a detailed biochemical and structural characterization of MutM variants containing either a F114A or M77A mutation, both of which showed significant decreases in the efficiency of oxoG repair. These data reveal that Met77 plays an important role in stabilizing the lesion-extruded conformation of the DNA. Phe114, on the other hand, appears to destabilize the intrahelical state of the oxoG lesion, primarily by buckling the target base pair. We report the observation of a completely unexpected interaction state, in which the target base pair is ruptured but remains fully intrahelical; this structure vividly illustrates the disruptive influence of MutM on the target base pair. PMID:23404556

  18. COMPARATIVE EVALUATION OF CULTURE MEDIA FOR PATHOGEN ISOLATION OF PURULENT BACTERIAL MENINGITIS

    Directory of Open Access Journals (Sweden)

    Ya. V. Podkopaev

    2016-01-01

    Full Text Available The State Research Center for Applied Microbiology and Biotechnology has designed two nutrient media — chocolate agar and PBM-agar to isolate pathogens of purulent bacterial meningitis (PBM. In our previous research using collected microbial strains the media were shown to be highly susceptible and to provide the growth of Neisseria meningiti-dis, Streptococcus pneumoniae and Haemophilus influenzae strains, when inoculated with microbial suspensions containing single cells. When isolating Haemophilus influenzae, meningococci, and pneumococci the use of selective additives in both media assures selective isolation of required microorganisms, inhibiting contaminants. The objective of this research was to assess the media in bacteriological tests of clinical samples collected from the upper and lower respiratory tract in humans. The bacteriological plating of throat smear specimens (n = 90 from children and adults at the age of 0 to 66 with disorder of the upper respiratory tract on chocolate agar, PBM-agar and on a control medium in the absence of selective additives resulted in the equal amount of microbial cultures isolated. Of 154 isolated cultures 2, 23 and 9 were attributed to Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae, respectively. The plating of throat smears (n = 10 from healthy people at the age of 30 to 55 on the analyzable and control media in the presence of additives allowed us to selectively isolate Haemophilus influenzae and Streptococcus pneumoniae cultures without a quantitative loss, with contaminants inhibited. By their growth characteristics chocolate agar and PBM-agar were highly competitive with reference media being used in clinical practice for isolating main causative agents of purulent bacterial meningitis.

  19. 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.

  20. [The isolation and partial purification of 2 DNA-dependent DNA polymerases from Acholeplasma laidlawii PG-8].

    Science.gov (United States)

    Bezuglyĭ, S V; Babichev, V V; Skripal', I G; Malinovskaia, L P

    1991-01-01

    Two forms of DNA-dependent DNA-polymerase have been isolated and partially purified from the limited amount of biomass of cells Acholeplasma laidlawii PG-8, a typical representative of genus Acholeplasmataceae, as a result of successive chromatography on the columns with DEAE-cellulose DE-52 and Green A-sepharose. The first form of DNA-polymerase is eluted from the ion-exchange column with NaCl concentration of 0.1 M from the column with Green A-sepharose of 0.27 M, while the second form-with NaCl concentrations of 0.6 and 0.4 M, respectively. The both enzymatic activities are able to implement DNA synthesis. The conditions of DNA-polymerase production proved to be rather convenient for isolation of the concentrated and highly active enzymes.

  1. 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. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    Zhang, Yongwei; Werling, Uwe; Edelmann, Winfried

    2012-01-01

    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. PMID:22241772

  3. 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.

  4. Determination of alkylation of bacterial DNA as a rapid test for toxicological evaluation of alkylating xenobiotic agents

    Energy Technology Data Exchange (ETDEWEB)

    Botzenhart, K.; Waldner-Sander, S.; Schweinsberg, F.

    1986-05-01

    Alkylated purine bases from hydrolized DNA can be separated by HPLC and quantified with a fluorescence detector. We applied this method to bacterial DNA. 7-methylguanine was detected after treatment of Serratia marcescens with iodoacetamide, dimethyl sulfate and with polluted air.

  5. 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)

  6. ‘Colibacter massiliensis’ gen. nov., sp. nov., a new bacterial species isolated from human left colon

    Directory of Open Access Journals (Sweden)

    M. Mailhe

    2017-05-01

    Full Text Available We present here the main characteristics of the strain Marseille-P2911 (= CSUR P2911=DSMZ 103304, a bacterial species isolated from the left colon liquid sample of a 60-year-old man.

  7. Chlorhexidine Digluconate Effects on Planktonic Growth and Biofilm Formation in Some Field Isolates of Animal Bacterial Pathogens

    Science.gov (United States)

    Ebrahimi, Azizollah; Hemati, Majid; Habibian Dehkordi, Saeed; Bahadoran, Shahab; Khoshnood, Sheida; Khubani, Shahin; Dokht Faraj, Mahdi; Hakimi Alni, Reza

    2014-01-01

    Background: To study chlorhexidine digluconate disinfectant effects on planktonic growth and biofilm formation in some bacterial field isolates from animals. Objectives: The current study investigated chlorhexidine digluconate effects on planktonic growth and biofilm formation in some field isolates of veterinary bacterial pathogens. Materials and Methods: Forty clinical isolates of Escherichia coli, Salmonella serotypes, Staphylococcus. aureus and Streptococcus agalactiae (10 isolates for each) were examined for chlorhexidine digluconate effects on biofilm formation and planktonic growth using microtiter plates. In all of the examined strains in the presence of chlorhexidine digluconate, biofilm development and planktonic growth were affected at the same concentrations of the disinfectant. Results: Chlorhexidine digluconate inhibited the planktonic growth of different bacterial species at sub-MICs. But they were able to induce biofilm development of the E. coli, Salmonella spp., S. aureus and Str. agalactiae strains. Conclusions: Bacterial resistance against chlorhexidine is increasing. Sub-MIC doses of chlorhexidine digluconate can stimulate the formation of biofilm strains. PMID:24872940

  8. The Role of the N-Terminal Domains of Bacterial Initiator DnaA in the Assembly and Regulation of the Bacterial Replication Initiation Complex

    Science.gov (United States)

    Zawilak-Pawlik, Anna; Nowaczyk, Małgorzata; Zakrzewska-Czerwińska, Jolanta

    2017-01-01

    The primary role of the bacterial protein DnaA is to initiate chromosomal replication. The DnaA protein binds to DNA at the origin of chromosomal replication (oriC) and assembles into a filament that unwinds double-stranded DNA. Through interaction with various other proteins, DnaA also controls the frequency and/or timing of chromosomal replication at the initiation step. Escherichia coli DnaA also recruits DnaB helicase, which is present in unwound single-stranded DNA and in turn recruits other protein machinery for replication. Additionally, DnaA regulates the expression of certain genes in E. coli and a few other species. Acting as a multifunctional factor, DnaA is composed of four domains that have distinct, mutually dependent roles. For example, C-terminal domain IV interacts with double-stranded DnaA boxes. Domain III drives ATP-dependent oligomerization, allowing the protein to form a filament that unwinds DNA and subsequently binds to and stabilizes single-stranded DNA in the initial replication bubble; this domain also interacts with multiple proteins that control oligomerization. Domain II constitutes a flexible linker between C-terminal domains III–IV and N-terminal domain I, which mediates intermolecular interactions between DnaA and binds to other proteins that affect DnaA activity and/or formation of the initiation complex. Of these four domains, the role of the N-terminus (domains I–II) in the assembly of the initiation complex is the least understood and appears to be the most species-dependent region of the protein. Thus, in this review, we focus on the function of the N-terminus of DnaA in orisome formation and the regulation of its activity in the initiation complex in different bacteria. PMID:28489024

  9. Plant growth-promoting activities for bacterial and fungal endophytes isolated from medicinal plant of Teucrium polium L.

    OpenAIRE

    Hassan, Saad El-Din

    2017-01-01

    Bacterial and fungal endophytes are widespread inhabitants inside plant tissues and have been shown to assist plant growth and health. However, little is known about plant growth-promoting endophytes (PGPE) of medicinal plants. Therefore, the aims of this study were to identify bacterial and fungal endophytes of Teucrium polium and to characterize plant growth-promoting (PGP) properties of these endophytes. Seven bacterial endophytes were isolated and identified as Bacillus cereus and Bacillu...

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

    Directory of Open Access Journals (Sweden)

    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

  11. A simple random amplified polymorphic DNA genotyping method for field isolates of Dermatophilus congolensis.

    Science.gov (United States)

    Larrasa, J; Garcia, A; Ambrose, N C; Alonso, J M; Parra, A; de Mendoza, M Hermoso; Salazar, J; Rey, J; de Mendoza, J Hermoso

    2002-04-01

    Dermatophilus congolensis is the pathogenic actinomycete that causes dermatophilosis in cattle, lumpy wool in sheep and rain scald in horses. Phenotypic variation between isolates has previously been described, but its genetic basis, extent and importance have not been investigated. Standard DNA extraction methods are not always successful for D. congolensis due to its complex life cycle, one stage of which is encapsulated. Here we describe the development of rapid and reliable DNA extraction and random amplified polymorphic DNA (RAPD) methods that can be used for genotyping D. congolensis field isolates. Our results suggest that genotypic variation between isolates correlates with host species. Several DNA extraction methods and RAPD protocols were compared. An extraction method based on incubation of the bacterium in lysozyme, sodium dodecyl sulphate (SDS) and proteinase K treatments and phenolic extraction yielded high-quality DNA, which was used to optimize RAPD-polymerase chain reaction (PCR) protocols for two random primers. An alternative rapid, non-phenolic extraction method based on proteinase K treatment and thermal shock was selected for routine RAPD typing of isolates. DNA extracted from reference strains from cattle, sheep and horse using either method gave reproducible banding patterns with different DNA batches and different thermal cyclers. The rapid DNA extraction method and RAPD-PCR were applied to 38 D. congolensis field isolates. The band patterns of the field and type isolates correlated with host species but not with geographical location.

  12. Radiation resistance of bacterial populations, isolated from the environment of the radiation sterilization plant type JS-6900, Debrecen

    International Nuclear Information System (INIS)

    Gazso, L.; Igali, S.; Daroczy, E.

    1978-01-01

    The radiation resistance of bacterial populations isolated from the air of an industrial sterilization plant loaded with an activity of 250000 Ci was investigated before loading and a year after loading with 60 Co. The mean D 10 value of the 23 strains isolated before loading was 73 krad with a maximum of 173 krad. The mean D 10 value of the 26 strains isolated a year after loading was 32 krad with a maximum of 156 krad. It was not possible to detect any increase in radiation resistance of bacterial populations isolated from the irradiation room after one year of running radiation sterilization of disposable medical supplies. (author)

  13. 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

  14. Biosynthesis of Bio surfactant by Egyptian Local Bacterial Isolates Using Different Agricultural Wastes

    International Nuclear Information System (INIS)

    El-Shahawy, M.R.

    2014-01-01

    Fifteen bacterial isolates were isolated from sea water from the coast of the General Petroleum Company on Suez Gulf. They were screened for bio surfactant production using emulsification activity and haemolytic activity. The most potent isolate B11 were selected according to two parameters: The ability to grow and produce surfactant and its haemolytic activity on blood agar plates. The isolate B11 was characterized and identified as Bacillus licheniformis according to API system. The isolate was subjected to different doses of gamma irradiation in a trial to improve its ability for bio surfactant production which resulted in a passive effect on bio surfactant production. Three types of agricultural wastes (Rice straw, Cane Bagasse, Corn straw) were used as fertilizers for bio surfactant biosynthesis by the promising isolate in concentrations of 1, 2, 3, 4, 5 g/l. At five g/l concentration cane bagasse gave high production of bio surfactant with maximum capacity at (32%) flowed by rice straw at 18% and corn straw at 9.8 %.

  15. cDNA cloning and bacterial expression of an endo-β-1,4-mannanase, AkMan, from Aplysia kurodai

    OpenAIRE

    Zahura, Umme Afsari; Rahman, Mohammad Matiur; Inoue, Akira; Tanaka, Hiroyuki; Ojima, Takao

    2011-01-01

    Previously we isolated an endo-β-1,4-mannanase (EC 3.2.1.78), AkMan, from the digestive fluid of a common sea hare Aplysia kurodai and demonstrated that this enzyme had a broad pH optimum spanning 4.0 to 7.5 and an appreciably high heat stability in this pH range (Zahura et al., Comp. Biochem. Physiol., B157, 137-148 (2010)). In the present study, we cloned the cDNA encoding AkMan and constructed a bacterial expression system for this enzyme to enrich information about the primary structure a...

  16. 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.

  17. 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...

  18. Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning

    International Nuclear Information System (INIS)

    Han, J.H.; Stratowa, C.; Rutter, W.J.

    1987-01-01

    The authors have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy using [ 32 P]-labelled nucleotides. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase

  19. Bacteriophages isolated from Lake Michigan demonstrate broad host-range across several bacterial phyla.

    Science.gov (United States)

    Malki, Kema; Kula, Alex; Bruder, Katherine; Sible, Emily; Hatzopoulos, Thomas; Steidel, Stephanie; Watkins, Siobhan C; Putonti, Catherine

    2015-10-09

    The study of bacteriophages continues to generate key information about microbial interactions in the environment. Many phenotypic characteristics of bacteriophages cannot be examined by sequencing alone, further highlighting the necessity for isolation and examination of phages from environmental samples. While much of our current knowledge base has been generated by the study of marine phages, freshwater viruses are understudied in comparison. Our group has previously conducted metagenomics-based studies samples collected from Lake Michigan - the data presented in this study relate to four phages that were extracted from the same samples. Four phages were extracted from Lake Michigan on the same bacterial host, exhibiting similar morphological characteristics as shown under transmission electron microscopy. Growth characteristics of the phages were unique to each isolate. Each phage demonstrated a host-range spanning several phyla of bacteria - to date, such a broad host-range is yet to be reported. Genomic data reveals genomes of a similar size, and close similarities between the Lake Michigan phages and the Pseudomonas phage PB1, however, the majority of annotated genes present were ORFans and little insight was offered into mechanisms for host-range. The phages isolated from Lake Michigan are capable of infecting several bacterial phyla, and demonstrate varied phenotypic characteristics despite similarities in host preference, and at the genomic level. We propose that such a broad host-range is likely related to the oligotrophic nature of Lake Michigan, and the competitive benefit that this characteristic may lend to phages in nature.

  20. Increasing Incidence of Multidrug Resistance Among Cystic Fibrosis Respiratory Bacterial Isolates.

    Science.gov (United States)

    Rutter, W Cliff; Burgess, Donna R; Burgess, David S

    2017-01-01

    Pseudomonas aeruginosa and Staphylococcus aureus are common pathogens in cystic fibrosis (CF) patients with increasing multidrug resistance (MDR). This study characterized antimicrobial susceptibility trends among organisms isolated from the respiratory tract of CF patients. Microbiological culture and sensitivity results for all CF patients were collected from January 2010 through December 2014. Minimum inhibitory concentrations were obtained using Phoenix ® and Etest ® methods. Clinical and Laboratory Standards Institute guidelines were used to remove duplicate isolates and develop antimicrobial susceptibility reports. MDR was defined as resistance to one agent in three or more antibiotic classes or oxacillin resistance in S. aureus. Overall, 542 bacterial isolates from 376 cultures were analyzed for trends. P. aeruginosa (41%), S. aureus (40%), and Stenotrophomonas maltophilia (8%) were the most commonly isolated organisms. Multidrug-resistant organism isolation increased from 39% to 49% (r = 0.76, p = 0.13), while representing 47.6% of all isolates. Multidrug-resistant P. aeruginosa incidence increased each year from 26% to 43% (r = 0.89, p = 0.046), while P. aeruginosa isolation decreased from 47% to 38% over the study period (r = -0.93, p = 0.02). MRSA accounted for 62.6% of all S. aureus isolated, while overall multidrug-resistant S. aureus incidence was 73.1% in all cultures. MDR among common pathogens in CF continues to increase. Empiric therapy for CF exacerbations should be targeted to previous antimicrobial susceptibility, and P. aeruginosa and S. aureus should be empirically covered.

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

    Directory of Open Access Journals (Sweden)

    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%.

  2. NORMAL VAGINAL BACTERIAL FLORA OF GIANT PANDAS (AILUROPODA MELANOLEUCA) AND THE ANTIMICROBIAL SUSCEPTIBILITY PATTERNS OF THE ISOLATES.

    Science.gov (United States)

    Yang, Xin; Yang, Jiang; Wang, Hongning; Li, Caiwu; He, Yongguo; Jin, SenYan; Zhang, Hemin; Li, Desheng; Wang, Pengyan; Xu, Yuesong; Xu, Changwen; Fan, Chengyun; Xu, Lulai; Huang, Shan; Qu, Chunmao; Li, Guo

    2016-06-01

    To study the typical vaginal bacterial flora of giant pandas (Ailuropoda melanoleuca), we took vaginal swabs for the sake of bacterial isolation, from 24 healthy female giant pandas. A total of 203 isolates were identified, representing a total of 17 bacterial species. The most common bacteria isolated were Lactobacillus spp. (54.2%, 13/24), followed by Staphylococcus epidermidis (41.7%, 10/24) and Escherichia coli (33.3%, 8/24). Some opportunistic pathogenic bacteria, such as Peptostreptococcus spp., Klebsiella pneumoniae , and Proteus mirabilis , were also isolated but showed no pathology. Antimicrobial susceptibility testing of aerobic bacterial isolates was performed with the disk diffusion method. Of the 152 isolates, resistance was most frequently observed with chloramphenicol (17.8%), followed by tetracycline (14.5%), ciprofloxacin (12.5%), streptomycin (11.8%), and florfenicol (11.8%), whereas 7.2% were multidrug resistant. This is the first report of the normal culturable vaginal bacterial flora of giant pandas and the antimicrobial susceptibility patterns of the isolates.

  3. Antimicrobial drug resistance among clinically relevant bacterial isolates in sub-Saharan Africa: a systematic review.

    Science.gov (United States)

    Leopold, Stije J; van Leth, Frank; Tarekegn, Hayalnesh; Schultsz, Constance

    2014-09-01

    Little is known about the prevalence of antimicrobial resistance (AMR) amongst bacterial pathogens in sub-Saharan Africa (sSA), despite calls for continent-wide surveillance to inform empirical treatment guidelines. We searched PubMed and additional databases for susceptibility data of key pathogens for surveillance, published between 1990 and 2013. Extracted data were standardized to a prevalence of resistance in populations of isolates and reported by clinical syndrome, microorganism, relevant antimicrobial drugs and region. We identified 2005 publications, of which 190 were analysed. Studies predominantly originated from east sSA (61%), were hospital based (60%), were from an urban setting (73%) and reported on isolates from patients with a febrile illness (42%). Quality procedures for susceptibility testing were described in resistance to chloramphenicol in Enterobacteriaceae, isolated from patients with a febrile illness, ranged between 31.0% and 94.2%, whilst MP of resistance to third-generation cephalosporins ranged between 0.0% and 46.5%. MP of resistance to nalidixic acid in Salmonella enterica Typhi ranged between 15.4% and 43.2%. The limited number of studies providing prevalence data on AMR in Gram-positive pathogens or in pathogens isolated from patients with a respiratory tract infection, meningitis, urinary tract infection or hospital-acquired infection suggested high prevalence of resistance to chloramphenicol, trimethoprim/sulfamethoxazole and tetracycline and low prevalence to third-generation cephalosporins and fluoroquinolones. Our results indicate high prevalence of AMR in clinical bacterial isolates to antimicrobial drugs commonly used in sSA. Enhanced approaches for AMR surveillance are needed to support empirical therapy in sSA. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  4. Bacterial succession during curing process of a skate (Dipturus batis) and isolation of novel strains.

    Science.gov (United States)

    Reynisson, E; Thornór Marteinsson, V; Jónsdóttir, R; Magnússon, S H; Hreggvidsson, G O

    2012-08-01

    To study the succession of cultivated and uncultivated microbes during the traditional curing process of skate. The microbial diversity was evaluated by sequencing 16Sr RNA clone libraries and cultivation in variety of media from skate samples taken periodically during a 9-day curing process. A pH shift was observed (pH 6·64-9·27) with increasing trimethylamine (2·6 up to 75·6 mg N per 100 g) and total volatile nitrogen (TVN) (from 58·5 to 705·8 mg N per 100 g) but with relatively slow bacterial growth. Uncured skate was dominated by Oceanisphaera and Pseudoalteromonas genera but was substituted after curing by Photobacterium and Aliivibrio in the flesh and Pseudomonas on the skin. Almost 50% of the clone library is derived from putative undiscovered species. Cultivation and enrichment strategies resulted in isolation of putatively new species belonging to the genera Idiomarina, Rheinheimera, Oceanisphaera, Providencia and Pseudomonas. The most abundant genera able to hydrolyse urea to ammonia were Oceanisphaera, Psychrobacter, Pseudoalteromonas and isolates within the Pseudomonas genus. The curing process of skate is controlled and achieved by a dynamic bacterial community where the key players belong to Oceanisphaera, Pseudoalteromonas, Photobacterium, Aliivibrio and Pseudomonas. For the first time, the bacterial population developments in the curing process of skate are presented and demonstrate a reservoir of many yet undiscovered bacterial species. No Claim to Norwegian Government works Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

  5. Pharmacogenetic testing after a simple DNA isolation method on buccal swab samples.

    Science.gov (United States)

    Wijnen, Petal A H M; Drent, Marjolein; van Dieijen-Visser, Marja P; Bekers, Otto

    2009-06-01

    To evaluate whether the quality and quantity of DNA isolated from noninvasively obtained buccal swab (BS) samples, using the previously described isolation method for dried blood spot (DBS) samples was satisfactory. From 25 healthy volunteers, DBS samples were obtained by the capillary finger prick method and BS samples were obtained by rubbing a sterile, dry cotton swab against the inside of their cheek. Thereafter, DNA was isolated. In addition, the quantity of the obtained DNA was measured and melting curve analyses for both sampling methods were performed to establish the quality of the obtained DNA from both the DBS and BS samples. The derivative melting curves of the DNA samples obtained from the capillary blood and BS were comparable and highly reproducible. The mean DNA concentrations measured were 16.0 ng/microl (12.6-19.4 ng/microl) and 70.2 ng/microl (57.3-83.1 ng/microl), respectively, for the DBS and BS samples (p < 0.001). The DBS DNA isolation method appeared to be extremely useful to discriminate between genotypes. This expands the possibilities of this quick and easy DNA isolation procedure. In particular, the noninvasive BS sampling method appeared to be a good alternative to invasive sampling methods.

  6. Isolation of genomic DNA suitable for community analysis from mature trees adapted to arid environment.

    Science.gov (United States)

    Gupta, Amit Kumar; Harish; Rai, Manoj Kumar; Phulwaria, Mahendra; Shekhawat, Narpat Singh

    2011-11-10

    Isolation of intact and pure genomic DNA (gDNA) is essential for many molecular biology applications. It is difficult to isolate pure DNA from mature trees of hot and dry desert regions because of the accumulation of high level of polysaccharides, phenolic compounds, tannins etc. We hereby report the standardized protocol for the isolation and purification of gDNA from seven ecologically and medically important tree species of Combretaceae viz. Anogeissus (Anogeissus sericea var. nummularia, Anogeissus pendula, and Anogeissus latifolia) and Terminalia (Terminalia arjuna, Terminalia bellirica, Terminalia catappa and Terminalia chebula). This method involves (i) washing the sample twice with Triton buffer (2%) then (ii) isolation of gDNA by modified-CTAB (cetyl trimethyl ammonium bromide) method employing a high concentration (4%) of PVP (Polyvinylpyrrolidone) and 50mM ascorbic acid, and (iii) purification of this CTAB-isolated gDNA by spin-column. gDNA isolated by modified CTAB or spin-column alone were not found suitable for PCR amplification. The Triton washing step is also critical. The quality of DNA was determined by the A(260)/A(280) absorbance ratio. gDNA was also observed for its intactness by running on 0.8% agarose gel. The suitability of extracted DNA for PCR was tested by amplification with RAPD primers, which was successful. Further, rbcLa (barcoding gene) was amplified and sequenced to check the quality of extracted gDNA for its downstream applications. Copyright © 2011 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    Malatinkova, Eva; Kiselinova, Maja; Bonczkowski, Pawel; Trypsteen, Wim; Messiaen, Peter; Vermeire, Jolien; Verhasselt, Bruno; Vervisch, Karen; Vandekerckhove, Linos; De Spiegelaere, Ward

    2014-01-01

    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. 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. 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). 2-LTR circles quantification in HIV-infected patients proved to be more

  8. Studies on the repair of damaged DNA in bacteriophage, bacterial and mammalian systems. Comprehensive report, 1 February 1981-15 September 1983

    International Nuclear Information System (INIS)

    Friedberg, E.C.

    1983-01-01

    We have explored the molecular mechanism of the repair of DNA at a number of different levels of biological organization, by investigating bacteriophage, bacterial, yeast and mammalian (including human) cells. We have demonstrated that uv endonuclease of phage T4 not only possesses pyrimidine dimer (PD)-DNA glycosylase activity but also apyrimidinic (AP) endonuclease activity. The demonstration of both activities provided an explanation for the specific endonucleosytic cleavage of DNA at sites of pyrimidine dimers catalyzed by this small protein. A new apurinic/apyrimidinic (AP) endonuclease, specific for sites of of base loss in single stranded DNA has been isolated from E. celi and presumably recognizes these lesions in single stranded regions of duplex DNA. We have partially purified this enzyme and have carried out a preliminary characterization of the activity. We treated xeroderma pigmentosum and normal cells with sodium butyrate in the hope of restoring normal levels of excision repair to the former. Although this result was not obtained, we established that all cells treated with sodium butyrate show enhanced levels of repair synthesis, thus providing a means for increasing the sensitivity of this commonly used technique for measuring DNA repair in mammalian cells in culture

  9. Impact of in Situ Isolated Bacterial Strains on Nitrogen Fixation in Alfalfa

    Directory of Open Access Journals (Sweden)

    Carmen Dragomir

    2013-05-01

    Full Text Available 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 isolated nitrogen fixing bacteria in alfalfa in two ways: rhizobia taken directly from the nodosities on the alfalfa roots and rhizobia taken from the alfalfa root system.

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

    Directory of Open Access Journals (Sweden)

    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

  11. Bacterial transposons are co-transferred with T-DNA to rice chromosomes during Agrobacterium-mediated transformation.

    Science.gov (United States)

    Kim, Sung-Ryul; An, Gynheung

    2012-06-01

    Agrobacterium tumefaciens is widely utilized for delivering a foreign gene into a plant's genome. We found the bacterial transposon Tn5393 in transgenic rice plants. Analysis of the flanking sequences of the transferred-DNA (T-DNA) identified that a portion of the Tn5393 sequence was present immediately next to the end of the T-DNA. Because this transposon was present in A. tumefaciens strain LBA4404, but not in EHA105 and GV3101, our findings indicated that Tn5393 was transferred from LBA4404 into the rice genome during the transformation process. We also noted that another bacterial transposon, Tn5563, is present in transgenic plants. Analyses of 331 transgenic lines revealed that 26.0% carried Tn5393 and 2.1% contained Tn5563. In most of the lines, an intact transposon was integrated into the T-DNA and transferred to the rice chromosome. More than one copy of T-DNA was introduced into the plants, often at a single locus. This resulted in T-DNA repeats of normal and transposon-carrying TDNA that generated deletions of a portion of the T-DNA, joining the T-DNA end to the bacterial transposon. Based on these data, we suggest that one should carefully select the appropriate Agrobacterium strain to avoid undesirable transformation of such sequences.

  12. Distribution, antibiotic susceptibility and tolerance of bacterial isolates in culture-positive cases of endocarditis in The Netherlands

    NARCIS (Netherlands)

    van der Meer, J. T.; van Vianen, W.; Hu, E.; van Leeuwen, W. B.; Valkenburg, H. A.; Thompson, J.; Michel, M. F.

    1991-01-01

    During a two-year period data were collected nationwide in The Netherlands on 438 episodes of bacterial endocarditis (BE) in 432 patients. Of the strains isolated in these patients 419 were available for analysis. Of these, 326 were isolated in native valve endocarditis (NVE) and 93 in prosthetic

  13. 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

    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...

  14. Systematic biases in DNA copy number originate from isolation procedures

    NARCIS (Netherlands)

    van Heesch, S.; Mokry, M.; Boskova, V.; Junker, W.; Mehon, R.; Toonen, P.; de Bruijn, E.; Shull, J.D.; Aitman, T.J.; Cuppen, E.; Guryev, V.

    2013-01-01

    BACKGROUND: The ability to accurately detect DNA copy number variation in both a sensitive and quantitative manner is important in many research areas. However, genome-wide DNA copy number analyses are complicated by variations in detection signal. RESULTS: While GC content has been used to correct

  15. A simple, rapid and efficient method of isolating DNA from ...

    African Journals Online (AJOL)

    Total DNA of Chokanan mango (Mangifera indica L.) was extracted from the leaf for the construction of total genomic library. However, the quality of the extracted DNA was often compromised by the presence of secondary metabolites, thus interfering with the analytical applications. Improvement on the quality of the ...

  16. 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.

  17. Uptake and expression of bacterial and cyanobacterial genes by isolated cucumber etioplasts

    Energy Technology Data Exchange (ETDEWEB)

    Daniell, H.; McFadden, B.A.

    1987-09-01

    The uptake and expression by plastids isolated from dark-grown cucumber cotyledons (etioplasts) of two pUC derivatives, pCS75 and pUC9-CM, respectively carrying genes for the large and small subunits of ribulose bisphosphate carboxylase/oxygenase of Anacystis nidulans or chloramphenicol acetyltransferase, is reported. Untreated etioplasts take up only 3% as much DNA as that taken up by EDTA-washed etioplasts after 2 hr of incubation with nick-translated (/sup 32/P)-pCS75. The presence or absence of light does not affect DNA uptake, binding, or breakdown by etioplasts. Calcium or magnesium ions inhibit DNA uptake by 86% but enhance binding and breakdown of donor DNA by EDTA-treated etioplasts. Uncouplers that abolish membrane potential, transmembrane proton gradient, or both do not affect DNA uptake, binding, or breakdown by etioplasts. However, both DNA uptake and binding are severely inhibited by ATP. After the incubation of EDTA-treated etioplasts with pCS75, immunoprecipitation using antiserum to the small subunit of ribulose bisphosphate carboxylase/oxygenase from A. nidulans reveals the synthesis of small subunits. Treatment of etioplasts with 10 mM EDTA shows a 10-min duration to be optimal for the expression of chloramphenicol acetyltransferase encoded by pUC9-CM. A progressive increase in the expression of this enzyme is observed with an increase in the concentration of pUC9-CM in the DNA uptake medium. The plasmid-dependent incorporation of (/sup 35/S) methionine by EDTA-treated organelles declines markedly during cotyledon greening in vivo.

  18. cDNA fingerprinting of osteoprogenitor cells to isolate differentiation stage-specific genes.

    OpenAIRE

    Candeliere, G A; Rao, Y; Floh, A; Sandler, S D; Aubin, J E

    1999-01-01

    A cDNA fingerprinting strategy was developed to identify genes based on their differential expression pattern during osteoblast development. Preliminary biological and molecular staging of cDNA pools prepared by global amplification PCR allowed discrim-inating choices to be made in selection of expressed sequence tags (ESTs) to be isolated. Sequencing of selected ESTs confirmed that both known and novel genes can be isolated from any developmental stage of interest, e.g. from primitive progen...

  19. 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

  20. Occurrence and antibacterial susceptibility pattern of bacterial pathogens isolated from diarrheal patients in Pakistan

    Directory of Open Access Journals (Sweden)

    Muhammad H. Rasool

    2016-03-01

    Full Text Available Objective: To determine the occurrence of bacterial pathogens responsible for diarrhea and to engender information regarding the effectiveness of commonly used antibiotic against diarrhea. Methods: This cross-sectional study was conducted between April and July 2014. Samples were collected from the Divisional Headquarter and Allied Hospital, Faisalabad, Pakistan. The differential and selective media were used to isolate bacterial pathogens, which were identified through cultural characteristics, microscopy, and biochemical tests. Disc diffusion assay was carried out using Muller Hinton agar medium, and minimum inhibitory concentration was determined using broth dilution method against isolated pathogens. Results: One hundred and forty-one (100% samples were positive for some bacteria. Frequency of occurrence was Bacillus cereus (B. cereus (66%, Escherichia coli (E. coli (48.5%, Salmonella typhi (S. Typhi (27.7%, Pseudomonas aeruginosa (P. aeruginosa (8.5%, and Staphylococcus aureus (S. aureus (4.3%. Single pathogen was detected in 20 (14.2% samples whereas combinations were found in 121 (85.8% samples. Bacillus cereus and E. coli were the most frequently detected pathogens followed by the S. Typhi, P. aeruginosa, and Staph. aureus. The percentage occurrence of isolated pathogens was 31% in B. cereus, 31% in E. coli, 18% in S. Typhi, 5% in P. aeruginosa, and 3% in Staph. aureus. Conclusion: Pseudomonas aeruginosa showed resistance against Amoxicillin and Cefotaxime, whereas S. aureus was found resistant against Cefotaxime. Statistical analysis using one way Analysis of Variance revealed that Ofloxacin and Gentamicin had significant (p<0.05 differences against all isolates as compared with other antibiotics used in this study.

  1. Isolation, screening and molecular identification of novel bacterial strain removing methylene blue from water solutions

    Science.gov (United States)

    Kilany, Mona

    2017-11-01

    The potentially deleterious effects of methylene blue (MB) on human health drove the interest in its removal promptly. Bioremediation is an effective and eco friendly for removing MB. Soil bacteria were isolated and examined for their potential to remove MB. The most potent bacterial candidate was characterized and identified using 16S rRNA sequence technique. The evolutionary history of the isolate was conducted by maximum likelihood method. Some physiochemical parameters were optimized for maximum decolorization. Decolorization mechanism and microbial toxicity study of MB (100 mg/l) and by-products were investigated. Participation of heat killed bacteria in color adsorption have been investigated too. The bacterial isolate was identified as Stenotrophomonas maltophilia strain Kilany_MB 16S ribosomal RNA gene with 99% sequence similarity. The sequence was submitted to NCBI (Accession number = KU533726). Phylogeny depicted the phylogenetic relationships between 16S ribosomal RNA gene, partial sequence (1442 bp), of the isolated strain and other strains related to Stenotrophomonas maltophilia in the GenBank database. The optimal conditions were investigated to be pH 5 at 30 °C, after 24 h using 5 mg/l MB showing optimum decolorization percentage (61.3%). Microbial toxicity study demonstrated relative reduction in the toxicity of MB decolorized products on test bacteria. Mechanism of color removal was proved by both biosorption and biodegradation, where heat-killed and live cells showed 43 and 52% of decolorization, respectively, as a maximum value after 24-h incubation. It was demonstrated that the mechanism of color removal is by adsorption. Therefore, good performance of S maltophilia in MB color removal reinforces the exploitation of these bacteria in environmental clean-up and restoration of the ecosystem.

  2. Results of the first external quality assessment scheme (EQA) for isolation and analysis of circulating tumour DNA (ctDNA).

    Science.gov (United States)

    Haselmann, Verena; Ahmad-Nejad, Parviz; Geilenkeuser, Wolf J; Duda, Angelika; Gabor, Merle; Eichner, Romy; Patton, Simon; Neumaier, Michael

    2018-01-26

    Circulating tumour DNA (ctDNA) is considered to have a high potential for future management of malignancies. This pilot external quality assessment (EQA) scheme aimed to address issues of analytical quality in this new area of laboratory diagnostics. The EQA scheme consisted of three 2-mL EDTA-plasma samples spiked with fragmented genomic DNA with a mutant allele frequency ranging from 0% to 10% dedicated to the analysis of nine known sequence variations in KRAS codon 12/13 and of BRAF V600E. Laboratories reported: (1) time elapsed for processing, (2) storage temperatures, (3) methods for extraction and quantification, (4) genotyping methodologies and (5) results. Specimens were sent to 42 laboratories from 10 European countries; 72.3% reported to isolate cell-free DNA (cfDNA) manually, 62.5% used the entire plasma volume for cfDNA isolation and 38.5% used >10% of cfDNA extracted for downstream genotyping. Of the methods used for quantification, PicoGreen demonstrated the lowest coefficient of variation (33.7%). For genotyping, 11 different methods were reported with the highest error rate observed for Sanger sequencing and the lowest for highly sensitive approaches like digital PCR. In total, 197 genotypes were determined with an overall error rate of 6.09%. This pilot EQA scheme illustrates the current variability in multiple phases of cfDNA processing and analysis of ctDNA resulting in an overall error rate of 6.09%. The areas with the greatest variance and clinical impact included specimen volume, cfDNA quantification method, and preference of genotyping platform. Regarding quality assurance, there is an urgent need for harmonisation of procedures and workflows.

  3. A protocol for large scale genomic DNA isolation for cacao genetics ...

    African Journals Online (AJOL)

    Advances in DNA technology, such as marker assisted selection, detection of quantitative trait loci and genomic selection also require the isolation of DNA from a large number of samples and the preservation of tissue samples for future use in cacao genome studies. The present study proposes a method for the ...

  4. Non liquid nitrogen-based-method for isolation of DNA from ...

    African Journals Online (AJOL)

    A simple, efficient, reliable and cost-effective method for isolation of total genomic DNA from fungi, suitable for polymerase chain reaction (PCR) amplification and other molecular applications was described. The main advantages of the method are: (1) does not require the use of liquid nitrogen for preparation of fungi DNA; ...

  5. A rapid and low-cost DNA extraction method for isolating ...

    African Journals Online (AJOL)

    The price of commercial DNA extraction methods makes the routine use of polymerase chain reaction amplification (PCR) based methods rather costly for scientists in developing countries. A guanidium thiocayante-based DNA extraction method was investigated in this study for the isolation of Escherichia coli (E. coli) DNA ...

  6. 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...

  7. Solid-phase DNA isolation from food matrices using hydrophilic magnetic microspheres

    Czech Academy of Sciences Publication Activity Database

    Trachtová, Š.; Španová, A.; Tóth, J.; Prettl, Z.; Horák, Daniel; Gyenis, J.; Rittich, B.

    2015-01-01

    Roč. 94, April (2015), s. 375-381 ISSN 0960-3085 R&D Projects: GA ČR GAP206/12/0381 Institutional support: RVO:61389013 Keywords : DNA compaction * magnetic microspheres * DNA isolation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.687, year: 2015

  8. A protocol for large scale genomic DNA isolation for cacao genetics ...

    African Journals Online (AJOL)

    aghomotsegin

    2014-02-12

    Feb 12, 2014 ... 3Universidade Estadual de Santa Cruz (UESC), 45662-900 Ilhéus-BA, Brazil. Accepted 5 January, 2014. Advances in DNA technology, such as marker assisted selection, detection of quantitative trait loci and genomic selection also require the isolation of DNA from a large number of samples and the.

  9. Chitosanase purified from bacterial isolate Bacillus licheniformis of ruined vegetables displays broad spectrum biofilm inhibition.

    Science.gov (United States)

    Muslim, Sahira Nsayef; Al-Kadmy, Israa M S; Hussein, Nadheema Hammood; Mohammed Ali, Alaa Naseer; Taha, Buthainah Mohammed; Aziz, Sarah Naji; Kheraif, Abdulaziz Abdullah Al; Divakar, Darshan Devang; Ramakrishnaiah, Ravikumar

    2016-11-01

    A number of bacterial species produces chitosanases which has variety of applications because of its high biodegradability, non-toxicity and antimicrobial assets. In the present study chitosanase is purified from new bacterial species Bacillus licheniformis from spoiled vegetable. This novel strain of Bacillus licheniformis isolated from spoilt cucumber and pepper samples has the ability to produce the chitosanase enzyme when grown on chitosan substrate. Study also examined its antibiofilm properties against diverse bacterial species with biofilm forming ability. The purified chitosanase inhibited the biofilm formation ability for all Gram-negative and Gram-positive biofilm-forming bacteria [biofilm producers] tested in this study in congo red agar and microtiter plate's methods. Highly antibiofilm activity of chitosanase was recorded against Pseudomonas aeruginosa followed by Klebsiella pneumoniae with reduction of biofilm formation upto 22 and 29%, respectively compared with [100] % of control. Biofilm formation has multiple role including ability to enhance resistance and self-protection from external stress. This chitosanase has promising benefit as antibiofilm agent against biofilm forming pathogenic bacteria and has promising application as alternative antibiofilm agents to combat the growing number of multidrug resistant pathogen-associated infections, especially in situation where biofilms are involved. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Dark fermentative biohydrogen production by mesophilic bacterial consortia isolated from riverbed sediments

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Sneha; Sudhakaran, Anu K.; Sarma, Priyangshu Manab; Subudhi, Sanjukta; Mandal, Ajoy Kumar; Lal, Banwari [Environmental and Industrial Biotechnology Division, The Energy and Resources Institute (TERI), Habitat Place, Darbari Seth Block, Lodhi Road, New Delhi 110003 (India); Gandham, Ganesh [Hindustan Petroleum Corporation Limited, Mumbai Refinery, B. D. Patil Marg, Mahul, Mumbai 400074 (India)

    2010-10-15

    Dark fermentative bacterial strains were isolated from riverbed sediments and investigated for hydrogen production. A series of batch experiments were conducted to study the effect of pH, substrate concentration and temperature on hydrogen production from a selected bacterial consortium, TERI BH05. Batch experiments for fermentative conversion of sucrose, starch, glucose, fructose, and xylose indicated that TERI BH05 effectively utilized all the five sugars to produce fermentative hydrogen. Glucose was the most preferred carbon source indicating highest hydrogen yields of 22.3 mmol/L. Acetic and butyric acid were the major soluble metabolites detected. Investigation on optimization of pH, temperature, and substrate concentration revealed that TERI BH05 produced maximum hydrogen at 37 C, pH 6 with 8 g/L of glucose supplementation and maximum yield of hydrogen production observed was 2.0-2.3 mol H{sub 2}/mol glucose. Characterization of TERI BH05 revealed the presence of two different bacterial strains showing maximum homology to Clostridium butyricum and Clostridium bifermentans. (author)

  11. Isolation and purification of the environmental DNA from the horizon of the capping: stratification study

    OpenAIRE

    Zahradník, Jiří

    2011-01-01

    According to our contemporary knowledge soil is the most abundant source of microbial biomass. Unfortunately, only one percent of the microorganism species is available by classical cultivation techniques. Soil metagenomic DNA is a collection of the whole DNA including also uncultivated microorganism in the soil sample and provides information to study molecular aspects of microorganism and their DNA sequences inaccessible by other techniques. This work is focused on characterization and isol...

  12. 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). Copyright © 2017 Elsevier B.V. All rights reserved.

  13. In vitro susceptibility of gram-negative bacterial isolates to chlorhexidine gluconate.

    Science.gov (United States)

    Mengistu, Y; Erge, W; Bellete, B

    1999-05-01

    To investigate the susceptibility of clinical isolates of gram-negative bacteria to chlorhexidine gluconate. Prospective laboratory study. Tikur Anbessa Hospital, Addis Ababa, Ethiopia. Clinical specimens from 443 hospital patients. Significant number of gram negative bacteria were not inhibited by chlorhexidine gluconate (0.02-0.05%) used for antisepsis. Four hundred and forty three strains of gram-negative bacteria were isolated from Tikur Anbessa Hospital patients. Escherichia coli (31.6%) and Klebsiella pneumoniae (23%) were the most frequently isolated bacteria followed by Proteus species (13.3%), Pseudomonas species (9.2%), and Citrobacter species (6.1%). Each organism was tested to chlorhexidine gluconate (CHG), minimum inhibitory concentration (MIC) ranging from 0.0001% to 1%w/v. All Salmonella species and E. coli were inhibited by CHG, MIC or = 0.1%). Our results showed that a significant number of the gram-negative bacterial isolates were not inhibited by CHG at the concentration used for disinfection of wounds or instruments (MIC 0.02-0.05% w/v). It is therefore important to select appropriate concentration of this disinfectant and rationally use it for disinfection and hospital hygiene. Continuing follow up and surveillance is also needed to detect resistant bacteria to chlorhexidine or other disinfectants in time.

  14. Cadmium Bio sorption by Some Bacterial Isolates and Their Mutants Induced by gamma Radiation

    International Nuclear Information System (INIS)

    Tawfik, Z.S.; Elsonbaty, S.M.; Abdalla, N.M.

    1999-01-01

    Cadmium bio sorption by bacterial cells is recognized as a potential alternative to existing recovery technologies. Bacterial strains under investigation were isolated from air surrounding gamma industrial facility Co 60 source of the NCRRT, Cairo. The effect of different concentrations of cadmium on the growth was determined for the spore forming bacteria B.coagulans, B.megaterium, B.pumilus, B.pantothenticus, and also for Staphylo coccus aureus, the reference standard strain used in these study for comparison was B.subtilis MERK 10646. The results indicated that, B.pantothenticus was the most tolerant isolate, and it can resist up to 400 ppm. Cadmium capacity for B.subtilis parent strain was increased through the influence of different doses of gamma radiation, selected mutant of B.subtilis show enhanced level of cadmium accumulation. The effect of environmental parameters as ph, temperature and also the effect of biomass factor on cadmium uptake by B.pantothenticus and B.subtilis (m) was traced

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

    Directory of Open Access Journals (Sweden)

    Christine Pourcel

    Full Text Available 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.

  16. Occurrence of plasmid linked multiple drug resistance in bacterial isolates of tannery effluent.

    Science.gov (United States)

    Naraian, R; Ram, S; Kaistha, S D; Srivastava, J

    2012-12-22

    Effluents of three different tanneries (T-1, T-2, & T-3) were investigated to isolate and scrutinize antibiotic, chromate and salinity resistant bacteria. Total 18 isolates of 9 different bacterial genera were screened out and identified; some strains established in all effluents. Amongst the three effluents tested; T-1 exhibited largest population of all isolates compared to T-2 and T-3 effluents. The T-1 effluent contained largest 4.4 x10(6) cfu/ml population of Pseudomonas aeruginosa followed by 3.9 x10(6) cfu/ml in T-2 effluent. The lowest 0.7 x10(6) cfu/ml count of Aeromonas spp. was recorded in T-3 effluent. Furthermore, antibiotic susceptibility tests were performed with 7 antibiotics which include ampicillin, sulfafurazole, ciprofloxacin, norfloxacin, tetracycline and amikacin. Three strains of P. aeruginosa and one strain of Escherichia coli deserved as multiple drug resistant (MDR). The P. aeruginosaT-3 and E. coliT-1 showed strongest MDR feature for 5 antibiotics. The response of chromate (50, 100, 200, 250 and 300 μg/ml) and NaCl concentrations (20, 40, 60 and 80 g/l) was incredible for 4 MDR isolates. Nearly each strain showed tolerance up to 300 μg/ml of chromate and 80 g/l of NaCl. The P. aeruginosaT-1, P. aeruginosaT-2, P. aeruginosaT-3 and E. coliT-1 were most tolerant isolates. Plasmid profiling of resistant strains was conducted with agarose gel electrophoresis. As consequence, plasmids from two strains of P. aeruginosa and E. coliT-1 represented different bands. At least for confirmation of plasmids nature; these were transformed and transformants were screened on medium having antibiotics. The study of plasmid transformation has confirmed the plasmid mediated resistance in isolates.

  17. Role of special cross-links in structure formation of bacterial DNA polymer

    Science.gov (United States)

    Agarwal, Tejal; Manjunath, G. P.; Habib, Farhat; Lakshmi Vaddavalli, Pavana; Chatterji, Apratim

    2018-01-01

    Using data from contact maps of the DNA-polymer of Escherichia coli (E. Coli) (at kilobase pair resolution) as an input to our model, we introduce cross-links between monomers in a bead-spring model of a ring polymer at very specific points along the chain. Via suitable Monte Carlo simulations, we show that the presence of these cross-links leads to a particular organization of the chain at large (micron) length scales of the DNA. We also investigate the structure of a ring polymer with an equal number of cross-links at random positions along the chain. We find that though the polymer does get organized at the large length scales, the nature of the organization is quite different from the organization observed with cross-links at specific biologically determined positions. We used the contact map of E. Coli bacteria which has around 4.6 million base pairs in a single circular chromosome. In our coarse-grained flexible ring polymer model, we used 4642 monomer beads and observed that around 80 cross-links are enough to induce the large-scale organization of the molecule accounting for statistical fluctuations caused by thermal energy. The length of a DNA chain even of a simple bacterial cell such as E. Coli is much longer than typical proteins, hence we avoided methods used to tackle protein folding problems. We define new suitable quantities to identify the large scale structure of a polymer chain with a few cross-links.

  18. 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.

  19. Bacterial isolates associated with pelvic inflammatory disease among female patients attending some hospitals in abuja, Nigeria.

    Science.gov (United States)

    Spencer, T H I; Umeh, P O; Irokanulo, E; Baba, M M; Spencer, B B; Umar, A I; Ardzard, S A; Oderinde, S; Onoja, O

    2014-01-01

    Pelvic inflammatory disease refers to any infection in the female lower reproductive tract that spreads to the upper reproductive tract. The disease comprises a spectrum of inflammatory disorders of the upper female genital tract, including any combination of endometritis, salpingitis, tubo-ovarian abscess and pelvic peritonitis. PID is not a notifiable disease in most countries, so accurate statistics are not available. This situation is not in any way different here in Nigeria and more so in the Federal Capital Territory, Abuja where this research was conducted, there had never been any published report so far on PID. It therefore became pertinent that such studies be carried out to evaluate the bacterial organisms which may be associated with the disease in this part of Nigeria so that health care providers could take a better look at this affliction in women. Endocervical swabs totalling 100 were aseptically collected from patients with confirmed Pelvic Inflammatory Disease (PID), attending some hospitals in Abuja, Nigeria for detection of bacterial pathogens based on cultural and biochemical characterisation tests. Antibiogram was also conducted on the identified bacterial isolates. Out of the 100 samples analysed, 43% yielded pure cultures of bacterial isolates, 2% yielded mixed cultures while no bacterial growths were recorded from the remaining 55% samples. Organisms encountered were Staphylococcus aureus (16%), Escherichia coli (10%), Streptococcus faecalis (8%), Pseudomonas aeruginosa (4%), Streptococcus pyogenes (3%), Klebsiella pneumoniae (3%), Proteus rettgeri (2%) and Proteus mirabilis (1%). The highest percentage occurrence of pathogenic isolates was observed in polygamous married patients (90%). The age group most affected falls within the mean age 30.5 years (68%) while the least affected group falls within the mean age 40.5 years (5%). There was a significant difference in the acquisition of PID in relation to marital status (P disease with

  20. Characterization of Botrytis cinerea isolates from chickpea: DNA ...

    African Journals Online (AJOL)

    Eight isolates of Botrytis cinerea, causal organism of chickpea from eight different locations of western and eastern Indo-Gangetic Plains (IGP) of India were analyzed for morphological, cultural, virulence and genetic variations. Characterization of virulence and genetic variations of the isolates was based on their ...

  1. Isolation of identical nitrilase genes from multiple bacterial strains and real-time PCR detection of the genes from soils provides evidence of horizontal gene transfer.

    Science.gov (United States)

    Coffey, Lee; Clarke, Adrienne; Duggan, Patrick; Tambling, Karen; Horgan, Serenia; Dowling, David; O'Reilly, Catherine

    2009-10-01

    Bacterial enzymes capable of nitrile hydrolysis have significant industrial potential. Microbacterium sp. AJ115, Rhodococcus erythropolis AJ270 and AJ300 were isolated from the same location in England and harbour identical nitrile hydratase/amidase gene clusters. Strain AJ270 has been well studied due to its nitrile hydratase and amidase activity. R. erythropolis ITCBP was isolated from Denmark and carries a very similar nitrile hydratase/amidase gene cluster. In this study, an identical nitrilase gene (nit1) was isolated from the four strains, and the nitrilase from strain AJ270 cloned and expressed in Escherichia coli. Analysis of the recombinant nitrilase has shown it to be functional with activity demonstrated towards phenylacetonitrile. A real-time PCR TaqMan assay was developed that allowed nit1 detection directly from soil enrichment cultures without DNA extraction, with nit1 detected in all samples tested. Real-time PCR screening of isolates from these soils resulted in the isolation of nit1 and also very similar nitrilase gene nit2 from a number of Burkholderia sp. The genes nit1 and nit2 have also been detected in many bacteria of different genera but are unstable in these isolates. It is likely that the genes were acquired by horizontal gene transfer and may be wide-spread in the environment.

  2. Influence of DNA isolation on Q-PCR-based quantification of methanogenic Archaea in biogas fermenters.

    Science.gov (United States)

    Bergmann, I; Mundt, K; Sontag, M; Baumstark, I; Nettmann, E; Klocke, M

    2010-03-01

    Quantitative real-time PCR (Q-PCR) is commonly applied for the detection of certain microorganisms in environmental samples. However, some environments, like biomass-degrading biogas fermenters, are enriched with PCR-interfering substances. To study the impact of the DNA extraction protocol on the results of Q-PCR-based analysis of the methane-producing archaeal community in biogas fermenters, nine different protocols with varying cell disruption and DNA purification approaches were tested. Differences in the quantities of the isolated DNA and the purity parameters were found, with the best cell lysis efficiencies being obtained by a combined lysozyme/SDS-based lysis. When DNA was purified by sephacryl columns, the amount of DNA decreased by one log cycle but PCR inhibitors were eliminated sufficiently. In the case of detection of methanogenic Archaea, the chosen DNA isolation protocol strongly influenced the Q-PCR-based determination of 16S rDNA copy numbers. For example, with protocols including mechanical cell disruption, the 16S rDNA of Methanobacteriales were predominantly amplified (81-90% of the total 16S rDNA copy numbers), followed by the 16S rDNA of Methanomicrobiales (9-18%). In contrast, when a lysozyme/SDS-based cell lysis was applied, the 16S rDNA copy numbers determined for these two orders were the opposite (Methanomicrobiales 82-95%, Methanobacteriales 4-18%). In extreme cases, the DNA isolation method led to discrimination of some groups of methanogens (e.g. members of the Methanosaetaceae). In conclusion, for extraction of high amounts of microbial DNA with high purity from samples of biogas plants, a combined lysozyme/SDS-based cell lysis followed by a purification step with sephacryl columns is recommended. Copyright 2010 Elsevier GmbH. All rights reserved.

  3. Selective microbial genomic DNA isolation using restriction endonucleases.

    Science.gov (United States)

    Barnes, Helen E; Liu, Guohong; Weston, Christopher Q; King, Paula; Pham, Long K; Waltz, Shannon; Helzer, Kimberly T; Day, Laura; Sphar, Dan; Yamamoto, Robert T; Forsyth, R Allyn

    2014-01-01

    To improve the metagenomic analysis of complex microbiomes, we have repurposed restriction endonucleases as methyl specific DNA binding proteins. As an example, we use DpnI immobilized on magnetic beads. The ten minute extraction technique allows specific binding of genomes containing the DpnI Gm6ATC motif common in the genomic DNA of many bacteria including γ-proteobacteria. Using synthetic genome mixtures, we demonstrate 80% recovery of Escherichia coli genomic DNA even when only femtogram quantities are spiked into 10 µg of human DNA background. Binding is very specific with less than 0.5% of human DNA bound. Next Generation Sequencing of input and enriched synthetic mixtures results in over 100-fold enrichment of target genomes relative to human and plant DNA. We also show comparable enrichment when sequencing complex microbiomes such as those from creek water and human saliva. The technique can be broadened to other restriction enzymes allowing for the selective enrichment of trace and unculturable organisms from complex microbiomes and the stratification of organisms according to restriction enzyme enrichment.

  4. Propionibacterium acnes bacteriophages display limited genetic diversity and broad killing activity against bacterial skin isolates.

    Science.gov (United States)

    Marinelli, Laura J; Fitz-Gibbon, Sorel; Hayes, Clarmyra; Bowman, Charles; Inkeles, Megan; Loncaric, Anya; Russell, Daniel A; Jacobs-Sera, Deborah; Cokus, Shawn; Pellegrini, Matteo; Kim, Jenny; Miller, Jeff F; Hatfull, Graham F; Modlin, Robert L

    2012-01-01

    Investigation of the human microbiome has revealed diverse and complex microbial communities at distinct anatomic sites. The microbiome of the human sebaceous follicle provides a tractable model in which to study its dominant bacterial inhabitant, Propionibacterium acnes, which is thought to contribute to the pathogenesis of the human disease acne. To explore the diversity of the bacteriophages that infect P. acnes, 11 P. acnes phages were isolated from the sebaceous follicles of donors with healthy skin or acne and their genomes were sequenced. Comparative genomic analysis of the P. acnes phage population, which spans a 30-year temporal period and a broad geographic range, reveals striking similarity in terms of genome length, percent GC content, nucleotide identity (>85%), and gene content. This was unexpected, given the far-ranging diversity observed in virtually all other phage populations. Although the P. acnes phages display a broad host range against clinical isolates of P. acnes, two bacterial isolates were resistant to many of these phages. Moreover, the patterns of phage resistance correlate closely with the presence of clustered regularly interspaced short palindromic repeat elements in the bacteria that target a specific subset of phages, conferring a system of prokaryotic innate immunity. The limited diversity of the P. acnes bacteriophages, which may relate to the unique evolutionary constraints imposed by the lipid-rich anaerobic environment in which their bacterial hosts reside, points to the potential utility of phage-based antimicrobial therapy for acne. Propionibacterium acnes is a dominant member of the skin microflora and has also been implicated in the pathogenesis of acne; however, little is known about the bacteriophages that coexist with and infect this bacterium. Here we present the novel genome sequences of 11 P. acnes phages, thereby substantially increasing the amount of available genomic information about this phage population

  5. Antimicrobial sensitivity and frequency of DRUG resistance among bacterial strains isolated from cancer patients

    International Nuclear Information System (INIS)

    Faiz, M.; Bashir, T.

    2004-01-01

    Blood stream infections (bacteremia) is potentially life threatening. Concomitant with a change in the incidence and epidemiology of infecting organisms, there has been an increase in resistance to many antibiotic compounds. The widespread emergence of resistance among bacterial pathogens has an impact on our ability to treat patients effectively. The changing spectrum of microbial pathogens and widespread emergence of microbial resistance to antibiotic drugs has emphasized the need to monitor the prevalence of resistance in these strains. In the present study frequency of isolation of clinically significant bacteria and their susceptibility and resistance pattern against a wide range of antimicrobial drugs from positive blood cultures collected during 2001-2003 was studied. A total of 102 consecutive isolates were found with 63% gram positive and 44% gram negative strains. The dominating pathogens were Staphylococcus aureus (51%), Streptococci (31%), Pseudomonas (40%), Proteus (13%), Klebsiella (13%). The isolated strains were tested against a wide range of antibiotics belonging to cephalosporins, aminoglycosides and quinolone derivative group by disk diffusion method. It has been observed that isolated strains among gram positive and negative strains showed different level of resistance against aminoglycosides and cephalosporin group of antibiotics with gram positives showing highest number and frequency of resistance against aminoglycosides (40-50%) and cephalosporins.(35-45%) whereas cephalosporins were found to be more effective against gram negatives with low frequency of resistant strains. Cabapenem and quinolone derivative drugs were found to be most effective among other groups in both gram positive and negative strains with 23-41% strains found sensitive to these two drugs. The frequency of sensitive strains against aminoglycoside and cephalosporin in gram negative and gram positive strains were found to be decreasing yearwise with a trend towards an

  6. Bacterial Microbiota of Rice Roots: 16S-Based Taxonomic Profiling of Endophytic and Rhizospheric Diversity, Endophytes Isolation and Simplified Endophytic Community.

    Science.gov (United States)

    Moronta-Barrios, Felix; Gionechetti, Fabrizia; Pallavicini, Alberto; Marys, Edgloris; Venturi, Vittorio

    2018-02-11

    Rice is currently the most important food crop in the world and we are only just beginning to study the bacterial associated microbiome. It is of importance to perform screenings of the core rice microbiota and also to develop new plant-microbe models and simplified communities for increasing our understanding about the formation and function of its microbiome. In order to begin to address this aspect, we have performed a 16S rDNA taxonomic bacterial profiling of the rhizosphere and endorhizosphere of two high-yield rice cultivars-Pionero 2010 FL and DANAC SD20A-extensively grown in Venezuela in 2014. Fifteen putative bacterial endophytes were then isolated from surface-sterilized roots and further studied in vitro and in planta . We have then performed inoculation of rice seedlings with a simplified community composed by 10 of the isolates and we have tracked them in the course of 30 days in greenhouse cultivation. The results obtained suggest that a set was able to significantly colonize together the rice endorhizospheres, indicating possible cooperation and the ability to form a stable multispecies community. This approach can be useful in the development of microbial solutions for a more sustainable rice production.

  7. Multi-locus DNA sequencing of Toxoplasma gondii isolated from Brazilian pigs identifies genetically divergent strains

    Science.gov (United States)

    Frazão-Teixeira, E.; Sundar, N.; Dubey, J. P.; Grigg, M. E.; de Oliveira, F. C. R.

    2010-01-01

    Five Toxoplasma gondii isolates (TgPgBr1–5) were isolated from hearts and brains of pigs freshly purchased at the market of Campos dos Goytacazes, Northern Rio de Janeiro State, Brazil. Four of the five isolates were highly pathogenic in mice. Four genotypes were identified. Multi-locus PCR-DNA sequencing showed that each strain possessed a unique combination of archetypal and novel alleles not previously described in South America. The data suggest that different strains circulate in pigs destined for human consumption from those previously isolated from cats and chickens in Brazil. Further, multi-locus PCR-RFLP analyses failed to accurately genotype the Brazilian isolates due to the high presence of atypical alleles. This is the first report of multi-locus DNA sequencing of T. gondii isolates in pigs from Brazil. PMID:21051148

  8. Microbiological and molecular identification of bacterial species isolated from nasal and oropharyngeal mucosa of fuel workers in Riyadh, Saudi Arabia.

    Science.gov (United States)

    AlWakeel, Suaad S

    2017-09-01

    This study aimed to determine the bacterial species colonizing the nasal and oropharyngeal mucosa of fuel workers in Central Riyadh, Saudi Arabia on a microbiological and molecular level. Throat and nasal swab samples were obtained from 29 fuel station attendants in the period of time extending from March to May 2014 in Riyadh, Saudi Arabia. Microbiological identification techniques were utilized to identify the bacterial species isolated. Antibiotic sensitivity was assessed for each of the bacterial isolates. Molecular identification techniques based on PCR analysis of specific genomic sequences was conducted and was the basis on which phylogeny representation was done for 10 randomly selected samples of the isolates. Blood was drawn and a complete blood count was conducted to note the hematological indices for each of the study participants. Nineteen bacterial species were isolated from both the nasal cavity and the oropharynx including Streptococcus thoraltensis , alpha-hemolytic streptococci, Staphylococcus hominis , coagulase-negative staphylococci, Leuconostoc mesenteroides , Erysipelothrix rhusiopathiae and several others. We found 100% sensitivity of the isolates to ciprofloxacin, cefuroxime and gentamicin. Whereas cefotaxime and azithromycin posted sensitivities of 85.7% and 91.4%, respectively. Low sensitivities (fuel products may be a contributing factor to bacterial colonization of the respiratory tract in fuel workers.

  9. Trends in antibiotic resistance in bacterial keratitis isolates from South India.

    Science.gov (United States)

    Lalitha, Prajna; Manoharan, Geetha; Karpagam, Rajaram; Prajna, Namperumalsamy V; Srinivasan, Muthiah; Mascarenhas, Jeena; Das, Manoranjan; Porco, Travis C; Lietman, Thomas M; Cevallos, Vicky; Keenan, Jeremy D

    2017-02-01

    To report trends in antibiotic resistance in cases of bacterial keratitis from a large eye hospital in South India. In this retrospective cross-sectional study, the microbiology laboratory records of patients with infectious keratitis diagnosed at an eye hospital in South India from 2002 to 2013 were reviewed to determine the proportion with antibiotic non-susceptibility. 3685 bacterial isolates had susceptibility testing performed over the 12-year period. The two most common organisms with resistance were Streptococcus pneumoniae (n=1204) and Pseudomonas aeruginosa (n=894). Antibiotic non-susceptibility was generally uncommon for these two organisms and no significant trends were detected over the course of the study. In contrast, Staphylococcus aureus (N=211) isolates demonstrated a significant increase in fluoroquinolone non-susceptibility over the 12-year study period. This coincided with a significant increase in methicillin-resistant S. aureus (MRSA) during the study period, though the increase in fluoroquinolone resistance was likewise seen in methicillin-sensitive S. aureus (MSSA). For example, ofloxacin resistance in MSSA increased from 11.1% in 2002 to 66.7% in 2013 (p=0.002). No trends were apparent for the aminoglycosides, cefazolin or vancomycin, for which in vitro non-susceptibility generally appeared to be low. Resistance to antibiotics was generally stable for infectious keratitis isolates from a large eye hospital in South India, except for S. aureus, which experienced a significant increase in fluoroquinolone resistance from 2002 to 2013. Fluoroquinolone antibiotics currently have poor in vitro activity against both MRSA and MSSA in South India and are therefore not the ideal therapy for Staphylococcal corneal ulcers. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  10. Presence of bacterial DNA and bacterial peptidoglycans in joints of patients with rheumatoid arthritis and other arthritides

    NARCIS (Netherlands)

    van der Heijden, I. M.; Wilbrink, B.; Tchetverikov, I.; Schrijver, I. A.; Schouls, L. M.; Hazenberg, M. P.; Breedveld, F. C.; Tak, P. P.

    2000-01-01

    The continuous presence of bacteria or their degraded antigens in the synovium may be involved in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to determine the presence of bacterial nucleic acids and bacterial cell wall constituents in the joints of patients with RA and

  11. 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...... to detect polymorphism within the isolates. Eleven distinct DNA profiles were obtained as follows: Of the 35 strains, 10 as profile 4; seven as profile 1; five as profile 3; three as profiles 2 and 9; two as profile 10; one as profiles 5, 6, 7, 8 and 11. Conclusions: Chicken carcasses sold in markets were...

  12. Characterization of biocontrol bacterial strains isolated from a suppressiveness-induced soil after amendment with composted almond shells.

    Science.gov (United States)

    Vida, Carmen; Cazorla, Francisco M; de Vicente, Antonio

    The improvement in soil quality of avocado crops through organic amendments with composted almond shells has a positive effect on crop yield and plant health, and enhances soil suppressiveness against the phytopathogenic fungus Rosellinia necatrix. In previous studies, induced soil suppressiveness against this pathogen was related to stimulation of Gammaproteobacteria, especially some members of Pseudomonas spp. with biocontrol-related activities. In this work, we isolated bacteria from this suppressiveness-induced amended soil using a selective medium for Pseudomonas-like microorganisms. We characterized the obtained bacterial collection to aid in identification, including metabolic profiles, antagonistic responses, hybridization to biosynthetic genes of antifungal compounds, production of lytic exoenzymatic activities and plant growth-promotion-related traits, and sequenced and compared amplified 16S rDNA genes from representative bacteria. The final selection of representative strains mainly belonged to the genus Pseudomonas, but also included the genera Serratia and Stenotrophomonas. Their biocontrol-related activities were assayed using the experimental avocado model, and results showed that all selected strains protected the avocado roots against R. necatrix. This work confirmed the biocontrol activity of these Gammaproteobacteria-related members against R. necatrix following specific stimulation in a suppressiveness-induced soil after a composted almond shell application. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  13. Mitochondrial DNA fingerprinting of Acanthamoeba spp. isolated from clinical and environmental sources.

    Science.gov (United States)

    Gautom, R K; Lory, S; Seyedirashti, S; Bergeron, D L; Fritsche, T R

    1994-04-01

    Restriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA fingerprinting) was evaluated as an epidemiologic tool for identifying potential reservoirs of Acanthamoeba infection. Fingerprints for 15 clinical isolates recovered by our affiliated laboratories were compared with those for 25 environmental isolates from western Washington State and 10 American Type Culture Collection (ATCC) strains. Seven different fingerprint groups emerged from the analysis of clinical isolates with six selected restriction enzymes (BamHI, BglII, EcoRI, HindIII, KpnI, and SalI). Fourteen (56%) environmental and 4 (40%) ATCC isolates displayed fingerprints similar to those of clinical isolates. In all, five of the seven groups contained one or more environmental and/or ATCC isolates. Comparisons with published mtDNA fingerprints for Acanthamoeba isolates showed that two groups have counterparts in Europe and Japan and in Europe and Australia. The inclusion of environmental isolates demonstrated that the most common clinical isolates do have counterparts readily recoverable from the surrounding environment and that some of these counterparts appear to be geographically widespread.

  14. 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.

  15. Bacterial Intoxication Evokes Cellular Senescence with Persistent DNA Damage and Cytokine Signaling

    DEFF Research Database (Denmark)

    Blazkova, Hana; Krejcikova, Katerina; Moudry, Pavel

    2009-01-01

    features shared by cells undergoing replicative or premature cellular senescence. We conclude that analogous to oncogenic, oxidative and replicative stresses, bacterial intoxication represents another pathophysiological stimulus that induces premature senescence, an intrinsic cellular response that may...... to such intoxication are mechanistically incompletely understood. Here we show that both normal and cancer cells (BJ, IMR-90 and WI-38 fibroblasts, HeLa and U2-OS cell lines) that survive the acute phase of intoxication by Haemophilus ducreyi CDT possess the hallmarks of cellular senescence. This characteristic...... phenotype included persistently activated DNA damage signaling (detected as 53BP1/gammaH2AX-positive foci), enhanced senescence-associated beta-galactosidase activity, expansion of PML nuclear compartments, and induced expression of several cytokines (especially interleukins IL-6, IL-8 and IL-24), overall...

  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. Effects of ionising radiation on isolated and cellular DNA

    International Nuclear Information System (INIS)

    Cadet, J.; Artignan, X.; Berger, M.; Douki, T.; Gromova, M.; Polverelli, M.; Ravanat, J.L.

    1997-01-01

    In the present survey, emphasis has been placed on mechanistic aspects of the radiation-induced decomposition of the base moities of DNA and model compounds. An almost complete description of the radical reactions mediated by both OH radicals (indirect effects) and one-electron oxidation (direct effects) is now possible for guanine compounds in aerated aqueous solution. In addition, the results of a comparison of a targeted assay (high performance liquid chromatography-electrochemical method) and a non specific method ('comet assay') for monitoring radiation-induced DNA damage within human cells are reported. (authors)

  18. Antimicrobial activity of endophytic bacterial populations isolated from medical plants of Iran.

    Science.gov (United States)

    Beiranvand, Maryam; Amin, Mansour; Hashemi-Shahraki, Abdolrazag; Romani, Bizhan; Yaghoubi, Sajad; Sadeghi, Parisa

    2017-02-01

    Endophytic actinobacteria colonize inside the plant tissues without causing damages to the host plant. Since these microorganisms colonize in the different parts of plants and can stop plant disease, they have been applied as biological agents for controlling human diseases. The aim of this study was molecular identification and measuring the antimicrobial activity of endophytic Actinomycete s isolated from medicinal plants of Iran. The total of 23 medicinal plant samples were collected, sterilized, and crushed. Small pieces of the crushed samples were then cultured directly on three selective media. Grown colonies were identified by 16S rRNA gene sequencing method. Each isolate was cultured in TSB medium and then antimicrobial compound was extracted using ethyl acetate and tested against the target bacteria. Sixteen out of 23 bacterial isolates (69%) exhibited antimicrobial activity against the selected pathogenic bacteria, such as Bacillus cereus, Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae, Citrobacter freundii, Proteus mirabilis, Shigella flexneri and Escherichia coli. Our Study showed a high phylogenetic diversity and the potent antibiotic activity of endophytic bacteria in medicinal plants of Iran.

  19. Antimicrobial activity of endophytic bacterial populations isolated from medical plants of Iran

    Science.gov (United States)

    Beiranvand, Maryam; Amin, Mansour; Hashemi-Shahraki, Abdolrazag; Romani, Bizhan; Yaghoubi, Sajad; Sadeghi, Parisa

    2017-01-01

    Background and Objectives: Endophytic actinobacteria colonize inside the plant tissues without causing damages to the host plant. Since these microorganisms colonize in the different parts of plants and can stop plant disease, they have been applied as biological agents for controlling human diseases. The aim of this study was molecular identification and measuring the antimicrobial activity of endophytic Actinomycetes isolated from medicinal plants of Iran. Materials and Methods: The total of 23 medicinal plant samples were collected, sterilized, and crushed. Small pieces of the crushed samples were then cultured directly on three selective media. Grown colonies were identified by 16S rRNA gene sequencing method. Each isolate was cultured in TSB medium and then antimicrobial compound was extracted using ethyl acetate and tested against the target bacteria. Results: Sixteen out of 23 bacterial isolates (69%) exhibited antimicrobial activity against the selected pathogenic bacteria, such as Bacillus cereus, Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae, Citrobacter freundii, Proteus mirabilis, Shigella flexneri and Escherichia coli. Conclusion: Our Study showed a high phylogenetic diversity and the potent antibiotic activity of endophytic bacteria in medicinal plants of Iran. PMID:28775818

  20. 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.

  1. Amylase production potentials of bacterial isolates obtained from the gut of Oryctes rhinoceros larvae

    Science.gov (United States)

    Aryati, P. C.; Pangastuti, A.; Sari, S. L. A.

    2017-04-01

    Amylase is one of the main enzymes used in industry, such as food, detergent, textile, and pharmaceutical industry. Amylase can be produced by plants, animals, and microorganisms. However, bacterial and fungal amylases have dominated application in industries. This research was aimed to determine amylolytic activity of bacteria isolated from the gut of Oryctes rhinoceros larvae. Based on clear zone formation, 9 from 11 isolates showed amylolytic activity. Isolates with the widest clear zone, i.e Bacillus subtilis GOR1, Bacillus cereus GOR3, and Bacillus pumilus GOR2, were screened for amylolytic activity based on reduction sugar production. The result showed that Bacillus subtilis GOR1 was the most potential as amylase producer, showed by the widest clear zone 5.224 cm2 and highest reduction sugar production 0.0235 mg/ml. Highest amylase specific activity (0.1447 U/mg protein) was obtained at 60°C and pH 7. Amylase activity was stable for 3 hours at 60°C with residual activity respectively was 59.7%.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. Antibiotic Resistance Pattern of Bacterial Pathogens Isolated from Cow Dung Used to Fertilize Nigerian Fish Ponds

    Directory of Open Access Journals (Sweden)

    Funso S. OMOJOWO

    2013-02-01

    Full Text Available This study was carried out to isolate and identify antibiotic resistant bacteria from cow dung used for pond fertilization. Cow dung from over 200 cows in NIFFR integrated farms, New-Bussa, Nigeria were collected. Six bacterial pathogens; Escherichia coli, E. coli O157:H7, Shigella dysentariae, Staphylococcus aureus, Salmonella typhi, and Aeromonas hydrophila were isolated. Antibiotic susceptibility testing by the disk diffusion method was conducted using ofloxacin, amoxicillin, tetracycline, ampicillin, erythromycin, gentamicin, nalidixic acid and chloramphenicol. All the isolated organisms were 100% sensitive to ofloxacin. The multiple resistance patterns revealed that 100% were resistant to tetracycline, ampicillin (85.6%, amoxicillin (83.3%, chloramphenicol (66%, gentamicin (47.6%, erythromycin (44.4% and nalidixic acid (18.3%. The Public Health risks posed by the cow dung manure include proliferation of ponds with these organisms that are pathogenic to fish and man, contamination of the environment and the possible retention of these organisms in the table size fish.

  4. 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.

  5. Identification of novel DNA binding proteins using DNA affinity chromatography-pulldown

    OpenAIRE

    Jutras, Brandon L; Verma, Ashutosh; Stevenson, Brian

    2012-01-01

    Methods are presented through which one may isolate and identify novel bacterial DNA-binding proteins. Briefly, the DNA sequence of interest is affixed to beads, then incubated with bacterial cytoplasmic extract. Washes with buffers containing non-specific DNA and low salt concentrations will remove non-adhering and low-specificity DNA-binding proteins, while subsequent washes with higher salt concentrations will elute more specific DNA-binding proteins. Eluted proteins may then be identified...

  6. 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...... 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...... 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...

  7. Optimization of FTA technology for large scale plant DNA isolation ...

    African Journals Online (AJOL)

    Conventional methods for DNA acquisition and storage require expensive reagents and equipments. Experimental fields located in remote areas and large sample size presents greater challenge to developing country institutions constrained financially. FTATM technology uses a single format utilizing basic tools found in ...

  8. Isolation and characterization of microsatellite DNA loci from Naihai ...

    Indian Academy of Sciences (India)

    The Naihai cutlassfish (Trichiurus nanhaiensis) is an impor- tant commercial fish species. In this study, we report 25 microsatellite DNA markers for T. nanhaiensis. The num- ber of alleles per locus ranged from 2 to 10 in a sample of. 37 individuals. Observed and expected heterozygosities per locus varied from 0.300 to ...

  9. Leaf storage conditions and genomic DNA isolation efficiency in ...

    African Journals Online (AJOL)

    SERVER

    2008-03-04

    Mar 4, 2008 ... 1Department of Botany, Jomo Kenyatta University of Agriculture and Technology, P. O. Box 62000, Nairobi, Kenya. 2Department of Biochemistry ... after field sampling. During the DNA extraction, four protocols were used; the ... The essential oils found in leaves, seeds, flowers and roots of Ocimum species ...

  10. Genomic DNA isolation from Artemisia species grown in cold desert ...

    African Journals Online (AJOL)

    ... protocol to extract pure genomic DNA from different Artemisia species was tailored. The protocol was based on the CTAB method with slight modifications. In the study, 1.6 M NaCl, 2% cetyltrimethyl ammonium bromide (CTAB), 3% polyvinylpyrrolidone (PVP) and 0.5% β-mercaptoethanol was used in the extraction buffer.

  11. Optimization of FTA technology for large scale plant DNA isolation ...

    African Journals Online (AJOL)

    GRACE

    2006-05-02

    May 2, 2006 ... Key words: FTATM, large-scale, DNA sampling, field set up, marker assisted selection. ... application. FTATM classic card (Whatman Inc., Clifton,. NJ) is a whatman paper that has been impregnated with a patented chemical formulation that lyses cells, .... bands for both normal agarose (data not shown) and.

  12. 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.

  13. Isolation and characterization of bacterial strains Paenibacillus sp. and Bacillus sp. for kraft lignin decolorization from pulp paper mill waste.

    Science.gov (United States)

    Chandra, Ram; Singh, Shail; Krishna Reddy, M M; Patel, D K; Purohit, Hemant J; Kapley, Atya

    2008-12-01

    Eight aerobic bacterial strains were isolated from pulp paper mill waste and screened for tolerance of kraft lignin (KL) using the nutrient enrichment technique in mineral salt media (MSM) agar plate (15 g/L) amended with different concentrations of KL (100, 200, 300, 400, 500, 600 ppm) along with 1% glucose and 0.5% peptone (w/v) as additional carbon and nitrogen sources. The strains ITRC S6 and ITRC S8 were found to have the most potential for tolerance of the highest concentration of KL. These organisms were characterized by biochemical tests and further 16S rRNA gene (rDNA) sequencing, which showed 96.5% and 95% sequence similarity of ITRC S(6) and ITRC S(8) and confirmed them as Paenibacillus sp. and Bacillus sp., respectively. KL decolorization was routinely monitored with a spectrophotometer and further confirmed by HPLC analysis. Among eight strains, ITRC S(6) and ITRC S(8) were found to degrade 500 mg/L of KL up to 47.97% and 65.58%, respectively, within 144 h of incubation in the presence of 1% glucose and 0.5% (w/v) peptone as a supplementary source of carbon and nitrogen. In the absence of glucose and peptone, these bacteria were unable to utilize KL. The analysis of lignin degradation products by GC-MS analysis revealed the formation of various acids as lignin monomers which resulted in a decrease in pH and a major change in the chromatographic profile of the bacterial degraded sample as compared to the control clear indications of biochemical modification of KL due to the bacterial ligninolytic system by ITRC S(6), namely, acetic acid, propanoic acid, butanoic acid, guaiacol, hexanoic acid, and ITRC S(8), namely acetic acid, propanoic acid, ethanedioic acid, furan carboxylic acid, 2-propanoic acid, butanoic acid, 3-acetoxybutyric acid, propanedioic acid, acetoguiacone, 1,2,3-thiadiazole, 5-carboxaldixime, 4-hydroxy-3,5-dimethoxyphenol, and dibutyl phthalate, indicating the bacterium characteristic to degrade G and S units of lignin polymer.

  14. DNA isolation by galactoacrylate-based nano-poly(HEMA-co-Gal-OPA) nanopolymers.

    Science.gov (United States)

    Türkcan Kayhan, Ceren; Zeynep Ural, Fulden; Koruyucu, Meryem; Gül Salman, Yeşim; Uygun, Murat; Aktaş Uygun, Deniz; Akgöl, Sinan; Denizli, Adil

    2017-10-01

    Isolation of DNA is one of the important processes for biotechnological applications such as investigation of DNA structures and functions, recombinant DNA preparations, identification of genetic factors and diagnosis and treatment of genetic disorders. The aim of this study was to synthesis and characterizes the galactoacrylate based nanopolymers with high surface area and to investigate the usability of these synthesized nanopolymers for DNA isolation studies. Nanopolymers were synthesized by the surfactant free emulsion polymerization technique by using the monomers of 2-hydroxyl ethylmethacrylate and 6-O-(2 ' -hydroxy-3 ' -acryloyloxypropyl)-1,2:3,4-di-O-isopropylidene-α-D-galactopyranose. Galactoacrylate origin of these newly synthesized nanopolymers increased the interaction between DNA and nanopolymers. Prepared nanopolymers were characterized by SEM, FT-IR and ZETA sizer analysis. Synthesized nanopolymers were spherical, and their average particle size was about 246.8 nm. Adsorption of DNA onto galactoacrylate based nanopolymers was investigated by using different pHs, temperatures, ionic strength, DNA concentrations and desorption studies and maximum DNA adsorption was found to be as 567.12 mg/g polymer at 25 °C, in pH 5.0 acetate buffer. Reusability was investigated for 5 successive reuse and DNA adsorption capacity decreased only about 10% at the end of the 5th reuse.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. Comparative study of Microsporum canis isolates by DNA fingerprinting.

    Science.gov (United States)

    Shafiee, Shabnam; Khosravi, Ali Reza; Ashrafi Tamai, Iradj

    2014-08-01

    Microsporum canis is a zoophilic fungus and it is an important agent of dermatophytosis. Cats act as important reservoirs. Clinically, it is too difficult to differentiate dermatophytosis caused by various species, also this fungus loses its morphological characteristics easily because of subculture; so using of rapid and accurate laboratory techniques for identifying the dermatophytes is important, therefore, RAPD-PCR was applied for the differentiation of the isolates. In this study, 10 M. canis isolates were detected in cats, dog, human, fox and rabbit at the Mycology Research Center, Faculty of Veterinary Medicine, University of Tehran. For running the RAPD-PCR, PCR set system and three random primers OPU 15, OPU 13 and OPA 04 were used. Then phylogenetic tree and similarity coefficient table were drawn. The results showed that there were some common bands between M. canis isolates. There were some specific bands for each isolates, as well. Our study showed, despite the typical morphology of the whole isolates, they were placed in different branches in molecular typing. © 2014 Blackwell Verlag GmbH.

  17. Evaluation of two real time PCR assays for the detection of bacterial DNA in amniotic fluid.

    Science.gov (United States)

    Girón de Velasco-Sada, Patricia; Falces-Romero, Iker; Quiles-Melero, Inmaculada; García-Perea, Adela; Mingorance, Jesús

    2018-01-01

    The aim of this study was to evaluate two non-commercial Real-Time PCR assays for the detection of microorganisms in amniotic fluid followed by identification by pyrosequencing. We collected 126 amniotic fluids from 2010 to 2015 for the evaluation of two Real-Time PCR assays for detection of bacterial DNA in amniotic fluid (16S Universal PCR and Ureaplasma spp. specific PCR). The method was developed in the Department of Microbiology of the University Hospital La Paz. Thirty-seven samples (29.3%) were positive by PCR/pyrosequencing and/or culture, 4 of them were mixed cultures with Ureaplasma urealyticum. The Universal 16S Real-Time PCR was compared with the standard culture (81.8% sensitivity, 97.4% specificity, 75% positive predictive value, 98% negative predictive value). The Ureaplasma spp. specific Real-Time PCR was compared with the Ureaplasma/Mycoplasma specific culture (92.3% sensitivity, 89.4% specificity, 50% positive predictive value, 99% negative predictive value) with statistically significant difference (p=0.005). Ureaplasma spp. PCR shows a rapid response time (5h from DNA extraction until pyrosequencing) when comparing with culture (48h). So, the response time of bacteriological diagnosis in suspected chorioamnionitis is reduced. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Microbiological and biochemical response of certain proteolytic bacterial isolates to varying levels of gamma irradiation

    International Nuclear Information System (INIS)

    El-Hifnawi, H.M.N.E.A.

    1997-01-01

    Amniotic membrane allo - and xeno grafts prepared from human foetal placenta, and their potential replacement of skin autotransplant, would significantly contribute to the success of clinical treatment of skin burns. Allo-and xenografts of human amniotic membrane should be ensured for their sterility, bio-mechanics and tissue antigenicity. The present study has been focused on sterilization and sterility assurance of the membrane grafts. Physico-chemical properties and antigenicity of the grafts await investigation. In the present study the isolation and identification of the bacteria contaminating the amniotic membrane allo-and xenografts prepared from human foetal placenta and the effect of gamma irradiation on it has been investigated. The proteolytic activity of these bacteria and the role of gamma irradiation in the control of bacterial activity were similarly investigated

  19. Profiling of antibiotic resistance of bacterial species recovered from routine clinical isolates in Ethiopia.

    Science.gov (United States)

    Ten Hove, Robert-Jan; Tesfaye, Melaku; Ten Hove, Witold Frederik; Nigussie, Mesfin

    2017-06-26

    With the alarming rise in antibiotic resistance in African countries, the need for a surveillance system in the region has become pressing. The rapid expansion of data networks makes it possible to set up healthcare applications that can be both cost-efficient and effective. Large data sets are available for assessment of current antibiotic resistance among Ethiopian patients. Based on the data-presentation, a practical approach is proposed on how diagnostic laboratories can participate remedial action against antibiotic resistance in Ethiopia. In Addis Ababa (Ethiopia), raw data comprising bacterial species name, specimen type and antibiograms covering the period January 2014 to May 2015 was accessed from the laboratory information management system. Using R code, the data was read and fitted into data-frames and analyzed to assess antibiotic resistance in the Ethiopian patient population. Susceptibility to an antibiotic was tested with 14.983 cultures of 54 different bacterial species or subgroups, isolated from 16 types of specimen. Half of the cultures (n = 6444) showed resistance to an antibiotic. Resistance against penicillin was highest with, on average, 91.1% of 79 bacterial cultures showing resistance. Very high resistance rates were also observed for ampicillin, whereas resistance was lowest with cefoxitin. Extraction and analysis of raw-data from the laboratory database is relatively simple and can provide valuable insight into the relationships between type of sample and drug-resistance in countries where such data is still scarce. With the largest number of antibiotic resistance tests described for Ethiopia, a tool is proposed for consistent data collection with specified core variables. Trends in antibiotic resistance can be revealed and treatment failures avoided when used as an easy accessible reference application for healthcare providers.

  20. Analysis of pyrimidine dimer content of isolated DNA by nuclease digestion

    International Nuclear Information System (INIS)

    Farland, W.H.; Sutherland, B.M.

    1980-01-01

    Isolated DNA is highly susceptible to degradation by exogenous nucleases. Complete digestion is possible with a number of well-characterized enzymes from a variety of sources. Treatment of DNA with a battery of enzymes including both phosphodiesterase and phosphatase activities yields a mixture of nucleosides and inorganic phosphate (P/sub i/) as a final product. Unlike native DNA, ultraviolet-irradiated DNA is resistant to complete digestion. Setlow et al. demonstrated that the structural changes in the DNA responsible for the nuclease resistance were the formation of cyclobutyl pyrimidine dimers, the major photoproduct in UV-irradiated DNA. Using venom phosphodiesterase, they demonstrated that UV irradiation of DNA affected both the rate and extent of enzymatic hydrolysis. In addition, it was demonstrated that the major nuclease-resistant product of this hydrolysis was an oligonucleotide containing dimerized pyrimidines. Treatment of the DNA to split the dimers, either photochemically or photoenzymatically, rendered the polymer more susceptible to hydrolysis by the phosphodiesterase. The specificity of photoreactivating enzyme for pyrimidine dimers lends support to the role of these structures in conferring nuclease resistance to UV-irradiated DNA. The nuclease resistance of DNA containing dimers has been the basis of several assays for the measurement of these photoproducts. Sutherland and Chamberlin reported the development of a rapid and sensitive assay for dimers in 32 P-labeled DNA

  1. Detection of bacterial DNA in serial synovial samples obtained during antibiotic treatment from patients with septic arthritis

    NARCIS (Netherlands)

    van der Heijden, I. M.; Wilbrink, B.; Vije, A. E.; Schouls, L. M.; Breedveld, F. C.; Tak, P. P.

    1999-01-01

    The management of septic arthritis could benefit from sensitive tests that detect the persistence of microorganisms in the joint. The aim of this study was to determine the feasibility of monitoring the presence of bacterial DNA in synovial samples from septic arthritis patients during antibiotic

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

    Directory of Open Access Journals (Sweden)

    Wei-wei SHEN

    2011-03-01

    Full Text Available 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 PXO71. The results indicate that ZR1 contains at least one functional bacterial blight resistance gene.

  3. Crude bacterial extracts of two new Streptomyces sp. isolates as bio-colorants for textile dyeing.

    Science.gov (United States)

    Kramar, Ana; Ilic-Tomic, Tatjana; Petkovic, Milos; Radulović, Niko; Kostic, Mirjana; Jocic, Dragan; Nikodinovic-Runic, Jasmina

    2014-08-01

    Renewed demand for incorporation of natural dyes (bio-colorants) in textile industry could be met through biotechnological production of bacterial pigments. Two new Streptomyces strains (NP2 and NP4) were isolated for the remarkable ability to produce diffusible deep blue and deep red pigment into fermentation medium. Crude mycelial extracts of both strains were used as bio-colorants in conventional textile dyeing procedures avoiding downstream purification procedures. The yields of bio-colorants obtained in this way were 62 and 84 mg per g of mycelia for Streptomyces sp. NP2 and Streptomyces sp. NP4, respectively. Through nuclear magnetic resonance analysis of crude extracts before and after dyeing procedures, it was shown that both extracts contained prodigiosin-like family of compounds that exhibited different dyeing capabilities towards different textile fibers. Polyamide and acrylic fibers were colored to the deepest shade, polyester and triacetate fibers to a noticeable, but much lower shade depth, while cotton and cellulosic fibers stained weakly. These results confirmed that crude bacterial extracts had the characteristics similar to those of ionic and disperse dyes, which was consistent with the identified polypyrrolic prodigiosin-like structures.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Related antimicrobial resistance genes detected in different bacterial species co-isolated from swine fecal samples.

    Science.gov (United States)

    Frye, Jonathan G; Lindsey, Rebecca L; Meinersmann, Richard J; Berrang, Mark E; Jackson, Charlene R; Englen, Mark D; Turpin, Jennifer B; Fedorka-Cray, Paula J

    2011-06-01

    A potential factor leading to the spread of antimicrobial resistance (AR) in bacteria is the horizontal transfer of resistance genes between bacteria in animals or their environment. To investigate this, swine fecal samples were collected on-farm and cultured for Escherichia coli, Salmonella enterica, Campylobacter spp., and Enterococcus spp. which are all commonly found in swine. Forty-nine of the samples from which all four bacteria were recovered were selected yielding a total of 196 isolates for analysis. Isolates were tested for antimicrobial susceptibility followed by hybridization to a DNA microarray designed to detect 775 AR-related genes. E. coli and Salmonella isolated from the same fecal sample had the most AR genes in common among the four bacteria. Genes detected encoded resistance to aminoglycosides (aac(3), aadA1, aadB, and strAB), β-lactams (ampC, ampR, and bla(TEM)), chloramphenicols (cat and floR), sulfanillic acid (sul1/sulI), tetracyclines (tet(A), tet(D), tet(C), tet(G), and tet(R)), and trimethoprim (dfrA1 and dfh). Campylobacter coli and Enterococcus isolated from the same sample frequently had tet(O) and aphA-3 genes detected in common. Almost half (47%) of E. coli and Salmonella isolated from the same fecal sample shared resistance genes at a significant level (χ², p genes between these bacteria or there may be a common source of AR genes in the swine environment for E. coli and Salmonella.

  7. Specificity of DNA import into isolated mitochondria from plants and mammals

    Directory of Open Access Journals (Sweden)

    Koulintchenko M. V.

    2014-01-01

    Full Text Available Aim. Investigation of different features of DNA import into plant and human mitochondria, for a better understanding of mitochondrial genetics and generation of biotechnological tools. Methods. DNA up-take experiments with isolated plant mitochondria, using as substrates various sequences associated or not with the specific terminal inverted repeats (TIRs present at each end of the plant mitochondrial linear plasmids. Results. It was established that the DNA import efficiency has a non-linear dependence on DNA size. It was shown that import into plant mitochondria of DNA molecules of «medium» sizes, i. e. between 4 and 7 kb, barely has any sequence specificity: neither TIRs from the 11.6 kb Brassica plasmid, nor TIRs from the Zea mays S-plasmids influenced DNA import into Solanum tuberosum mitochondria. Conclusions. The data obtained support the hypothesis about species-specific import mechanism operating under the mitochondrial linear plasmids transfer into plant mitochondria.

  8. 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

  9. Isolation and characterization of microsatellite DNA loci from Sillago ...

    Indian Academy of Sciences (India)

    In China, it is a common marine food fish species, and the supply of this fish relies on capture from the wild. So far, studies of this species have focussed on ... genomic library of S. sihama using the fast isolation by. AFLP of sequences containing repeats (FIASCO) protocol of Zane et al. (2002). These markers should provide ...

  10. Promising Biological Indicator of Heavy Metal Pollution: Bioluminescent Bacterial Strains Isolated and Characterized from Marine Niches of Goa, India.

    Science.gov (United States)

    Thakre, Neha A; Shanware, Arti S

    2015-09-01

    In present study, several marine water samples collected from the North Goa Beaches, India for isolation of luminescent bacterial species. Isolates obtained labelled as DP1-5 and AB1-6. Molecular characterization including identification of a microbial culture using 16S rRNA gene based molecular technique and phylogenetic analysis confirmed that DP3 & AB1 isolates were Vibrio harveyi. All of the isolates demonstrated multiple metal resistances in terms of growth, with altered luminescence with variable metal concentration. Present investigations were an attempt towards exploring and reporting an updated diversity of bioluminescent bacterial species from various sites around the Goa, India which would be explored in future for constructing luminescence based biosensor for efficiently monitoring the level of hazardous metals in the environment.

  11. 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

  12. Molecular Characterization and Analysis of 16S Ribosomal DNA in some Isolates of Demodex folliculorum

    Directory of Open Access Journals (Sweden)

    Afrooz DANESHPARVAR

    2017-06-01

    Full Text Available Background: Demodicosis is one of the most prevalent skin diseases resulting from infestation by Demodex mites. This parasite usually inhabits in follicular infundibulum or sebaceous duct transmitted through close contact with an infested host.Methods: This study was carried from September 2014 to January 2016 at Tehran University of Medical Sciences, Tehran, Iran. DNA extraction and amplification of 16S ribosomal RNA was performed on four isolates, obtained from four patients and identified morphologically through clearing with 10% Potassium hydroxide (KOH and microscopical examination. Amplified fragments from the isolates were compared with GenBank database and phylogenetic analysis was carried out using MEGA6 software.Results: A 390 bp fragment of 16S rDNA was obtained in all isolates and analysis of generated sequences showed high similarity with those submitted to GenBank, previously. Intra-species similarity and distance also showed 99.983% and 0.017, respectively, for the studied isolates. Multiple alignments of the isolates showed Single Nucleotide Polymorphisms (SNPs in 16S rRNA fragment. Phylogenetic analysis revealed that all 4 isolates clustered with other D. folliculorum, recovered from GenBank database. Our accession numbers KF875587 and KF875589 showed more similarity together in comparison with two other studied isolates. Conclusion: Mitochondrial 16S rDNA is one of the most suitable molecular barcodes for identification D. folliculorum and this fragment can use for intra-species characterization of the most human-infected mites.

  13. Microbiological and molecular identification of bacterial species isolated from nasal and oropharyngeal mucosa of fuel workers in Riyadh,

    Directory of Open Access Journals (Sweden)

    Suaad S. AlWakeel

    2017-09-01

    Full Text Available This study aimed to determine the bacterial species colonizing the nasal and oropharyngeal mucosa of fuel workers in Central Riyadh, Saudi Arabia on a microbiological and molecular level. Throat and nasal swab samples were obtained from 29 fuel station attendants in the period of time extending from March to May 2014 in Riyadh, Saudi Arabia. Microbiological identification techniques were utilized to identify the bacterial species isolated. Antibiotic sensitivity was assessed for each of the bacterial isolates. Molecular identification techniques based on PCR analysis of specific genomic sequences was conducted and was the basis on which phylogeny representation was done for 10 randomly selected samples of the isolates. Blood was drawn and a complete blood count was conducted to note the hematological indices for each of the study participants. Nineteen bacterial species were isolated from both the nasal cavity and the oropharynx including Streptococcus thoraltensis, alpha-hemolytic streptococci, Staphylococcus hominis, coagulase-negative staphylococci, Leuconostoc mesenteroides, Erysipelothrix rhusiopathiae and several others. We found 100% sensitivity of the isolates to ciprofloxacin, cefuroxime and gentamicin. Whereas cefotaxime and azithromycin posted sensitivities of 85.7% and 91.4%, respectively. Low sensitivities (<60% sensitivity to the antibiotics ampicillin, erythromycin, clarithromycin and norfloxacin were observed. Ninety-seven percent similarity to the microbial bank species was noted when the isolates were compared to it. Most hematological indices recorded were within the normal range. In conclusion, exposure to toxic fumes and compounds within fuel products may be a contributing factor to bacterial colonization of the respiratory tract in fuel workers.

  14. DNA Sequence of Cutinase Gene Isolated from Spilocaea oleagina as Determined by PCR

    OpenAIRE

    Matsuda, Yoshinori; Toyoda, Hideyoshi; Morii, Naho; Graniti, Antonio; Ouchi, Seiji

    1998-01-01

    [Synopsis] In this study, partial cutinase genes were cloned from Spilocaea oleagina and four different formae specials of Fusarium oxysporum (lycopersici, melonis, spinaciae and fragariae) by using PCR method. The reported amino acid sequences were aligned with DNA Data Bank of Japan (DDBJ), and highly conserved amino acid regions were selected to construct specific primers of cutinase genes. Chromosomal DNA of S. oleagina was isolated and used for template of PCR to amplify cutinase genes. ...

  15. Isolation, identification, and pathological effects of beach sand bacterial extract on human skin keratinocytesin vitro.

    Science.gov (United States)

    Subhan, Fazli; Shahzad, Raheem; Tauseef, Isfahan; Haleem, Kashif Syed; Rehman, Atta-Ur; Mahmood, Sajid; Lee, In-Jung

    2018-01-01

    Beaches are recreational spots for people. However, beach sand contains harmful microbes that affect human health, and there are no established methods for either sampling and identifying beach-borne pathogens or managing the quality of beach sand. This study was conducted with the aim of improving human safety at beaches and augmenting the quality of the beach experience. Beach sand was used as a resource to isolate bacteria due to its distinctive features and the biodiversity of the beach sand biota. A selected bacterial isolate termed FSRS was identified as Pseudomonas stutzeri using 16S rRNA sequencing and phylogenetic analysis, and the sequence was deposited in the NCBI GenBank database under the accession number MF599548. The isolated P. stutzeri bacterium was cultured in Luria-Bertani growth medium, and a crude extract was prepared using ethyl acetate to examine the potential pathogenic effect of P. stutzeri on human skin. A human skin keratinocyte cell line (HaCaT) was used to assess cell adhesion, cell viability, and cell proliferation using a morphological analysis and a WST-1 assay. The crude P. stutzeri extract inhibited cell adhesion and decreased cell viability in HaCaT cells. We concluded that the crude extract of P. stutzeri FSRS had a strong pathological effect on human skin cells. Beach visitors frequently get skin infections, but the exact cause of the infections is yet to be determined. The beach sand bacterium P. stutzeri may, therefore, be responsible for some of the dermatological problems experienced by people visiting the beach.

  16. Prevalence and antibiotic resistance of bacterial pathogens isolated from childhood diarrhoea in four provinces of Kenya.

    Science.gov (United States)

    Sang, Willie Kipkemboi; Oundo, Valerie; Schnabel, David

    2012-07-23

    Diarrhoea is one of the main causes of morbidity and mortality among children in sub-Saharan Africa, and one of the main causes of hospital admissions in rural areas of Kenya. In Kenya, antimicrobial resistance surveillance has been conducted only at the institutional levels, with limited sharing of information and analysis of data. As a result, the actual scale of regional or national antimicrobial drug resistance is not well defined. Stool samples were collected between 1 October 2007 and 30 September 2008 from a total of 651 outpatients with diarrhoea who were under five years of age in four provinces of Kenya.  Conventional, biochemical methods, multiplex PCR and antimicrobial susceptibility were conducted to identify the bacterial causes and virulence factors in the isolates, respectively.  Of the 651 patients screened, we identified the causes of 115 cases (17.7%) as follows: Pathogenic E. coli (11.2%) [enteroaggregative (8.9%), enterotoxigenic (1.2%), enteroinvasive (0.6%), shigatoxigenic (0.5%)], Salmonella (3.5%), Shigella (2%) and Vibrio cholera O1 (0.7%). The highest levels of resistance among the E. coli isolates were observed in ampicillin and trimethoprim/sulphamethoxazole each at 95% followed by tetracycline at 81%. Shigella isolate levels of resistance ranged from 80% to 100% for ampicillin, tetracycline and trimethoprim/sulphamethoxazole. The highest prevalence of antimicrobial resistance was to ampicillin followed by trimethoprim/sulphamethoxazole and tetracycline. Though still at low levels, the major concern from our findings is the emerging resistance of enteric pathogens that was observed to quinolones (ciprofloxacin, nalidixic acid, norfloxacin) and gentamycin.

  17. Towards Defining the Ecological Niches of Novel Coastal Gulf of Mexico Bacterial Isolates

    Science.gov (United States)

    Henson, M. W.; Thrash, C.; Nall, E.

    2016-02-01

    The study of microbial contributions to biogeochemistry is critical to understanding the cycles of fundamental compounds and gain predictive capabilities in a changing environment. Such study requires observation of microbial communities and genetics in nature, coupled with experimental testing of hypotheses both in situ and in laboratory settings. This study combines dilution-to-extinction based high-throughput culturing (HTC) with cultivation-independent and geochemical measurements to define potential ecological niches of novel bacterial isolates from the coastal northern Gulf of Mexico (cnGOM). Here we report findings from the first of a three-year project. In total, 43 cultures from seven HTC experiments were capable of being repeatedly transferred. Sanger sequencing of the 16S rRNA gene identified these isolates as belonging to the phyla Gammaproteobacteria, Alphaproteobacteria, Actinobacteria, and Betaproteobacteria. Eight are being genome sequenced, with two selected for further physiological characterization due to their phylogenic novelty and potential ecological significance. Strain LSUCC101 likely represents a novel family of Gammaproteobacteria (best blast hit to a cultured representative showed 91% sequence identity) and strain LSUCC96 belongs to the OM252 clade, with the Hawaiian isolate HIMB30 as its closest relative. Both are small (0.3-0.5 µm) cocci. The environmental importance of both LSUCC101 and LSUCC96 was illustrated by their presence within the top 30 OTU0.03 of cnGOM 16S rRNA gene datasets as well as within clone libraries from coastal regions around the world. Ongoing work is determining growth efficiencies, substrate utilization profiles, and metabolic potential to elucidate the roles of these organisms in the cnGOM. Comparative genomics will examine the evolutionary divergence of these organisms from their closest neighbors, and metagenomic recruitment to genomes will help identify strain-based variation from different coastal regions.

  18. Isolation, identification, and pathological effects of beach sand bacterial extract on human skin keratinocytes in vitro

    Directory of Open Access Journals (Sweden)

    Fazli Subhan

    2018-01-01

    Full Text Available Background Beaches are recreational spots for people. However, beach sand contains harmful microbes that affect human health, and there are no established methods for either sampling and identifying beach-borne pathogens or managing the quality of beach sand. Method This study was conducted with the aim of improving human safety at beaches and augmenting the quality of the beach experience. Beach sand was used as a resource to isolate bacteria due to its distinctive features and the biodiversity of the beach sand biota. A selected bacterial isolate termed FSRS was identified as Pseudomonas stutzeri using 16S rRNA sequencing and phylogenetic analysis, and the sequence was deposited in the NCBI GenBank database under the accession number MF599548. The isolated P. stutzeri bacterium was cultured in Luria–Bertani growth medium, and a crude extract was prepared using ethyl acetate to examine the potential pathogenic effect of P. stutzeri on human skin. A human skin keratinocyte cell line (HaCaT was used to assess cell adhesion, cell viability, and cell proliferation using a morphological analysis and a WST-1 assay. Result The crude P. stutzeri extract inhibited cell adhesion and decreased cell viability in HaCaT cells. We concluded that the crude extract of P. stutzeri FSRS had a strong pathological effect on human skin cells. Discussion Beach visitors frequently get skin infections, but the exact cause of the infections is yet to be determined. The beach sand bacterium P. stutzeri may, therefore, be responsible for some of the dermatological problems experienced by people visiting the beach.

  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. ‘Lactomassilus timonensis,’ a new anaerobic bacterial species isolated from the milk of a healthy African mother

    Directory of Open Access Journals (Sweden)

    A.H. Togo

    2018-01-01

    Full Text Available We here report the main characteristics of a new anaerobic bacterial genus and species ‘Lactomassilus timonensis,’ strain Marseille-P4641T (CSUR = P4641, isolated by microbial culturomics from the milk of a 35-year-old healthy lactating mother from Mali.

  1. ‘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.

  2. Diversity of Hindgut Bacterial Population in Subterranean Termite, Reticulitermes flavipes

    Science.gov (United States)

    Olanrewaju Raji; Dragica Jeremic-Nikolic; Juliet D. Tang

    2017-01-01

    The termite hindgut contains a bacterial community that symbiotically aids in digestion of cellulosic materials. For this paper, a species survey of bacterial hindgut symbionts in termites collected from Saucier, Mississippi was examined. Two methods were tested for optimal genetic material isolation. Genomic DNA was isolated from the hindgut luminal contents of five...

  3. SISPA-Seq for rapid whole genome surveys of bacterial isolates.

    Science.gov (United States)

    Wright, Meredith S; Stockwell, Timothy B; Beck, Erin; Busam, Dana A; Bajaksouzian, Saralee; Jacobs, Michael R; Bonomo, Robert A; Adams, Mark D

    2015-06-01

    Whole genome sequencing (WGS) of large isolate collections has many applications, yet sequencing costs are still significant. We sought to develop a rapid and cost efficient WGS method to address fundamental questions in clinical microbiology. We evaluated the performance of SISPA (Sequence-Independent, Single-Primer Amplification) combined with next-generation sequencing (SISPA-Seq) of 75 clinical isolates of Acinetobacter baumannii to establish whether SISPA-Seq resulted in sufficient coverage and quality to (1) determine strain phylogenetic placement and (2) and carriage of known antibiotic resistance (AbR) genes. Strains for which whole genome sequences were available were included for validation. Two libraries for each strain were constructed from separate SISPA reactions with different barcoded primers, using genomic DNA prepared from either high quality or rapid heat-lysis preparations. SISPA-Seq resulted in a median of 65× genome coverage when reads from both primer sets were combined. Coverage and quality were sufficient for detection of AbR genes by comparison of reads to the ARG-ANNOT database and were often sufficient to distinguish between different allelic variants of the same gene. kSNP and RAxML were used to construct a robust phylogeny based on single-nucleotide variants (SNVs) that showed that the SISPA-Seq data was sufficient for sensitive and accurate phylogenetic placement. Advantages of the SISPA-Seq method include inexpensive and rapid DNA preparation and a typical total cost less than one-half that of standard genome sequencing. In summary, SISPA-Seq can be used to survey whole genomes of a large strain collection and identify strains that should be targeted for additional sequencing. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Endophytic bacterial flora in root and stem tissues of black pepper (Piper nigrum L.) genotype: isolation, identification and evaluation against Phytophthora capsici.

    Science.gov (United States)

    Aravind, R; Kumar, A; Eapen, S J; Ramana, K V

    2009-01-01

    To isolate and identify black pepper (Piper nigrum L) associated endophytic bacteria antagonistic to Phytophthora capsici causing foot rot disease. Endophytic bacteria (74) were isolated, characterized and evaluated against P. capsici. Six genera belong to Pseudomonas spp (20 strains), Serratia (1 strain), Bacillus spp. (22 strains), Arthrobacter spp. (15 strains), Micrococcus spp. (7 strains), Curtobacterium sp. (1 strain) and eight unidentified strains were isolated from internal tissues of root and stem. Three isolates, IISRBP 35, IISRBP 25 and IISRBP 17 were found effective for Phytophthora suppression in multilevel screening assays which recorded over 70% disease suppression in greenhouse trials. A species closest match (99% similarity) of IISRBP 35 was established as Pseudomonas aeruginosa (Pseudomonas EF568931), IISRBP 25 as P. putida (Pseudomonas EF568932), and IISRBP 17 as Bacillus megaterium (B. megaterium EU071712) based on 16S rDNA sequencing. Black pepper associated P. aeruginosa, P. putida and B. megaterium were identified as effective antagonistic endophytes for biological control of Phytophthora foot rot in black pepper. This work provides the first evidence for endophytic bacterial diversity in black pepper stem and roots, with biocontrol potential against P. capsici infection.

  5. 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.

  6. Simple and rapid protocol for the isolation of PCR-amplifiable DNA from medicinal plants.

    Science.gov (United States)

    Alatar, A A; Mahmoud, M A; Al-Sohaibani, S A; Abd-Elsalam, K A

    2012-02-13

    Medicinal plant species has a valuable economic importance because of its usage as pharmaceuticals, nutritional, as well as its use in popular medication. For DNA-based techniques, nanogram quantities of the purified DNA are requisite to amplify and yield sufficient amounts of PCR products. SDS-based DNA isolation method was used to extract DNA from 11 species of different aromatic and medicinal plants collected from Saudi Arabia. Three hundred milligrams of fresh shredded plant material was necessary. The DNA purity was further confirmed by agarose gel, restriction endonuclease digestion and microsatellite primed-polymerase chain reaction (MP-PCR). DNA yields ranged from 10-20 μg (in 100-μL elution volumes) from all plant material evaluated. The DNA obtained was free of any contaminating proteins, polysaccharides and colored pigments. The extracted genomic DNA was found suitable for restriction digestion and PCR amplification. Our experimental procedure provides an easy, suitable, non-toxic, cheap, and quick process for the amplification of DNA from medical plant tissue.

  7. Comparative Analysis of the Genomic DNA Isolation Methods on Inula sp. (Asteraceae

    Directory of Open Access Journals (Sweden)

    Emre SEVİNDİK

    2016-12-01

    Full Text Available Simple, fast, low-cost and high throughput protocols are required for DNA isolation of plant species. In this study, phenol chloroform isoamyl alcohol and commercial (Sigma DNA isolation kit methods were applied on some Inula species that belong to Asteraceae family. Genomic DNA amounts, A260, A280, A260/A230 and purity degrees (A260/A280 that were obtained through both methods were measured through electrophoresis and spectrophotometer. Additionally, PCR amplification was realized by primer pairs specific to nrDNA ITS, cpDNA ndhF (972F-1603R and trnL-F regions. Results showed that maximum genomic DNA in nanograms obtained by phenol chloroform isoamyl alcohol method. The study also revealed that I. macrocephala had the maximum DNA and I. heterolepis had the minimum DNA amount. A260/A280 purity degrees showed that the highest and lowest purity in gDNAs obtained through phenol-choloform isoamyl alcohol method were in I.aucheriana and I. salicina, respectively. The highest and lowest purity degrees of gDNAs obtained through commercial kit was observed in I. fragilis and I. macrocephala samples, respectively. PCR amplification results showed that while band profiles of each three regions (ITS, trnL-F and ndhF did not yield positive results in PCR amplifications using phenol-choloform isoamyl alcohol method; PCR band profiles obtained through commercial kit yielded positive results. As a result, it is fair to say that the relation of genomic DNA with PCR was found to be more efficient although the maximum amount of genomic DNA was obtained through phenol chloroform isoamyl alcohol method.

  8. Reliable genotyping of the koala (Phascolarctos cinereus) using DNA isolated from a single faecal pellet.

    Science.gov (United States)

    Wedrowicz, Faye; Karsa, Mawar; Mosse, Jennifer; Hogan, Fiona E

    2013-07-01

    The koala, an Australian icon, has been added to the threatened species list. Rationale for the listing includes proposed declines in population size, threats to populations (e.g. disease) and loss and fragmentation of habitat. There is now an urgent need to obtain accurate data to assess the status of koala populations in Australia, to ensure the long-term viability of this species. Advances in genetic techniques have enabled DNA analysis to study and inform the management of wild populations; however, sampling of individual koalas is difficult in tall, often remote, eucalypt forest. The collection of faecal pellets (scats) from the forest floor presents an opportunistic sampling strategy, where DNA can be collected without capturing or even sighting an individual. Obtaining DNA via noninvasive sampling can be used to rapidly sample a large proportion of a population; however, DNA from noninvasively collected samples is often degraded. Factors influencing DNA quality and quantity include environmental exposure, diet and methods of sample collection, storage and DNA isolation. Reduced DNA quality and quantity can introduce genotyping errors and provide inaccurate DNA profiles, reducing confidence in the ability of such data to inform management/conservation strategies. Here, we present a protocol that produces a reliable individual koala genotype from a single faecal pellet and highlight the importance of optimizing DNA isolation and analysis for the species of interest. This method could readily be adapted for genetic studies of mammals other than koalas, particularly those whose diet contains high proportions of volatile materials that are likely to induce DNA damage. © 2013 John Wiley & Sons Ltd.

  9. Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens.

    Science.gov (United States)

    Saeidi, Saman; McKain, Michael R; Kellogg, Elizabeth A

    2018-03-08

    Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

  10. 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

    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.

  11. 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.

  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. 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...

  14. Isolating human DNA repair genes using rodent-cell mutants

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-03-23

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab.

  15. Isolation of genomic DNA using magnetic nanoparticles as a solid-phase support

    Energy Technology Data Exchange (ETDEWEB)

    Saiyed, Z M; Ramchand, C N [Kemin Nutritional Technologies (India) Pvt Ltd, Trapezium, Nelson Manickam Road, Chennai 600 029 (India); Telang, S D [Department of Biochemistry, Faculty of Science, M S University of Baroda, Sayajigunj, Vadodara 390 002 (India)], E-mail: cnramchand@yahoo.com, E-mail: ramchand.cn@kemin.com

    2008-05-21

    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.

  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. Stabilization of chromosomes by DNA intercalators for flow karyotyping and identification by banding of isolated chromosomes

    NARCIS (Netherlands)

    Aten, J. A.; Buys, C. H.; van der Veen, A. Y.; Mesa, J. R.; Yu, L. C.; Gray, J. W.; Osinga, J.; Stap, J.

    1987-01-01

    A number of structurally unrelated DNA intercalators have been studied as stabilizers of mitotic chromosomes during isolation from rodent and human metaphase cells. Seven out of the nine intercalators tested were found to be useful as chromosome stabilizing agents. Chromosome suspensions prepared in

  18. 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

    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...

  19. [The random amplified polymorphic DNA identification of 9 Taenia saginata isolates from four provinces].

    Science.gov (United States)

    Zhang, Ke; Yang, Ming; Bao, Huai-En

    2006-12-01

    To make molecular identification for 9 isolates of Taenia saginata from 4 provinces. Genomic DNA was extracted from the segments of adult tapeworms collected from Taoyuan of Taiwan (TW1), Duyun of Guizhou (DY1, DY2), Congjiang of Guizhou (CJ1, CJ2, CJ3, CJ4), Dali of Yunnan (DL1) and Wushi of Xinjiang (XJ1) respectively. PCRs were carried out with 13 random primers. A phylogenetic tree of different geographical strains was constructed. 331 DNA fragments were amplified. The number of DNA fragments amplified by single primer was between 3 and 28. The average number of amplified DNA fragments by the 13 primers was 14.15. The average number of fragments from the 9 isolates of T. saginata was 14.08. Phylogenetic tree revealed that there were two branches in the tree, DY1, DY2, DL1 and TW1 occupied one branch, while CJ1, CJ2, CJ3, CJ4 and XJ1 occupied the other one. By the RAPD analysis, the isolates DY1, DY2, DL1 and TW1 belong to Taenia saginata asiatica, and the isolates CJ1, CJ2, CJ3, CJ4 and XJ1 belong to T. saginata saginata.

  20. 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)

  1. 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.

  2. Indoleacetic acid production and plant growth promoting potential of bacterial endophytes isolated from rice (Oryza sativa L.) seeds.

    Science.gov (United States)

    Shahzad, Raheem; Waqas, Muhammad; Khan, Abdul Latif; Al-Hosni, Khadija; Kang, Sang-Mo; Seo, Chang-Woo; Lee, In-Jung

    2017-06-01

    Bacterial endophytes from the phyllosphere and rhizosphere have been used to produce bioactive metabolites and to promote plant growth. However, little is known about the endophytes residing in seeds. This study aimed to isolate and identify seed-borne bacterial endophytes from rice and elucidate their potential for phytohormone production and growth enhancement. The isolated endophytes included Micrococcus yunnanensis RWL-2, Micrococcus luteus RWL-3, Enterobacter soli RWL-4, Leclercia adecarboxylata RWL-5, Pantoea dispersa RWL-6, and Staphylococcus epidermidis RWL-7, which were identified using 16S rRNA sequencing and phylogenetic analysis. These strains were analyzed for indoleacetic acid (IAA) production by using GC-MS and IAA was found in the range of 11.50 ± 0.77 μg ml -1 to 38.80 ± 1.35 μg ml -1 . We also assessed the strains for plant growth promoting potential because these isolates were able to produce IAA in pure culture. Most of the growth attributes of rice plants (shoot and root length, fresh and dry biomass, and chlorophyll content) were significantly increased by bacterial endophytes compared to the controls. These results show that IAA producing bacterial endophytes can improve hostplant growth traits and can be used as bio-fertilizers.

  3. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. DNA repair in Haemophilus influenzae: isolation and characterization of an ultraviolet sensitive mutator mutant

    International Nuclear Information System (INIS)

    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

  5. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

    International Nuclear Information System (INIS)

    Kennedy, Edward M.; Cullen, Bryan R.

    2015-01-01

    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

  6. 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

  7. Homogeneity of the 16S rDNA sequence among geographically disparate isolates of Taylorella equigenitalis.

    Science.gov (United States)

    Matsuda, M; Tazumi, A; Kagawa, S; Sekizuka, T; Murayama, O; Moore, J E; Millar, B C

    2006-01-06

    At present, six accessible sequences of 16S rDNA from Taylorella equigenitalis (T. equigenitalis) are available, whose sequence differences occur at a few nucleotide positions. Thus it is important to determine these sequences from additional strains in other countries, if possible, in order to clarify any anomalies regarding 16S rDNA sequence heterogeneity. Here, we clone and sequence the approximate full-length 16S rDNA from additional strains of T. equigenitalis isolated in Japan, Australia and France and compare these sequences to the existing published sequences. Clarification of any anomalies regarding 16S rDNA sequence heterogeneity of T. equigenitalis was carried out. When cloning, sequencing and comparison of the approximate full-length 16S rDNA from 17 strains of T. equigenitalis isolated in Japan, Australia and France, nucleotide sequence differences were demonstrated at the six loci in the 1,469 nucleotide sequence. Moreover, 12 polymorphic sites occurred among 23 sequences of the 16S rDNA, including the six reference sequences. High sequence similarity (99.5% or more) was observed throughout, except from nucleotide positions 138 to 501 where substitutions and deletions were noted.

  8. Homogeneity of the 16S rDNA sequence among geographically disparate isolates of Taylorella equigenitalis

    Directory of Open Access Journals (Sweden)

    Moore JE

    2006-01-01

    Full Text Available Abstract Background At present, six accessible sequences of 16S rDNA from Taylorella equigenitalis (T. equigenitalis are available, whose sequence differences occur at a few nucleotide positions. Thus it is important to determine these sequences from additional strains in other countries, if possible, in order to clarify any anomalies regarding 16S rDNA sequence heterogeneity. Here, we clone and sequence the approximate full-length 16S rDNA from additional strains of T. equigenitalis isolated in Japan, Australia and France and compare these sequences to the existing published sequences. Results Clarification of any anomalies regarding 16S rDNA sequence heterogeneity of T. equigenitalis was carried out. When cloning, sequencing and comparison of the approximate full-length 16S rDNA from 17 strains of T. equigenitalis isolated in Japan, Australia and France, nucleotide sequence differences were demonstrated at the six loci in the 1,469 nucleotide sequence. Moreover, 12 polymorphic sites occurred among 23 sequences of the 16S rDNA, including the six reference sequences. Conclusion High sequence similarity (99.5% or more was observed throughout, except from nucleotide positions 138 to 501 where substitutions and deletions were noted.

  9. 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…

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

    International Nuclear Information System (INIS)

    Concha-Guerrero, S.I.; Pinon-Castillo, H.A.; Luna-Velasco, A.; Orrantia-Borunda, E.; Brito, E.M.S.; Tarango-Rivero, S.H.; Caretta, C.A.; Duran, R.

    2014-01-01

    The increased use of the nanoparticles (NPs) on several processes is notorious. In contrast the eco toxicological 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 Gamma proteobacteria (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 later osporus 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

  11. In silico design of PHA synthase and its validation by PHAs producing bacterial isolates

    Directory of Open Access Journals (Sweden)

    Susrita Sahoo

    2017-10-01

    Full Text Available Biopolymers are important alternatives to the petroleum-based plastics due to environment friendly manufacturing processes, biodegradability and biocompatibility. Therefore use of novel biopolymers such as polylactide, polysaccharides, aliphatic polyesters and polyhydroxyalkonoates (PHAs is of interest. PHAs are biodegradable polyesters of hydroxyalkanoates (HA produced from renewable resources by using microorganisms as intracellular carbon and energy storage compounds.  Even though PHAs are promising candidate for biodegradable polymers, however, the production cost limits their application on an industrial scale. Therefore an attempt was made to model different PHAs synthases which are the key enzyme in the biosynthesis of Polyhydroxyalkanoates as the structural information of this enzyme is in dark veil.Then molecular docking  of class I  PHA  Synthase from Ralstonia Eutrophia was done to study the PHA synthase activity. As there are lots of strain which needs to explore for the production of PHA. This investigation leads to find out the most industrial applicable microbes. Few bacterial isolates from soil sample were screened for production of PHA followed by the validation of the enzymatic activity and its product characterization to understand its structural properties.

  12. Comparative Genomics of Facultative Bacterial Symbionts Isolated from European Orius Species Reveals an Ancestral Symbiotic Association

    Directory of Open Access Journals (Sweden)

    Xiaorui Chen

    2017-10-01

    Full Text Available Pest control in agriculture employs diverse strategies, among which the use of predatory insects has steadily increased. The use of several species within the genus Orius in pest control is widely spread, particularly in Mediterranean Europe. Commercial mass rearing of predatory insects is costly, and research efforts have concentrated on diet manipulation and selective breeding to reduce costs and improve efficacy. The characterisation and contribution of microbial symbionts to Orius sp. fitness, behaviour, and potential impact on human health has been neglected. This paper provides the first genome sequence level description of the predominant culturable facultative bacterial symbionts associated with five Orius species (O. laevigatus, O. niger, O. pallidicornis, O. majusculus, and O. albidipennis from several geographical locations. Two types of symbionts were broadly classified as members of the genera Serratia and Leucobacter, while a third constitutes a new genus within the Erwiniaceae. These symbionts were found to colonise all the insect specimens tested, which evidenced an ancestral symbiotic association between these bacteria and the genus Orius. Pangenome analyses of the Serratia sp. isolates offered clues linking Type VI secretion system effector–immunity proteins from the Tai4 sub-family to the symbiotic lifestyle.

  13. 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.

  14. Effect of gut bacterial isolates fromApis mellifera jemeniticaonPaenibacillus larvaeinfected bee larvae.

    Science.gov (United States)

    Al-Ghamdi, Ahmad; Ali Khan, Khalid; Javed Ansari, Mohammad; Almasaudi, Saad B; Al-Kahtani, Saad

    2018-02-01

    The probiotic effects of seven newly isolated gut bacteria, from the indegenous honey bees of Saudi Arabia were investigated. In vivo bioassays were used to investigate the effects of each gut bacterium namely, Fructobacillus fructosus (T1), Proteus mirabilis (T2), Bacillus licheniformis (T3), Lactobacillus kunkeei (T4), Bacillus subtilis (T5), Enterobacter kobei (T6), and Morganella morganii (T7) on mortality percentage of honey bee larvae infected with P. larvae spores along with negative control (normal diet) and positive control (normal diet spiked with P. larvae spores). Addition of gut bacteria to the normal diet significantly reduced the mortality percentage of the treated groups. Mortality percentage in all treated groups ranged from 56.67% up to 86.67%. T6 treated group exhibited the highest mortality (86.67%), whereas T4 group showed the lowest mortality (56.67%). Among the seven gut bacterial treatments, T4 and T3 decreased the mortality 56.67% and 66.67%, respectively, whereas, for T2, T6, and T7 the mortality percentage was equal to that of the positive control (86.67%). Mortality percentages in infected larval groups treated with T1, and T5 were 78.33% and 73.33% respectively. Most of the mortality occurred in the treated larvae during days 2 and 3. Treatments T3 and T4 treatments showed positive effects and reduced mortality.

  15. Effect of gut bacterial isolates from Apis mellifera jemenitica on Paenibacillus larvae infected bee larvae

    Directory of Open Access Journals (Sweden)

    Ahmad Al-Ghamdi

    2018-02-01

    Full Text Available The probiotic effects of seven newly isolated gut bacteria, from the indegenous honey bees of Saudi Arabia were investigated. In vivo bioassays were used to investigate the effects of each gut bacterium namely, Fructobacillus fructosus (T1, Proteus mirabilis (T2, Bacillus licheniformis (T3, Lactobacillus kunkeei (T4, Bacillus subtilis (T5, Enterobacter kobei (T6, and Morganella morganii (T7 on mortality percentage of honey bee larvae infected with P. larvae spores along with negative control (normal diet and positive control (normal diet spiked with P. larvae spores. Addition of gut bacteria to the normal diet significantly reduced the mortality percentage of the treated groups. Mortality percentage in all treated groups ranged from 56.67% up to 86.67%. T6 treated group exhibited the highest mortality (86.67%, whereas T4 group showed the lowest mortality (56.67%. Among the seven gut bacterial treatments, T4 and T3 decreased the mortality 56.67% and 66.67%, respectively, whereas, for T2, T6, and T7 the mortality percentage was equal to that of the positive control (86.67%. Mortality percentages in infected larval groups treated with T1, and T5 were 78.33% and 73.33% respectively. Most of the mortality occurred in the treated larvae during days 2 and 3. Treatments T3 and T4 treatments showed positive effects and reduced mortality.

  16. Increasing antibiotic resistance in preservative-tolerant bacterial strains isolated from cosmetic products.

    Science.gov (United States)

    Orús, Pilar; Gomez-Perez, Laura; Leranoz, Sonia; Berlanga, Mercedes

    2015-03-01

    To ensure the microbiological quality, consumer safety and organoleptic properties of cosmetic products, manufacturers need to comply with defined standards using several preservatives and disinfectants. A drawback regarding the use of these preservatives is the possibility of generating cross-insusceptibility to other disinfectants or preservatives, as well as cross resistance to antibiotics. Therefore, the objective of this study was to understand the adaptive mechanisms of Enterobacter gergoviae, Pseudomonas putida and Burkholderia cepacia that are involved in recurrent contamination in cosmetic products containing preservatives. Diminished susceptibility to formaldehyde-donors was detected in isolates but not to other preservatives commonly used in the cosmetics industry, although increasing resistance to different antibiotics (β-lactams, quinolones, rifampicin, and tetracycline) was demonstrated in these strains when compared with the wild-type strain. The outer membrane protein modifications and efflux mechanism activities responsible for the resistance trait were evaluated. The development of antibiotic-resistant microorganisms due to the selective pressure from preservatives included in cosmetic products could be a risk for the emergence and spread of bacterial resistance in the environment. Nevertheless, the large contribution of disinfection and preservation cannot be denied in cosmetic products. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.

  17. Whole-Genome Sequencing and Concordance Between Antimicrobial Susceptibility Genotypes and Phenotypes of Bacterial Isolates Associated with Bovine Respiratory Disease

    Directory of Open Access Journals (Sweden)

    Joseph R. Owen

    2017-09-01

    Full Text Available Extended laboratory culture and antimicrobial susceptibility testing timelines hinder rapid species identification and susceptibility profiling of bacterial pathogens associated with bovine respiratory disease, the most prevalent cause of cattle mortality in the United States. Whole-genome sequencing offers a culture-independent alternative to current bacterial identification methods, but requires a library of bacterial reference genomes for comparison. To contribute new bacterial genome assemblies and evaluate genetic diversity and variation in antimicrobial resistance genotypes, whole-genome sequencing was performed on bovine respiratory disease–associated bacterial isolates (Histophilus somni, Mycoplasma bovis, Mannheimia haemolytica, and Pasteurella multocida from dairy and beef cattle. One hundred genomically distinct assemblies were added to the NCBI database, doubling the available genomic sequences for these four species. Computer-based methods identified 11 predicted antimicrobial resistance genes in three species, with none being detected in M. bovis. While computer-based analysis can identify antibiotic resistance genes within whole-genome sequences (genotype, it may not predict the actual antimicrobial resistance observed in a living organism (phenotype. Antimicrobial susceptibility testing on 64 H. somni, M. haemolytica, and P. multocida isolates had an overall concordance rate between genotype and phenotypic resistance to the associated class of antimicrobials of 72.7% (P < 0.001, showing substantial discordance. Concordance rates varied greatly among different antimicrobial, antibiotic resistance gene, and bacterial species combinations. This suggests that antimicrobial susceptibility phenotypes are needed to complement genomically predicted antibiotic resistance gene genotypes to better understand how the presence of antibiotic resistance genes within a given bacterial species could potentially impact optimal bovine respiratory

  18. Reproductive Fitness and Random Amplified Polymorphic DNA Variation among Isolates of Pratylenchus vulnus.

    Science.gov (United States)

    Pinochet, P; Cenis, J L; Fernández, C; Doucet, M; Maruli, J

    1994-09-01

    The reproductive fitness of seven isolates of Pratylenchus vulnus from different geographical areas and hosts was assessed in monoxenic cultures (carrot), and greenhouse cultures (plum, sour orange, and quince). The genetic makeup of the different isolates was compared by Random Amplified Polymorphic DNA (RAPD-PCR). The apple (PvAP-S) and apricot (PvAT-F) isolates reproduced less in monoxenic cultures than the rose (PvRO-S) and walnut (PvWA-A and PvWA-U) isolates. On plum, the rose isolate (PvRO-S) reproduced better than the apple (PvAP-S) and walnut isolate from the United States (PvWA-U). On sour orange, the apple (PvAP-S), unknown origin (PvU-UK), and walnut isolate from Argentina (PvWA-A) multiplied well, whereas the walnut isolate from the United States (PvWA-U), apricot (PvAT-F), and rose (PvRO-S) did not. On quince, the apple (PvAP-S) and walnut (PvWA-U) isolates showed a higher reproduction than the one from unknown origin (PvU-UK). RAPD-PCR patterns among the seven P. vulnus isolates were similar, although high intraspecific varibility was detected. Very few bands of P. neglectus were shared by any population of P. vulnus. A high degree of similarity was found among the patterns corresponding to the rose (PvRO-S), apple (PvAP-S), walnut from the United States (PvWA-U), and unknown origin (PvUK-U) isolates. The apricot isolate (PvAT-F) was the most dissimilar among the seven isolates. No correlation could be established between the genetic variation of P. vulnus detected by RAPD-PCR and reproductive fitness. Results demonstrate high genetic varibility between geographically separated populations of P. vulnus.

  19. 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

  20. Identification of a Cryptic Bacterial Promoter in Mouse (mdr1a P-Glycoprotein cDNA.

    Directory of Open Access Journals (Sweden)

    Kristen M Pluchino

    Full Text Available The efflux transporter P-glycoprotein (P-gp is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse mdr1a cDNA display significant genetic instability during cloning in bacteria, indicating that mdr1a cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse mdr1a cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of mdr1a DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of mdr1a cDNA capable of initiating bacterial protein expression. An mdr1a M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of mdr1a in E. coli while retaining transport properties similar to wild-type P-gp. This mutant mdr1a cDNA may prove useful for efficient cloning of mdr1a in E. coli.

  1. A rapid and simple method for DNA extraction from yeasts and fungi isolated from Agave fourcroydes.

    Science.gov (United States)

    Tapia-Tussell, Raul; Lappe, Patricia; Ulloa, Miguel; Quijano-Ramayo, Andrés; Cáceres-Farfán, Mirbella; Larqué-Saavedra, Alfonso; Perez-Brito, Daisy

    2006-05-01

    A simple and easy protocol for extracting high-quality DNA from different yeast and filamentous fungal species is described. This method involves two important steps: first, the disruption of cell walls by mechanical means and freezing; and second, the extraction, isolation, and precipitation of genomic DNA. The absorbance ratios (A(260)/A(280)) obtained ranged from 1.6 to 2.0. The main objective of this procedure is to extract pure DNA from yeast and filamentous fungi, including those with high contents of proteins, polysaccharides, and other complex compounds in their cell walls. The yield and quality of the DNAs obtained were suitable for micro/minisatellite primer-polymerase chain reaction (MSP-PCR) fingerprinting as well as for the sequence of the D1/D2 domain of the 26S rDNA.

  2. 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

    collected in the same way and processed using the same blood isolation procedure. The inter-laboratory variation was the prominent contributor to the overall variation. The inter-laboratory coefficient of variation decreased for both DNA strand breaks (from 68 to 26%) and FPG sensitive sites (from 57 to 12......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......%) 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...

  3. 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.

  4. Effect of oxygen and misonidazole on radiation damage in biologically active DNA dissolved in a bacterial extract. Influence of cytochrome c

    International Nuclear Information System (INIS)

    Lafleur, M.V.M.; Pluijmackers-Westmijze, E.J.; Loman, H.

    1982-01-01

    The effect of radiation on biologically active DNA, dissolved in a bacterial extract was studied. The results show that oxygen affects DNA inactivation for both double-stranded (RF) and single-stranded phiX174 DNA. Misonidazole induces an increased radiosensitivity in this system as found for single-stranded DNA. These effects disappear with ageing of the extract, but can be recovered by the addition of 10 -6 M cytochrome c. (author)

  5. Plasmid DNA Analysis of Pasteurella multocida Serotype B isolated from Haemorrhagic Septicaemia outbreaks in Malaysia

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    Jamal, H.

    2005-01-01

    Full Text Available A total of 150 purified isolates of Pasteurella multocida serotype B were used (Salmah, 2004 for plasmid DNA curing experiment to determine hyaluronidase activity, antibiotic resistance pattern (ARP and mice lethality test (LD50 for their role of pathogenicity. A plasmid curing experiment was carried out by using the intercalating agent; ethidium bromide and rifampicin, where it was found all the plasmids had been cured (plasmidless from Pasteurella multocida. All of these plasmidless isolates maintained their phenotypic characteristics. They showed the same antibiotic resistancepattern as before curing, produced hyaluronidase and possessed lethality activity in mice when injected intraperitoneally(i.p. Based on this observation, the antibiotic resistance, hyaluronidase activity and mice virulence could probably be chromosomal-mediated. Plasmids were detected 100% in all P. multocida isolates with identical profile of 2 plasmids size 3.0 and 5.5 kb. No large plasmids could be detected in all isolates. Since all the isolates appeared to have identicalplasmid profiles, they were subjected to restriction enzyme(RE analysis. From RE analysis results obtained, it can be concluded that the plasmid DNA in serotype B isolates are identical. Only 4 of 32 REs were found to cleave these plasmids with identical restriction fingerprints; BglII, HaeIII, RsaI and SspI. From RE analysis results, it can be concluded that the plasmid DNA isolates are identical. This plasmid might not played any role in pathogenicity of Pasteurella multocida serotype B, however this information is important for the construction of shuttle vectors in genetic studies of the pathogenicity of haemorrhagic septicaemia(HS.

  6. Characterization of novel extracellular protease produced by marine bacterial isolate from the Indian Ocean

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    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. Biocontrol of verticillium wilt and colonization of cotton plants by an endophytic bacterial isolate.

    Science.gov (United States)

    Li, C-H; Shi, L; Han, Q; Hu, H-L; Zhao, M-W; Tang, C-M; Li, S-P

    2012-09-01

    To explore biocontrol potential of 39 DAEB isolates (doubly antagonistic towards both Verticillium dahliae Kleb and Fusarium oxysporum) against verticillium wilt of cotton and to elucidate colonization and category characteristics of an endophytic bacterium with significant biocontrol activity. Thirty-nine antagonistic endophytic bacteria strains were tested for their ability to control verticillium wilt in cotton plants caused by a defoliating pathotype of V. dahliae 107 in cotton under controlled conditions. The biocontrol trial revealed that an endophytic bacterium, designated HA02, showed a significant biocontrol activity to V. dahliae 107. After cotton seedlings were inoculated with a gfp gene-tagged HA02 (HA02-gfp), HA02-gfp populations were higher in the root than in the stem; in addition, the HA02-gfp was distributed in the maturation zone of cotton root. Furthermore, HA02-gfp also colonized seedlings of maize, rape and soybean after the bacteria inoculation. Phylogenetic trees based on 16S rDNA sequences combined with morphological, physiological and identification showed that the bacterium belongs to the Enterobacter genus. Our results showed that only 1 of 39 DAEB isolates demonstrated more efficient biocontrol potential towards V. dahliae 107 in greenhouse and field trials. HA02-gfp mainly colonized cotton in roots. In addition, we quantitatively observed HA02 colonization in other hosts. HA02 belongs to the Enterobacter genus. This is the first study on biocontrol to defoliating pathotype of V. dahliae Kleb by endophytic bacteria. The HA02 showed effective biocontrol to V. dahliae 107 in greenhouse and field trials. Furthermore, we assessed the quantitative and qualitative colonization of HA02 in cotton seedlings. Our study provides basic information to further explore managing strategies to control this critical disease. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

  8. Isolation and partial characterization of bacterial strains on low organic carbon medium from soils fertilized with different organic amendments.

    Science.gov (United States)

    Senechkin, Ilya V; Speksnijder, Adrianus G C L; Semenov, Alexander M; van Bruggen, Ariena H C; van Overbeek, Leonard S

    2010-11-01

    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 µg C ml(-1)) traditionally used for isolation of oligotrophs. Organic amendments in combination with field history resulted in differences in dissolved organic carbon contents in these soils. There were negative correlations between total and dissolved organic carbon content and the number of isolates on low-C agar medium, whereas these correlations were absent for bacterial strains isolated from the same soil on high-C agar medium (1,000 µg C ml(-1)). Repeated transfers (up to ten times) of the isolates from low-C agar medium to fresh low- and high-C agar media were done to test for exclusive growth under oligotrophic conditions. The number of isolates exclusively growing under oligotrophic conditions dropped after each subsequent transfer from 241 after the first to 98 after the third transfer step. Identification on the basis of partial 16S rRNA gene sequences revealed that most of the 241 isolates (as well as the subset of 98 isolates) belong to widespread genera such as Streptomyces, Rhizobium, Bradyrhizobium, and Mesorhizobium, and the taxonomic composition of dominant genera changed from the first transfer step to the third. A selected subset of 17 isolates were further identified and characterized for exclusive growth on low-C agar medium. Two isolates continued to grow only on low-C agar medium up to the tenth transfer step and matched most closely with Rhizobium sullae and an uncultured bacterium on the basis of the almost full-length 16S rRNA gene. It was concluded that the vast majority of strains which are isolated on low-C agar media belong to the trophic group of microorganisms adapted to a "broad range" of carbon concentrations, including well-known and widespread bacterial genera. Oligotrophy is a physiological, not a taxonomic property, and can only be identified by cultural means so far. We

  9. Comparison of Methods for Isolating High Quality DNA and RNA from an Oleaginous Fungus Cunninghamella bainieri Strain 2a1

    OpenAIRE

    Noor Adila, A. K.; Farah Diba, A. B.; Zamri, Z.; Wan Mohtar, W. Y.; Aidil, A. H.; Mahadi, N. M.; Murad, A. M. A.

    2007-01-01

    A number of protocols have been reported for efficient fungal DNA and RNA isolation. However, many of these methods are often designed for certain groups or morphological forms of fungi and, in some cases, are species dependent. In this report, we compared four published protocols for DNA isolation from a locally isolated oleaginous fungus, Cunninghamella bainieri strain 2a1. These protocols either involved the use of polyvinyl pyrrolidone (PVP), hexacetyltrimethylammonium bromide (CTAB) or w...

  10. Evaluation of DNA extraction methods for Bacillus anthracis spores isolated from spiked food samples.

    Science.gov (United States)

    Thomas, M C; Shields, M J; Hahn, K R; Janzen, T W; Goji, N; Amoako, K K

    2013-07-01

    Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10-fold serial dilutions of Bacillus anthracis spores using quantitative real-time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Positive; MoBio PowerFood; ABI PrepSeq) were subsequently tested for their ability to isolate DNA from trace amounts of B. anthracis spores (approx. 6·5 × 10(1) and 1·3 × 10(2)  CFU in 25 ml or 50 g of food sample) spiked into complex food samples including apple juice, ham, whole milk and bagged salad and recovered with immunomagnetic separation (IMS). The MasterPure kit effectively and consistently isolated DNA from low amounts of B. anthracis spores captured from food samples. Detection was achieved from apple juice, ham, whole milk and bagged salad from as few as 65 ± 14, 68 ± 8, 66 ± 4 and 52 ± 16 CFU, respectively, and IMS samples were demonstrated to be free of PCR inhibitors. Detection of B. anthracis spores isolated from food by IMS differs substantially between commercial DNA extraction kits; however, sensitive results can be obtained with the MasterPure Gram Positive kit. The extraction protocol identified herein combined with IMS is novel for B. anthracis and allows detection of low levels of B. anthracis spores from contaminated food samples. © Her Majesty the Queen in Right of Canada [2013]. Reproduced with the permission of the Canadian Food Inspection Agency.

  11. 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.

  12. Identification of a DNA restriction-modification system in Pectobacterium carotovorum strains isolated from Poland.

    Science.gov (United States)

    Waleron, K; Waleron, M; Osipiuk, J; Podhajska, A J; Lojkowska, E

    2006-02-01

    Polish isolates of pectinolytic bacteria from the species Pectobacterium carotovorum were screened for the presence of a DNA restriction-modification (R-M) system. Eighty-nine strains of P. carotovorum were isolated from infected potato plants. Sixty-six strains belonged to P. carotovorum ssp. atrosepticum and 23 to P. carotovorum ssp. carotovorum. The presence of restriction enzyme Pca17AI, which is an isoschizomer of EcoRII endonuclease, was observed in all isolates of P. c. atrosepticum but not in P. c. carotovorum. The biochemical properties, PCR amplification, and sequences of the Pca17AI restriction endonuclease and methyltransferase genes were compared with the prototype EcoRII R-M system genes. Only when DNA isolated from cells of P. c. atrosepticum was used as a template, amplification of a 680 bp homologous to the gene coding EcoRII endonuclease. Endonuclease Pca17AI, having a relatively low temperature optimum, was identified. PCR amplification revealed that the nucleotide sequence of genes for EcoRII and Pca17AI R-M are different. Dcm methylation was observed in all strains of Pectobacterium and other Erwinia species tested. The sequence of a DNA fragment coding Dcm methylase in P. carotovorum was different from that of Escherichia coli. Pca17AI is the first psychrophilic isoschizomer of EcoRII endonuclease. The presence of specific Dcm methylation in chromosomal DNA isolated from P. carotovorum is described for the first time. A 680 bp PCR product, unique for P. c. atrosepticum strains, could serve as a molecular marker for detection of these bacteria in environmental samples.

  13. Ionome changes in Xylella fastidiosa-infected Nicotiana tabacum correlate with virulence and discriminate between subspecies of bacterial isolates.

    Science.gov (United States)

    Oliver, J E; Sefick, S A; Parker, J K; Arnold, T; Cobine, P A; De La Fuente, L

    2014-10-01

    Characterization of ionomes has been used to uncover the basis of nutrient utilization and environmental adaptation of plants. Here, ionomic profiles were used to understand the phenotypic response of a plant to infection by genetically diverse isolates of Xylella fastidiosa, a gram-negative, xylem-limited bacterial plant pathogen. In this study, X. fastidiosa isolates were used to infect a common model host (Nicotiana tabacum 'SR1'), and leaf and sap concentrations of eleven elements together with plant colonization and symptoms were assessed. Multivariate statistical analysis revealed that changes in the ionome were significantly correlated with symptom severity and bacterial populations in host petioles. Moreover, plant ionome modification by infection could be used to differentiate the X. fastidiosa subspecies with which the plant was infected. This report establishes host ionome modification as a phenotypic response to infection.

  14. 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.

  15. Molecular basis for the functions of a bacterial MutS2 in DNA repair and recombination.

    Science.gov (United States)

    Wang, Ge; Maier, Robert J

    2017-09-01

    Bacterial MutS2 proteins, consisting of functional domains for ATPase, DNA-binding, and nuclease activities, play roles in DNA recombination and repair. Here we observe a mechanism for generating MutS2 expression diversity in the human pathogen Helicobacter pylori, and identify a unique MutS2 domain responsible for specific DNA-binding. H. pylori strains differ in mutS2 expression due to variations in the DNA upstream sequence containing short sequence repeats. Based on Western blots, mutS2 in some strains appears to be co-translated with the upstream gene, but in other strains (e.g. UA948) such translational coupling does not occur. Accordingly, strain UA948 had phenotypes similar to its ΔmutS2 derivative, whereas expression of MutS2 at a separate locus in UA948 (the genetically complemented strain) displayed a lower mutation rate and lower transformation frequency than did ΔmutS2. A series of truncated HpMutS2 proteins were purified and tested for their specific abilities to bind 8-oxoG-containing DNA (GO:C) and Holiday Junction structures (HJ). The specific DNA binding domain was localized to an area adjacent to the Smr nuclease domain, and it encompasses 30-amino-acid-residues containing a "KPPKNKFKPPK" motif. Gel shift assays and competition assays supported that a truncated version of HpMutS2-C12 (∼12kDa protein containing the specific DNA-binding domain) has much greater capacity to bind to HJ or GO:C DNA than to normal double stranded DNA. By studying the in vivo roles of the separate domains of HpMutS2, we observed that the truncated versions were unable to complement the ΔmutS2 strain, suggesting the requirement for coordinated function of all the domains in vivo. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Surviving chytridiomycosis: differential anti-Batrachochytrium dendrobatidis activity in bacterial isolates from three lowland species of Atelopus.

    Directory of Open Access Journals (Sweden)

    Sandra V Flechas

    Full Text Available In the Neotropics, almost every species of the stream-dwelling harlequin toads (genus Atelopus have experienced catastrophic declines. The persistence of lowland species of Atelopus could be explained by the lower growth rate of Batrachochytrium dendrobatidis (Bd at temperatures above 25 °C. We tested the complementary hypothesis that the toads' skin bacterial microbiota acts as a protective barrier against the pathogen, perhaps delaying or impeding the symptomatic phase of chytridiomycosis. We isolated 148 cultivable bacterial strains from three lowland Atelopus species and quantified the anti-Bd activity through antagonism assays. Twenty-six percent (38 strains representing 12 species of the bacteria inhibited Bd growth and just two of them were shared among the toad species sampled in different localities. Interestingly, the strongest anti-Bd activity was measured in bacteria isolated from A. elegans, the only species that tested positive for the pathogen. The cutaneous bacterial microbiota is thus likely a fitness-enhancing trait that may (adaptation or not (exaptation have appeared because of natural selection mediated by chytridiomycosis. Our findings reveal bacterial strains for development of local probiotic treatments against chytridiomycosis and also shed light on the mechanisms behind the frog-bacteria-pathogen interaction.

  17. Plant-bacteria interactions: identification, characterization and localization of beneficial bacterial endophytes isolated from Vitis vinifera cv. Glera

    OpenAIRE

    Nigris, Sebastiano

    2015-01-01

    This thesis reports the results obtained during the three years PhD course focused on the study of culturable bacterial endophytes of Vitis vinifera Glera and their beneficial activities. The study, part of a large project named “EndoFlorVit project” (FEARS-UE and Regione Del Veneto), aims at investigate the biodiversity and the plant growth promoting activities of culturable endophytes isolated from Glera grapevine in vineyards of Conegliano-Valdobbiadene DOCG production area. This thesis re...

  18. 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

    2015-01-01

    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.

  19. Culturable bacterial endophytes isolated from Mangrove tree (Rhizophora apiculata Blume) enhance seedling growth in Rice

    OpenAIRE

    Deivanai, Subramanian; Bindusara, Amitraghata Santhanam; Prabhakaran, Guruswamy; Bhore, Subhash Janardhan

    2014-01-01

    Background: Endophytic bacteria do have several potential applications in medicine and in other various sectors of biotechnology including agriculture. Bacterial endophytes need to be explored for their potential applications in agricultural biotechnology. One of the potential applications of bacterial endophytes in agricultural is to enhance the growth of the agricultural crops. Hence, this study was undertaken to explore the plant growth promoting potential application of bacterial endophyt...

  20. Bacterial toxin's DNA vaccine serves as a strategy for the treatment of cancer, infectious and autoimmune diseases.

    Science.gov (United States)

    Behzadi, Elham; Halabian, Raheleh; Hosseini, Hamideh Mahmoodzadeh; Fooladi, Abbas Ali Imani

    2016-11-01

    DNA vaccination -a third generation vaccine-is a modern approach to stimulate humoral and cellular responses against different diseases such as infectious diseases, cancer and autoimmunity. These vaccines are composed of a gene that encodes sequences of a desired protein under control of a proper (eukaryotic or viral) promoter. Immune response following DNA vaccination is influenced by the route and the dose of injection. In addition, antigen presentation following DNA administration has three different mechanisms including antigen presentation by transfected myocytes, transfection of professional antigen presenting cells (APCs) and cross priming. Recently, it has been shown that bacterial toxins and their components can stimulate and enhance immune responses in experimental models. A study demonstrated that DNA fusion vaccine encoding the first domain (DOM) of the Fragment C (FrC) of tetanus neurotoxin (CTN) coupled with tumor antigen sequences is highly immunogenic against colon carcinoma. DNA toxin vaccines against infectious and autoimmune diseases are less studied until now. All in all, this novel approach has shown encouraging results in animal models, but it has to go through adequate clinical trials to ensure its effectiveness in human. However, it has been proven that these vaccines are safe, multifaceted and simple and can be used widely in organisms which may be of advantage to public health in the near future. This paper outlines the mechanism of the action of DNA vaccines and their possible application for targeting infectious diseases, cancer and autoimmunity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Efficacy of fresh leaf extracts of Spondias mombin against some clinical bacterial isolates from typhoid patients

    Directory of Open Access Journals (Sweden)

    Olukemi Aromolaran

    2014-12-01

    Full Text Available Objective: To evaluate the phytochemical properties and antibacterial activity of methanol, acetone, ethanol and aqueous extracts of fresh leaves of Spondias mombin (S. mombin on some clinical bacterial isolates. Methods: Clean and fresh leaves of S. mombin were collected in Ondo, Southwestern Nigeria. The leaves were blended, extracted with methanol, acetone, ethanol and water. The extracts were evaporated to dryness using rotary evaporator and tested for the presence of saponins, tannins, cardiac glycoside, terpenoids, flavonoids, reducing sugars, volatile oils, alkaloids and glycoside. The extract were tested against Gram-negative bacteria Klebsiella pneumonia, Serratia marcescens, Salmonella typhi and Enterobacter aerogens; Gram-positive bacteria Staphylococcus aureus by observing the zones of inhibition using agar well diffusion assay. Results: The study showed that the leaves contained saponins, tannins, flavonoids, alkaloids and glycoside. All the solvent extracts showed activity against all the test bacteria. The methanol extract also showed the highest activity against Enterobacter aerogens, zone of diameter (15.00 依1.89 mm, while the ethanol extract showed the highest activity against Staphylococcus aureus with zone of diameter (12.50依1.50 mm. The acetone extract showed the highest activity against Salmonella typhi, zone of diameter (17.50依0.29 mm followed by methanol extract showing zone of diameter (15.67依1.01 mm. The acetone extract showed the highest activity against Klebsiella pneumonia (15.17依0.67 mm, while the aqueous extract shows the highest activity against Serratia marcescens (14.67依2.68 mm. The minimum inhibitory concentration of the leaf extracts ranged between 10-90 mg/mL. Conclusions: This study showed that the aqueous and organic solvents extract of fresh leaves of S. mombin has anti-microbial activity against all the tested organisms.

  2. 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. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  3. Biocontrol of Sugarcane Smut Disease by Interference of Fungal Sexual Mating and Hyphal Growth Using a Bacterial Isolate.

    Science.gov (United States)

    Liu, Shiyin; Lin, Nuoqiao; Chen, Yumei; Liang, Zhibin; Liao, Lisheng; Lv, Mingfa; Chen, Yufan; Tang, Yingxin; He, Fei; Chen, Shaohua; Zhou, Jianuan; Zhang, Lianhui

    2017-01-01

    Sugarcane smut is a fungal disease caused by Sporisorium scitamineum , which can cause severe economic losses in sugarcane industry. The infection depends on the mating of bipolar sporida to form a dikaryon and develops into hyphae to penetrate the meristematic tissue of sugarcane. In this study, we set to isolate bacterial strains capable of blocking the fungal mating and evaluate their potential in control of sugarcane smut disease. A bacterial isolate ST4 from rhizosphere displayed potent inhibitory activity against the mating of S. scitamineum bipolar sporida and was selected for further study. Phylogenetic analyses and biochemical characterization showed that the isolate was most similar to Pseudomonas guariconensis . Methanol extracts from minimum and potato dextrose agar (PDA) agar medium, on which strain ST4 has grown, showed strong inhibitory activity on the sexual mating of S. scitamineum sporida, without killing the haploid cells MAT-1 or MAT-2. Further analysis showed that only glucose, but not sucrose, maltose, and fructose, could support strain ST4 to produce antagonistic chemicals. Consistent with the above findings, greenhouse trials showed that addition of 2% glucose to the bacterial inoculum significantly increased the strain ST4 biocontrol efficiency against sugarcane smut disease by 77% than the inoculum without glucose. The results from this study depict a new strategy to screen for biocontrol agents for control and prevention of the sugarcane smut disease.

  4. The effect of rhamnolipid biosurfactant produced by Pseudomonas fluorescens on model bacterial strains and isolates from industrial wastewater.

    Science.gov (United States)

    Vasileva-Tonkova, Evgenia; Sotirova, Anna; Galabova, Danka

    2011-02-01

    In this study, the effect of rhamnolipid biosurfactant produced by Pseudomonas fluorescens on bacterial strains, laboratory strains, and isolates from industrial wastewater was investigated. It was shown that biosurfactant, depending on the concentration, has a neutral or detrimental effect on the growth and protein release of model Gram (+) strain Bacillus subtilis 168. The growth and protein release of model Gram (-) strain Pseudomonas aeruginosa 1390 was not influenced by the presence of biosurfactant in the medium. Rhamnolipid biosurfactant at the used concentrations supported the growth of some slow growing on hexadecane bacterial isolates, members of the microbial community. Changes in cell surface hydrophobicity and permeability of some Gram (+) and Gram (-) isolates in the presence of rhamnolipid biosurfactant were followed in experiments in vitro. It was found that bacterial cells treated with biosurfactant became more or less hydrophobic than untreated cells depending on individual characteristics and abilities of the strains. For all treated strains, an increase in the amount of released protein was observed with increasing the amount of biosurfactant, probably due to increased cell permeability as a result of changes in the organization of cell surface structures. The results obtained could contribute to clarify the relationships between members of the microbial community as well as suggest the efficiency of surface properties of rhamnolipid biosurfactant from Pseudomonas fluorescens making it potentially applicable in bioremediation of hydrocarbon-polluted environments.

  5. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Plant growth-promoting activities for bacterial and fungal endophytes isolated from medicinal plant of Teucrium polium L.

    Directory of Open Access Journals (Sweden)

    Saad El-Din Hassan

    2017-11-01

    Full Text Available Bacterial and fungal endophytes are widespread inhabitants inside plant tissues and have been shown to assist plant growth and health. However, little is known about plant growth-promoting endophytes (PGPE of medicinal plants. Therefore, the aims of this study were to identify bacterial and fungal endophytes of Teucrium polium and to characterize plant growth-promoting (PGP properties of these endophytes. Seven bacterial endophytes were isolated and identified as Bacillus cereus and Bacillus subtilis, where five endophytic fungi were obtained and assigned to Penicillium chrysogenum and Penicillium crustosum. The isolated endophytes differentially produced indole acetic acid (IAA and ammonia, and in addition to their enzymatic and antimicrobial activities, they exhibited variable capacity for phosphate solubilization. In order to investigate the effect of endophytes on plant growth, four representative endophytes and their consortiums were selected concerning to their potential ability to promote plant growth. The results indicated that microbial endophytes isolated from medicinal plants possessing a vital role to improve plant growth and could be used as inoculants to establish a sustainable crop production system.

  7. Plant growth-promoting activities for bacterial and fungal endophytes isolated from medicinal plant of Teucrium polium L.

    Science.gov (United States)

    Hassan, Saad El-Din

    2017-11-01

    Bacterial and fungal endophytes are widespread inhabitants inside plant tissues and have been shown to assist plant growth and health. However, little is known about plant growth-promoting endophytes (PGPE) of medicinal plants. Therefore, the aims of this study were to identify bacterial and fungal endophytes of Teucrium polium and to characterize plant growth-promoting (PGP) properties of these endophytes. Seven bacterial endophytes were isolated and identified as Bacillus cereus and Bacillus subtilis , where five endophytic fungi were obtained and assigned to Penicillium chrysogenum and Penicillium crustosum . The isolated endophytes differentially produced indole acetic acid (IAA) and ammonia, and in addition to their enzymatic and antimicrobial activities, they exhibited variable capacity for phosphate solubilization. In order to investigate the effect of endophytes on plant growth, four representative endophytes and their consortiums were selected concerning to their potential ability to promote plant growth. The results indicated that microbial endophytes isolated from medicinal plants possessing a vital role to improve plant growth and could be used as inoculants to establish a sustainable crop production system.

  8. Ceftaroline activity against bacterial organisms isolated from acute bacterial skin and skin structure infections in United States medical centers (2009-2011).

    Science.gov (United States)

    Pfaller, Michael A; Flamm, Robert K; Sader, Helio S; Jones, Ronald N

    2014-04-01

    Ceftaroline, the active metabolite of the prodrug ceftaroline fosamil, is a new cephalosporin with bactericidal activity against resistant Gram-positive organisms including methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant strains of Streptococcus pneumoniae, and commonly isolated Gram-negative organisms, including ceftriaxone-susceptible Enterobacteriaceae. We evaluated the in vitro activity of ceftaroline and selected comparator agents against bacterial isolates collected from patients with acute bacterial skin and skin structure infections (ABSSSIs) in the USA. A total of 6222 isolates were collected from 67 medical centers distributed across all nine USA census regions between 2009 and 2011 and tested for susceptibility by reference broth microdilution methods. Ceftaroline was very active against S. aureus (MIC50/90, 0.5/1 μg/mL; 99.6% susceptible), including MRSA (MIC50/90, 0.5/1 μg/mL; 99.1% susceptible). Against β-hemolytic streptococci, the activity of ceftaroline (MIC50/90, ≤0.015/0.03 μg/mL; 100.0% susceptible) was comparable to that of both penicillin (MIC50/90, ≤0.06/≤0.06 μg/mL; 100.0% susceptible) and ceftriaxone (MIC50/90, ≤0.25/≤0.25 μg/mL; 100.0% susceptible). Ceftaroline was also highly active against viridans group streptococci (MIC50/90, 0.03/0.06 μg/mL). Similar to ceftriaxone and ceftazidime, ceftaroline was active against wild-type strains of Escherichia coli (MIC50/90, 0.12/0.25 μg/mL; 94.0% susceptible) and Klebsiella pneumoniae (MIC50/90, 0.12/0.25 μg/mL; 96.8% susceptible); however, the ceftaroline activity was compromised among strains with an extended-spectrum β-lactamase-phenotype (MIC50/90, >32/>32 μg/mL for both E. coli and K. pneumoniae). In summary, ceftaroline showed potent activity against a large contemporary collection (6222) of bacterial isolates associated with ABSSSI in the USA. © 2014.

  9. Construction of Infectious cDNA Clone of a Chrysanthemum stunt viroid Korean Isolate

    Science.gov (United States)

    Yoon, Ju-Yeon; Cho, In-Sook; Choi, Gug-Seoun; Choi, Seung-Kook

    2014-01-01

    Chrysanthemum stunt viroid (CSVd), a noncoding infectious RNA molecule, causes seriously economic losses of chrysanthemum for 3 or 4 years after its first infection. Monomeric cDNA clones of CSVd isolate SK1 (CSVd-SK1) were constructed in the plasmids pGEM-T easy vector and pUC19 vector. Linear positive-sense transcripts synthesized in vitro from the full-length monomeric cDNA clones of CSVd-SK1 could infect systemically tomato seedlings and chrysanthemum plants, suggesting that the linear CSVd RNA transcribed from the cDNA clones could be replicated as efficiently as circular CSVd in host species. However, direct inoculation of plasmid cDNA clones containing full-length monomeric cDNA of CSVd-SK1 failed to infect tomato and chrysanthemum and linear negative-sense transcripts from the plasmid DNAs were not infectious in the two plant species. The cDNA sequences of progeny viroid in systemically infected tomato and chrysanthemum showed a few substitutions at a specific nucleotide position, but there were no deletions and insertions in the sequences of the CSVd progeny from tomato and chrysanthemum plants. PMID:25288987

  10. Isolation and identification of bacterial populations of zoonotic importance from captive non-venomous snakes in Malaysia.

    Science.gov (United States)

    Abba, Yusuf; Ilyasu, Yusuf Maina; Noordin, Mustapha Mohamed

    2017-07-01

    Captivity of non-venomous snakes such as python and boa are common in zoos, aquariums and as pets in households. Poor captivity conditions expose these reptiles to numerous pathogens which may result in disease conditions. The purpose of this study was to investigate the common bacteria isolated from necropsied captive snakes in Malaysia over a five year period. A total of 27 snake carcasses presented for necropsy at the Universiti Putra Malaysia (UPM) were used in this survey. Samples were aseptically obtained at necropsy from different organs/tissues (lung, liver, heart, kindey, oesophagus, lymph node, stomach, spinal cord, spleen, intestine) and cultured onto 5% blood and McConkey agar, respectively. Gram staining, morphological evaluation and biochemical test such as oxidase, catalase and coagulase were used to tentatively identify the presumptive bacterial isolates. Pythons had the highest number of cases (81.3%) followed by anaconda (14.8%) and boa (3.7%). Mixed infection accounted for 81.5% in all snakes and was highest in pythons (63%). However, single infection was only observed in pythons (18.5%). A total of 82.7%, 95.4% and 100% of the bacterial isolates from python, anaconda and boa, respectively were gram negative. Aeromonas spp was the most frequently isolated bacteria in pythons and anaconda with incidences of 25 (18%) and 8 (36.6%) with no difference (p > 0.05) in incidence, respectively, while Salmonella spp was the most frequently isolated in boa and significantly higher (p < 0.05) than in python and anaconda. Bacteria species were most frequently isolated from the kidney of pythons 35 (25.2%), intestines of anacondas 11 (50%) and stomach of boa 3 (30%). This study showed that captive pythons harbored more bacterial species than anaconda or boa. Most of the bacterial species isolated from these snakes have public health importance and have been incriminated in human infections worldwide. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. 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.

  12. The Unculturables: targeted isolation of bacterial species associated with canine periodontal health or disease from dental plaque.

    Science.gov (United States)

    Davis, Ian J; Bull, Christopher; Horsfall, Alexander; Morley, Ian; Harris, Stephen

    2014-08-01

    The current inability to culture the entirety of observed bacteria is well known and with the advent of ever more powerful molecular tools, that can survey bacterial communities at previously unattainable depth, the gap in our capacity to culture and define all of these species increases exponentially. This gap has essentially become the rate limiting step in determining how the knowledge of which species are present in a sample can be applied to understand the role of these species in an ecosystem or disease process. A case in point is periodontal disease, which is the most widespread oral disease in dogs. If untreated the disease results in significant pain, eventual loss of the dentition and potentially an increased risk of systemic diseases. Previous molecular based studies have identified the bacterial species associated with periodontal disease in dogs; however without cultured strains from many of these species it has not been possible to study whether they play a role in the disease process. Using a quantitative polymerase chain reaction (qPCR) directed approach a range of microbiological media were screened and optimized to enrich for previously uncultivated target species. A systematic screening methodology was then employed to isolate the species of interest. In cases where the target species were not cultivable in isolation, helper strains grown underneath a nitrocellulose membrane were used to provide the necessary growth factors. This guided media optimization approach enabled the purification of 14 species, 8 of which we had previously been unable to cultivate in isolation. It is also applicable to the targeted isolation of isolates from species that have previously been cultured (for example to study intra-species variation) as demonstrated by the successful isolation of 6 targeted isolates of already cultured species. To our knowledge this is the first time this combination of qPCR guided media optimization, strategic screening and helper strain

  13. Characterization of methicillin-resistant Staphylococcus aureus isolated from healthy turkeys and broilers using DNA microarrays

    Directory of Open Access Journals (Sweden)

    Hosny El-Adawy

    2016-12-01

    Full Text Available Methicillin-resistant Staphylococcus aureus (MRSA is a major human health problem and recently, domestic animals, in particular pigs and poultry are discussed as carriers and possible reservoirs of MRSA. 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

  14. 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.

  15. Carboxyl-functionalized magnetic microparticle carrier for isolation and identification of DNA in dairy products

    Science.gov (United States)

    Horák, Daniel; Rittich, Bohuslav; Španová, Alena

    2007-04-01

    Magnetite nanoparticles about 14 nm 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 μm 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 KMnO4. 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.

  16. Carboxyl-functionalized magnetic microparticle carrier for isolation and identification of DNA in dairy products

    International Nuclear Information System (INIS)

    Horak, Daniel; Rittich, Bohuslav; Spanova, Alena

    2007-01-01

    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 1μm 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 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

  17. [Genotyping of Vaginal Candida glabrata Isolates Using Microsatellite Marker Analysis and DNA Sequencing to Identify Mutations Associated with Antifungal Resistance].

    Science.gov (United States)

    Döğen, Aylin; Durukan, Hüseyin; Güzel, Ahmet Barış; Oksüz, Zehra; Kaplan, Engin; Serin, Mehmet Sami; Serin, Ayşe; Emekdaş, Gürol; Aslan, Gönül; Tezcan, Seda; Kalkancı, Ayşe; Ilkit, Macit

    2013-01-01

    Vulvovaginal candidosis is the second most common cause of vaginitis (17-39%) after bacterial vaginosis (22-50%). Since the diagnosis of vulvovaginal candidosis mainly depends on clinical findings without mycologic confirmatory tests and treated empirically, the actual incidence rate of vulvovaginal candidosis is unknown. Approximately 70-90% of vulvovaginal candidosis cases are caused by Candida albicans, however the increasing incidence of C.glabrata infections and its reduced susceptibility to azole drug therapy have generated increasing attention. The epidemiology and population structure of vulvovaginal candidosis due to C.glabrata are poorly characterized. This study was aimed to genotype the C.glabrata strains isolated from vaginal samples in Cukurova region, Turkey by microsatellite markers, to investigate the antifungal susceptibility profiles of the strains and to determine the molecular mechanisms leading to phenotypical azole resistance. A total of 34 unrelated vaginal C.glabrata strains isolated from patients with acute (n= 11) and recurrent (n= 14) vulvovaginal candidosis, control group (n= 9) without vaginitis symptoms, and a reference strain of C.glabrata CBS 138 (ATCC 2001) were included in the study. These isolates were genotyped using multiple-locus variable number tandem repeat analysis of three microsatellite markers (RPM2, MTI, and Cg6). Analysis of microsatellite markers was performed by fragment size determination of RPM2, MTI, and Cg6 PCR products through capillary electrophoresis. For each of the evaluated strains, DNA sequence analysis was performed for one gene (CgERG11) and four loci (CgPDR1, NTM1, TRP1, and URA3) to detect mutations possibly associated with antifungal resistance in each strain. In vitro susceptibility profiles of the strains to 13 antifungals and boric acid were determined according to CLSI document M27-A3 to investigate possible relationships between detected mutations and phenotypic resistance. C.glabrata CBS 138

  18. Aspiration in head and neck cancer patients: a single centre experience of clinical profile, bacterial isolates and antibiotic sensitivity pattern.

    Science.gov (United States)

    Lakshmaiah, K C; Sirsath, Nagesh T; Subramanyam, Jayshree R; Govind, Babu K; Lokanatha, D; Shenoy, Ashok M

    2013-07-01

    Most patients with head and neck cancer have dysphagia and are at increased risk of having aspiration and subsequent pneumonia. It can cause prolonged hospitalization, treatment delay and/or interruption and mortality in cancer patients. The treatment of these infections often relies on empirical antibiotics based on local microbiology and antibiotic sensitivity patterns. The aim of present study is to analyse respiratory tract pathogens isolated by sputum culture in head and neck cancer patients undergoing treatment at a tertiary cancer centre in South India who presented with features of aspiration. The study is carried out to establish empirical antibiotic policy for head and neck cancer patients who present with features of aspiration. This was a retrospective study. The study included sputum samples sent for culture and sensitivity from January 2011 to December 2012. Analysis of microbiologic species isolated in sputum specimen and the antibiotic sensitivity pattern of the bacterial isolates was performed. A detailed study of case files of all patients was done to find out which is the most common site prone for producing aspiration. There were 47 (31.54 %) gram positive isolates and 102 (68.45 %) gram negative isolates. The most common bacterial isolates were Klebsiella pneumoniae (25.50 %), Pseudomonas aeruginosa (16.77 %) and Haemophilus influenzae (15.43 %). Levofloxacin was the most effective antibiotic with excellent activity against both gram positive and gram negative isolates. Most patients with aspiration had laryngeal cancer (34.89 %). Aspiration pneumonia was present in 14 (9.39 %) patients. Gram negative bacteria are common etiologic agents in head and neck cancer patients presenting with features of aspiration. Levofloxacin should be started as empirical antibiotic in these patients while awaiting sputum culture sensitivity report. As aspiration in head and neck cancer is an underreported event such institutional antibiotic sensitivity

  19. Enhancing DNA electro-transformation efficiency on a clinical Staphylococcus capitis isolate.

    Science.gov (United States)

    Cui, Bintao; Smooker, Peter M; Rouch, Duncan A; Deighton, Margaret A

    2015-02-01

    Clinical staphylococcus isolates possess a stronger restriction-modification (RM) barrier than laboratory strains. Clinical isolates are therefore more resistant to acceptance of foreign genetic material than laboratory strains, as their restriction systems more readily recognize and destroy foreign DNA. This stronger barrier consequently restricts genetic studies to a small number of domestic strains that are capable of accepting foreign DNA. In this study, an isolate of Staphylococcus capitis, obtained from the blood of a very low birth-weight baby, was transformed with a shuttle vector, pBT2. Optimal conditions for electro-transformation were as follows: cells were harvested at mid-log phase, electro-competent cells were prepared; cells were pre-treated at 55°C for 1min; 3μg of plasmid DNA was mixed with 70-80μL of competent cells (3-4×10(10)cells/mL) at 20°C in 0.5M sucrose, 10% glycerol; and electroporation was conducted using 2.1kV/cm field strength with a 0.1cm gap. Compared to the conventional method, which involves DNA electroporation of Staphylococcus aureus RN4220 as an intermediate strain to overcome the restriction barrier, our proposed approach exhibits a higher level (3 log10 units) of transformation efficiency. Heat treatment was used to temporarily inactivate the recipient RM barrier. Other important parameters contributing to improved electro-transformation efficiency were growth stage for cell harvesting, the quantity of DNA, the transformation temperature and field strength. The approach described here may facilitate genetic manipulations of this opportunistic pathogen. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Magnetic hydrophilic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) microspheres for DNA isolation from faeces

    Czech Academy of Sciences Publication Activity Database

    Trachtová, Š.; Obermajer, T.; Španová, A.; Matijašić, B. B.; Rogelj, I.; Horák, Daniel; Rittich, B.

    2012-01-01

    Roč. 555, č. 1 (2012), s. 263-270 ISSN 1542-1406. [International Conference on Frontiers of Polymers and Advanced Materials /11./. Pretoria, 22.05.2011-27.05.2011] R&D Projects: GA MŠk 2B06053 Institutional research plan: CEZ:AV0Z40500505 Keywords : DNA isolation * magnetic microspheres * mouse faeces Subject RIV: EE - Microbiology, Virology Impact factor: 0.530, year: 2012

  1. Occurrence, isolation and DNA identification of streptococcus thermophilus involved in algerian traditional butter 'Smen'

    OpenAIRE

    Labtar, Asmaa; Delorme, Christine; Renault, Pierre

    2011-01-01

    Streptococcus thermophilus isolates from traditional butter 'Smen', a fermented product from cow's and ewe's milk in arid area was subjected to taxonomical investigations. The identification procedure included phenotypic approaches, molecular characterization by using genus polymerase chain reaction (PCR) amplifications for sodA gene encoding the manganese-dependant superoxide dismutase A, and species-specific primers from gene encoding glucose kinase (glcK), gene encoding DNA polymerase III ...

  2. Pasteurella multocida isolated from wild birds of North America: a serotype and DNA fingerprint study of isolates from 1978 to 1993

    Science.gov (United States)

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

    1995-01-01

    Serotype and DNA fingerprint methods were used to study Pasteurella multocida isolated from 320 wild birds of North America. Isolates were collected during 1978-93. The HhaI profiles of 314 isolates matched the HhaI profile of somatic reference type 1, strain X-73; somatic type 1 antigen was expressed by 310 isolates, and the serotype of four isolates was undetected. Differentiation of the 314 isolates was observed by digestion of DNA with HpaII. None of the HpaII profiles matched the HpaII profile of X-73 (designated HhaI 001/HpaII 001). Three HpaII profiles were recognized among the somatic type 1 isolates: HpaII 002 (n = 18), HpaII 003 (n = 122), and HpaII 004 (n = 174). Profile HpaII 002 was found among isolates collected during 1979-83. Profile HpaII 003 was identified from isolates collected during 1979-89, with the exception of two isolates in 1992. The HpaII 004 profile was identified from isolates collected during 1983-93. Of the six remaining isolates, four expressed somatic type 4 and had HhaI profiles identical to the somatic type 4 reference strain P-1662 profile (designated HhaI 004); these isolates were differentiated by digestion of DNA with HpaII. One isolate was identified as serotype F:11, and another was serotype A:3,4. In the present study, 314 of 316 (99.4%) isolates from wild birds in the Central, Mississippi, and Pacific flyways during 1978-93, were P. multocida somatic type 1.

  3. Molecular Mechanisms of the Whole DNA Repair System: A Comparison of Bacterial and Eukaryotic Systems

    Directory of Open Access Journals (Sweden)

    Rihito Morita

    2010-01-01

    Full Text Available DNA is subjected to many endogenous and exogenous damages. All organisms have developed a complex network of DNA repair mechanisms. A variety of different DNA repair pathways have been reported: direct reversal, base excision repair, nucleotide excision repair, mismatch repair, and recombination repair pathways. Recent studies of the fundamental mechanisms for DNA repair processes have revealed a complexity beyond that initially expected, with inter- and intrapathway complementation as well as functional interactions between proteins involved in repair pathways. In this paper we give a broad overview of the whole DNA repair system and focus on the molecular basis of the repair machineries, particularly in Thermus thermophilus HB8.

  4. Two efficient methods for isolation of high-quality genomic DNA from entomopathogenic fungi.

    Science.gov (United States)

    Serna-Domínguez, María G; Andrade-Michel, Gilda Y; Arredondo-Bernal, Hugo C; Gallou, Adrien

    2018-03-27

    Conventional and commercial methods for isolation of nucleic acids are available for fungal samples including entomopathogenic fungi (EPF). However, there is not a unique optimal method for all organisms. The cell wall structure and the wide range of secondary metabolites of EPF can broadly interfere with the efficiency of the DNA extraction protocol. This study compares three commercial protocols: DNeasy® Plant Mini Kit (Qiagen), Wizard® Genomic DNA Purification Kit (Promega), and Axygen™ Multisource Genomic DNA Miniprep Kit (Axygen) and three conventional methods based on different buffers: SDS, CTAB/PVPP, and CTAB/β-mercaptoethanol versus three cell lysis procedures: liquid nitrogen homogenization and two bead-beating materials (i.e., tungsten-carbide and stainless-steel) for four representative species of EPF (i.e., Beauveria bassiana, Hirsutella citriformis, Isaria javanica, and Metarhizium anisopliae). Liquid nitrogen homogenization combined with DNeasy® Plant Mini Kit (i.e., QN) or SDS buffer (i.e., SN) significantly improved the yield with a good purity (~1.8) and high integrity (>20,000 bp) of genomic DNA in contrast with other methods, also, these results were better when compared with the two bead-beating materials. The purified DNA was evaluated by PCR-based techniques: amplification of translation elongation factor 1-α (TEF) and two highly sensitive molecular markers (i.e., ISSR and AFLP) with reliable and reproducible results. Despite a variation in yield, purity, and integrity of extracted DNA across the four species of EPF with the different DNA extraction methods, the SN and QN protocols maintained a high-quality of DNA which is required for downstream molecular applications. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Isolation and characterization of cDNA clones for human erythrocyte β-spectrin

    International Nuclear Information System (INIS)

    Prchal, J.T.; Morley, B.J.; Yoon, S.H.; Coetzer, T.L.; Palek, J.; Conboy, J.G.; Kan, Y.W.

    1987-01-01

    Spectrin is an important structural component of the membrane skeleton that underlies and supports the erythrocyte plasma membrane. It is composed of nonidentical α (M/sub r/ 240,000) and β (M/sub r/ 220,000) subunits, each of which contains multiple homologous 106-amino acid segments. The authors report here the isolation and characterization of a human erythroid-specific β-spectrin cDNA clone that encodes parts of the β-9 through β-12 repeat segments. This cDNA was used as a hybridization probe to assign the β-spectrin gene to human chromosome 14 and to begin molecular analysis of the gene and its mRNA transcripts. RNA transfer blot analysis showed that the reticulocyte β-spectrin mRNA is 7.8 kilobases in length. Southern blot analysis of genomic DNA revealed the presence of restriction fragment length polymorphisms (RFLPs) within the β-spectrin gene locus. The isolation of human spectrin cDNA probes and the identification of closely linked RFLPs will facilitate analysis of mutant spectrin genes causing congenital hemolytic anemias associated with quantitative and qualitative spectrin abnormalities

  6. Isolation/separation of plasmid DNA using hemoglobin modified magnetic nanocomposites as solid-phase adsorbent.

    Science.gov (United States)

    Chen, Xu-Wei; Mao, Quan-Xing; Liu, Jia-Wei; Wang, Jian-Hua

    2012-10-15

    Hemoglobin (Hb) modified magnetic nanocomposites are prepared by immobilization of Hb onto the surface of amino-functionalized Fe(3)O(4)@SiO(2) magnetic nanoparticles via covalent bonding with glutaraldehyde as cross-linker. The obtained nanocomposites are characterized with FT-IR, SEM, XRD and surface charge analysis. A direct solid-phase extraction procedure for the isolation/separation of plasmid DNA using this nanocomposite as a novel adsorbent is thus developed. Some important experimental parameters governing the sorption efficiency, i.e., the pH of sample solution and the ionic strength, are investigated. The Hb modified magnetic nanocomposites provide a sorption capacity of 27.86 mg g(-1) for DNA. By using 2.0mg of the nanocomposites as sorption medium and a suitable acidity of pH 6.1, a sorption efficiency of 93% is achieved for 25 μg mL(-1) of DNA in 1.0 mL of sample solution. Afterwards, the absorbed DNA could be readily recovered by using 1.0 mL of Tris-HCl buffer (pH 8.9, 0.01 mol L(-1)), giving rise to a recovery of ca. 68.3%. The present solid-phased extraction protocol is applied for the isolation of plasmid DNA from Escherichia coli culture, resulting in comparable yield and purity of plasmid DNA with respect to those obtained by using commercial kits. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Antibiotic Resistance and Virulence Phenotypes of Recent Bacterial Strains Isolated from Urinary Tract Infections in Elderly Patients with Prostatic Disease.

    Science.gov (United States)

    Delcaru, Cristina; Podgoreanu, Paulina; Alexandru, Ionela; Popescu, Nela; Măruţescu, Luminiţa; Bleotu, Coralia; Mogoşanu, George Dan; Chifiriuc, Mariana Carmen; Gluck, Marinela; Lazăr, Veronica

    2017-05-31

    Acute bacterial prostatitis is one of the frequent complications of urinary tract infection (UTI). From the approximately 10% of men having prostatitis, 7% experience a bacterial prostatitis. The purpose of this study was to investigate the prevalence of uropathogens associated with UTIs in older patients with benign prostatic hyperplasia and to assess their susceptibility to commonly prescribed antibiotics as well as the relationships between microbial virulence and resistance features. Uropathogenic Escherichia coli was found to be the most frequent bacterial strain isolated from patients with benign prostatic hyperplasia, followed by Enterococcus spp., Enterobacter spp., Klebsiella spp., Proteus spp., Pseudomonas aeruginosa , and Serratia marcescens . Increased resistance rates to tetracyclines, quinolones, and sulfonamides were registered. Besides their resistance profiles, the uropathogenic isolates produced various virulence factors with possible implications in the pathogenesis process. The great majority of the uropathogenic isolates revealed a high capacity to adhere to HEp-2 cell monolayer in vitro, mostly exhibiting a localized adherence pattern. Differences in the repertoire of soluble virulence factors that can affect bacterial growth and persistence within the urinary tract were detected. The Gram-negative strains produced pore-forming toxins-such as hemolysins, lecithinases, and lipases-proteases, siderophore-like molecules resulted from the esculin hydrolysis and amylases, while Enterococcus sp. strains were positive only for caseinase and esculin hydrolase. Our study demonstrates that necessity of investigating the etiology and local resistance patterns of uropathogenic organisms, which is crucial for determining appropriate empirical antibiotic treatment in elderly patients with UTI, while establishing correlations between resistance and virulence profiles could provide valuable input about the clinical evolution and recurrence rates of UTI.

  8. Antibiotic Resistance and Virulence Phenotypes of Recent Bacterial Strains Isolated from Urinary Tract Infections in Elderly Patients with Prostatic Disease

    Directory of Open Access Journals (Sweden)

    Cristina Delcaru

    2017-05-01

    Full Text Available Acute bacterial prostatitis is one of the frequent complications of urinary tract infection (UTI. From the approximately 10% of men having prostatitis, 7% experience a bacterial prostatitis. The purpose of this study was to investigate the prevalence of uropathogens associated with UTIs in older patients with benign prostatic hyperplasia and to assess their susceptibility to commonly prescribed antibiotics as well as the relationships between microbial virulence and resistance features. Uropathogenic Escherichia coli was found to be the most frequent bacterial strain isolated from patients with benign prostatic hyperplasia, followed by Enterococcus spp., Enterobacter spp., Klebsiella spp., Proteus spp., Pseudomonas aeruginosa, and Serratia marcescens. Increased resistance rates to tetracyclines, quinolones, and sulfonamides were registered. Besides their resistance profiles, the uropathogenic isolates produced various virulence factors with possible implications in the pathogenesis process. The great majority of the uropathogenic isolates revealed a high capacity to adhere to HEp-2 cell monolayer in vitro, mostly exhibiting a localized adherence pattern. Differences in the repertoire of soluble virulence factors that can affect bacterial growth and persistence within the urinary tract were detected. The Gram-negative strains produced pore-forming toxins—such as hemolysins, lecithinases, and lipases—proteases, siderophore-like molecules resulted from the esculin hydrolysis and amylases, while Enterococcus sp. strains were positive only for caseinase and esculin hydrolase. Our study demonstrates that necessity of investigating the etiology and local resistance patterns of uropathogenic organisms, which is crucial for determining appropriate empirical antibiotic treatment in elderly patients with UTI, while establishing correlations between resistance and virulence profiles could provide valuable input about the clinical evolution and

  9. 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.

  10. 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.

  11. Loss of the DNA Damage Repair Kinase ATM Impairs Inflammasome-Dependent Anti-Bacterial Innate Immunity.

    Science.gov (United States)

    Erttmann, Saskia F; Härtlova, Anetta; Sloniecka, Marta; Raffi, Faizal A M; Hosseinzadeh, Ava; Edgren, Tomas; Rofougaran, Reza; Resch, Ulrike; Fällman, Maria; Ek, Torben; Gekara, Nelson O

    2016-07-19

    The ATM kinase is a central component of the DNA damage repair machinery and redox balance. ATM dysfunction results in the multisystem disease ataxia-telangiectasia (AT). A major cause of mortality in AT is respiratory bacterial infections. Whether ATM deficiency causes innate immune defects that might contribute to bacterial infections is not known. Here we have shown that loss of ATM impairs inflammasome-dependent anti-bacterial innate immunity. Cells from AT patients or Atm(-/-) mice exhibited diminished interleukin-1β (IL-1β) production in response to bacteria. In vivo, Atm(-/-) mice were more susceptible to pulmonary S. pneumoniae infection in a manner consistent with inflammasome defects. Our data indicate that such defects were due to oxidative inhibition of inflammasome complex assembly. This study reveals an unanticipated function of reactive oxygen species (ROS) in negative regulation of inflammasomes and proposes a theory for the notable susceptibility of AT patients to pulmonary bacterial infection. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Isolation and characterization of the murine alpha-L-iduronidase cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Clarke, L.A.; Zhang, H. Nasir, J. [Univ. of British Columbia, Vancouver (Canada)] [and others

    1994-09-01

    Mucopolysaccharidosis I (MPS I) are a group of disorders caused by deficiency of the lysosomal enzyme alpha-L-iduronidase. The characterization of the human gene and the identification of mutations underlying MPS I in humans has led to the delineation of the molecular basis of this disorder. Model systems are now needed for the evaluation and development of therapeutics for this disorder. Both canine and feline models for MPS type I have been described but only the canine gene has been isolated and characterized. We report here the cloning and expression of the murine alpha-L-iduronidase cDNA. The murine cDNA was obtained by screening a mouse liver cDNA library with a probe from the human cDNA. The full length murine cDNA is 3120 base pairs in length and thus is considerably larger than both the human and canine transcripts. The increase in size is due to a 1.2 kb 3{prime} untranslated region in the murine cDNA that contains a CA dinucleotide repeat. Within the coding region the murine cDNA shows sequences. At the protein level the murine protein shows 77% similarity with the human protein and 75% similarity with the canine protein. There are significant differences in both the start and stop sites with the murine protein 9 amino acids shorter at both the N terminal signal peptide region and the C terminus. Expression of the murine cDNA in COS-1 cells resulted in a 20 fold increase in intracellular alpha-L-iduronidase activity as well as the detection of considerable enzyme activity in the culture medium. Comparison of the reported missense mutations underlying MPS I in humans (A75T, H82P, R89Q, L218P, P533R, Q310X, T366P) has shown conservation of these amino acid residues in the murine protein. The isolation of the murine iduronidase cDNA will now allow for the development of a murine model for MPS I.

  13. CHROMagar COL-APSE: a selective bacterial culture medium for the isolation and differentiation of colistin-resistant Gram-negative pathogens

    DEFF Research Database (Denmark)

    Abdul Momin, Muhd Haziq F; Bean, David C; Hendriksen, Rene S.

    2017-01-01

    Purpose. A selective chromogenic culture medium for the laboratory isolation and differentiation of colistin resistant Acinetobacter, Pseudomonas, Stenotrophomonas and Enterobacteriaceae spp. (CHROMagar COL-APSE) was developed, evaluated and compared to an existing selective bacterial culture...

  14. First Isolation and Molecular Characterization of Bacteriophages Infecting Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch

    Directory of Open Access Journals (Sweden)

    Aryan Rahimi-Midani

    2018-02-01

    Full Text Available Bacteriophages of Acidovorax citrulli, the causal agent of bacterial fruit blotch, were isolated from 39 watermelon, pumpkin, and cucumber leaf samples collected from various regions of Korea and tested against 18 A. citrulli strains. Among the six phages isolated, ACP17 forms the largest plaque, and exhibits the morphology of phages in the Myoviridae family with a head diameter of 100 ± 5 nm and tail length of 150 ± 5 nm. ACP17 has eclipse and latent periods of 25 ± 5 min and 50 ± 5 min, respectively, and a burst size of 120. The genome of ACP17 is 156,281 base pairs with a G + C content of 58.7%, 263 open reading frames, and 4 transfer RNA genes. Blast search and phylogenetic analysis of the major capsid protein showed that ACP17 has limited homology to two Stentrophomonas phages, suggesting that ACP17 is a new type of Myoviridae isolated from A. citrulli.

  15. Conformational Diversity of Single-Stranded DNA from Bacterial Repetitive Extragenic Palindromes: Implications for the DNA Recognition Elements of Transposases

    Czech Academy of Sciences Publication Activity Database

    Charnavets, Tatsiana; Nunvář, Jaroslav; Nečasová, Iva; Voelker, J.; Breslauer, K.J.; Schneider, Bohdan

    2015-01-01

    Roč. 103, č. 10 (2015), s. 585-596 ISSN 0006-3525 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109; GA ČR GAP305/12/1801; GA MŠk(CZ) EE2.3.30.0020 Institutional support: RVO:86652036 Keywords : bacterial repetitive extragenic palindromes (REP) * circular dichroism spectroscopy * REP associated tyrosine transposases (RAYTs) Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.248, year: 2015

  16. 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.

  17. Use of Repetitive DNA Sequences and the PCR To Differentiate Escherichia coli Isolates from Human and Animal Sources

    Science.gov (United States)

    Dombek, Priscilla E.; Johnson, LeeAnn K.; Zimmerley, Sara T.; Sadowsky, Michael J.

    2000-01-01

    The rep-PCR DNA fingerprint technique, which uses repetitive intergenic DNA sequences, was investigated as a way to differentiate between human and animal sources of fecal pollution. BOX and REP primers were used to generate DNA fingerprints from Escherichia coli strains isolated from human and animal sources (geese, ducks, cows, pigs, chickens, and sheep). Our initial studies revealed that the DNA fingerprints obtained with the BOX primer were more effective for grouping E. coli strains than the DNA fingerprints obtained with REP primers. The BOX primer DNA fingerprints of 154 E. coli isolates were analyzed by using the Jaccard band-matching algorithm. Jackknife analysis of the resulting similarity coefficients revealed that 100% of the chicken and cow isolates and between 78 and 90% of the human, goose, duck, pig, and sheep isolates were assigned to the correct source groups. A dendrogram constructed by using Jaccard similarity coefficients almost completely separated the human isolates from the nonhuman isolates. Multivariate analysis of variance, a form of discriminant analysis, successfully differentiated the isolates and placed them in the appropriate source groups. Taken together, our results indicate that rep-PCR performed with the BOX A1R primer may be a useful and effective tool for rapidly determining sources of fecal pollution. PMID:10831440

  18. Poly-β-hydroxybutyrate and exopolysaccharide biosynthesis by bacterial isolates from pigeonpea [Cajanus cajan (L.) Millsp] root nodules.

    Science.gov (United States)

    Fernandes, Paulo Ivan; de Oliveira, Paulo Jansen; Rumjanek, Norma Gouvêa; Xavier, Gustavo Ribeiro

    2011-02-01

    The bacterial strains that are able to produce biopolymers that are applied in industrial sectors present a source of renewable resources. Some microorganisms are already applied at several industrial sectors, but the prospecting of new microbes must bring microorganisms that are feasible to produce interesting biopolymers more efficiently and in cheaper conditions. Among the biopolymers applied industrially, polyhydroxybutyrate (PHB) and exopolysaccharides (EPS) stand out because of its applications, mainly in biodegradable plastic production and in food industry, respectively. In this context, the capacity of bacteria isolated from pigeonpea root nodules to produce EPS and PHB was evaluated, as well as the cultural characterization of these isolates. Among the 38 isolates evaluated, the majority presented fast growth and ability to acidify the culture media. Regarding the biopolymer production, five isolates produced more than 10 mg PHB per liter of culture medium. Six EPS producing bacteria achieved more than 200 mg EPS per liter of culture medium. Evaluating different carbon sources, the PHB productivity of the isolate 24.6b reached 69% of cell dry weight when cultured with starch as sole carbon source, and the isolate 8.1c synthesized 53% PHB in dry cell biomass and more than 1.3 g L⁻¹ of EPS when grown using xylose as sole carbon source.

  19. Comparison of three methods of parasitoid polydnavirus genomic DNA isolation to facilitate polydnavirus genomic sequencing.

    Science.gov (United States)

    Rodríguez-Pérez, Mario A; Beckage, Nancy E

    2008-04-01

    A major long-term goal of polydnavirus (PDV) genome research is to identify novel virally encoded molecules that may serve as biopesticides to target insect pests that threaten agriculture and human health. As PDV viral replication in cell culture in vitro has not yet been achieved, several thousands of wasps must be dissected to yield enough viral DNA from the adult ovaries to carry out PDV genomic sequencing. This study compares three methods of PDV genomic DNA isolation for the PDV of Cotesia flavipes, which parasitizes the sugarcane borer, Diatraea saccharalis, preparatory to sequencing the C. flavipes bracovirus genome. Two of these protocols incorporate phenol-chloroform DNA extraction steps in the procedure and the third protocol uses a modified Qiagen DNA kit method to extract viral DNA. The latter method proved significantly less time-consuming and more cost-effective. Efforts are currently underway to bioengineer insect pathogenic viruses with PDV genes, so that their gene products will enhance baculovirus virulence for agricultural insect pests, either via suppression of the immune system of the host or by PDV-mediated induction of its developmental arrest. Sequencing a growing number of complete PDV genomes will enhance those efforts, which will be facilitated by the study reported here. (c) 2008 Wiley-Liss, Inc.

  20. Filtration Isolation of Nucleic Acids: A Simple and Rapid DNA Extraction Method.

    Science.gov (United States)

    McFall, Sally M; Neto, Mário F; Reed, Jennifer L; Wagner, Robin L

    2016-08-06

    FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 µl whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.